Effect of Chromatin Structure on the Extent and Distribution of DNA Double Strand Breaks Produced by Ionizing Radiation; Comparative Study of hESC and Differentiated Cells Lines.

PubMed

Venkatesh, Priyanka; Panyutin, Irina V; Remeeva, Evgenia; Neumann, Ronald D; Panyutin, Igor G

2016-01-02

Chromatin structure affects the extent of DNA damage and repair. Thus, it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here, we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC, heterochromatin is confined to distinct regions, while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives, normal human fibroblasts; and one cancer cell line. At the same time, the number of DNA repair foci were not statistically different among these cells. We showed that in hESC, DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells, and to a lesser extent in IMR90 normal fibroblasts, but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC.

Sub-micrometer 20MeV protons or 45MeV lithium spot irradiation enhances yields of dicentric chromosomes due to clustering of DNA double-strand breaks.

PubMed

Schmid, T E; Friedland, W; Greubel, C; Girst, S; Reindl, J; Siebenwirth, C; Ilicic, K; Schmid, E; Multhoff, G; Schmitt, E; Kundrát, P; Dollinger, G

2015-11-01

In conventional experiments on biological effects of radiation types of diverse quality, micrometer-scale double-strand break (DSB) clustering is inherently interlinked with clustering of energy deposition events on nanometer scale relevant for DSB induction. Due to this limitation, the role of the micrometer and nanometer scales in diverse biological endpoints cannot be fully separated. To address this issue, hybrid human-hamster AL cells have been irradiated with 45MeV (60keV/μm) lithium ions or 20MeV (2.6keV/μm) protons quasi-homogeneously distributed or focused to 0.5×1μm(2) spots on regular matrix patterns (point distances up to 10.6×10.6μm), with pre-defined particle numbers per spot to provide the same mean dose of 1.7Gy. The yields of dicentrics and their distribution among cells have been scored. In parallel, track-structure based simulations of DSB induction and chromosome aberration formation with PARTRAC have been performed. The results show that the sub-micrometer beam focusing does not enhance DSB yields, but significantly affects the DSB distribution within the nucleus and increases the chance to form DSB pairs in close proximity, which may lead to increased yields of chromosome aberrations. Indeed, the experiments show that focusing 20 lithium ions or 451 protons per spot on a 10.6μm grid induces two or three times more dicentrics, respectively, than a quasi-homogenous irradiation. The simulations reproduce the data in part, but in part suggest more complex behavior such as saturation or overkill not seen in the experiments. The direct experimental demonstration that sub-micrometer clustering of DSB plays a critical role in the induction of dicentrics improves the knowledge on the mechanisms by which these lethal lesions arise, and indicates how the assumptions of the biophysical model could be improved. It also provides a better understanding of the increased biological effectiveness of high-LET radiation. Copyright © 2015 Elsevier B.V. All rights reserved.

Estimation of the radiation-induced DNA double-strand breaks number by considering cell cycle and absorbed dose per cell nucleus

PubMed Central

Mori, Ryosuke; Matsuya, Yusuke; Yoshii, Yuji; Date, Hiroyuki

2018-01-01

Abstract DNA double-strand breaks (DSBs) are thought to be the main cause of cell death after irradiation. In this study, we estimated the probability distribution of the number of DSBs per cell nucleus by considering the DNA amount in a cell nucleus (which depends on the cell cycle) and the statistical variation in the energy imparted to the cell nucleus by X-ray irradiation. The probability estimation of DSB induction was made following these procedures: (i) making use of the Chinese Hamster Ovary (CHO)-K1 cell line as the target example, the amounts of DNA per nucleus in the logarithmic and the plateau phases of the growth curve were measured by flow cytometry with propidium iodide (PI) dyeing; (ii) the probability distribution of the DSB number per cell nucleus for each phase after irradiation with 1.0 Gy of 200 kVp X-rays was measured by means of γ-H2AX immunofluorescent staining; (iii) the distribution of the cell-specific energy deposition via secondary electrons produced by the incident X-rays was calculated by WLTrack (in-house Monte Carlo code); (iv) according to a mathematical model for estimating the DSB number per nucleus, we deduced the induction probability density of DSBs based on the measured DNA amount (depending on the cell cycle) and the calculated dose per nucleus. The model exhibited DSB induction probabilities in good agreement with the experimental results for the two phases, suggesting that the DNA amount (depending on the cell cycle) and the statistical variation in the local energy deposition are essential for estimating the DSB induction probability after X-ray exposure. PMID:29800455

Estimation of the radiation-induced DNA double-strand breaks number by considering cell cycle and absorbed dose per cell nucleus.

PubMed

Mori, Ryosuke; Matsuya, Yusuke; Yoshii, Yuji; Date, Hiroyuki

2018-05-01

DNA double-strand breaks (DSBs) are thought to be the main cause of cell death after irradiation. In this study, we estimated the probability distribution of the number of DSBs per cell nucleus by considering the DNA amount in a cell nucleus (which depends on the cell cycle) and the statistical variation in the energy imparted to the cell nucleus by X-ray irradiation. The probability estimation of DSB induction was made following these procedures: (i) making use of the Chinese Hamster Ovary (CHO)-K1 cell line as the target example, the amounts of DNA per nucleus in the logarithmic and the plateau phases of the growth curve were measured by flow cytometry with propidium iodide (PI) dyeing; (ii) the probability distribution of the DSB number per cell nucleus for each phase after irradiation with 1.0 Gy of 200 kVp X-rays was measured by means of γ-H2AX immunofluorescent staining; (iii) the distribution of the cell-specific energy deposition via secondary electrons produced by the incident X-rays was calculated by WLTrack (in-house Monte Carlo code); (iv) according to a mathematical model for estimating the DSB number per nucleus, we deduced the induction probability density of DSBs based on the measured DNA amount (depending on the cell cycle) and the calculated dose per nucleus. The model exhibited DSB induction probabilities in good agreement with the experimental results for the two phases, suggesting that the DNA amount (depending on the cell cycle) and the statistical variation in the local energy deposition are essential for estimating the DSB induction probability after X-ray exposure.

Activity and interactions of methane seep microorganisms assessed by parallel transcription and FISH-NanoSIMS analyses

PubMed Central

Dekas, Anne E; Connon, Stephanie A; Chadwick, Grayson L; Trembath-Reichert, Elizabeth; Orphan, Victoria J

2016-01-01

To characterize the activity and interactions of methanotrophic archaea (ANME) and Deltaproteobacteria at a methane-seeping mud volcano, we used two complimentary measures of microbial activity: a community-level analysis of the transcription of four genes (16S rRNA, methyl coenzyme M reductase A (mcrA), adenosine-5′-phosphosulfate reductase α-subunit (aprA), dinitrogenase reductase (nifH)), and a single-cell-level analysis of anabolic activity using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS). Transcript analysis revealed that members of the deltaproteobacterial groups Desulfosarcina/Desulfococcus (DSS) and Desulfobulbaceae (DSB) exhibit increased rRNA expression in incubations with methane, suggestive of ANME-coupled activity. Direct analysis of anabolic activity in DSS cells in consortia with ANME by FISH-NanoSIMS confirmed their dependence on methanotrophy, with no 15NH4+ assimilation detected without methane. In contrast, DSS and DSB cells found physically independent of ANME (i.e., single cells) were anabolically active in incubations both with and without methane. These single cells therefore comprise an active ‘free-living' population, and are not dependent on methane or ANME activity. We investigated the possibility of N2 fixation by seep Deltaproteobacteria and detected nifH transcripts closely related to those of cultured diazotrophic Deltaproteobacteria. However, nifH expression was methane-dependent. 15N2 incorporation was not observed in single DSS cells, but was detected in single DSB cells. Interestingly, 15N2 incorporation in single DSB cells was methane-dependent, raising the possibility that DSB cells acquired reduced 15N products from diazotrophic ANME while spatially coupled, and then subsequently dissociated. With this combined data set we address several outstanding questions in methane seep microbial ecosystems and highlight the benefit of measuring microbial activity in the context of spatial associations. PMID:26394007

Activity and interactions of methane seep microorganisms assessed by parallel transcription and FISH-NanoSIMS analyses

DOE PAGES

Dekas, Anne E.; Connon, Stephanie A.; Chadwick, Grayson L.; ...

2015-09-22

To characterize the activity and interactions of methanotrophic archaea (ANME) and Deltaproteo-bacteria at a methane-seeping mud volcano, we used two complimentary measures of microbial activity: a community-level analysis of the transcription of four genes (16S rRNA, methyl coenzyme M reductase A (mcrA), adenosine-5'-phosphosulfate reductase α-subunit (aprA), dinitrogenase reductase (nifH)), and a single-cell-level analysis of anabolic activity using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS). Transcript analysis revealed that members of the deltaproteobacterial groups Desulfosarcina/Desulfococcus (DSS) and Desulfobulbaceae (DSB) exhibit increased rRNA expression in incubations with methane, suggestive of ANME-coupled activity. Direct analysis of anabolic activity in DSS cells in consortia with ANME by FISH-NanoSIMS confirmed their dependence on methanotrophy, with no 15NHmore » $$+\\atop{4}$$ assimilation detected without methane. In contrast, DSS and DSB cells found physically independent of ANME (i.e., single cells) were anabolically active in incubations both with and without methane. These single cells therefore comprise an active ‘free-living’ population, and are not dependent on methane or ANME activity. We investigated the possibility of N 2 fixation by seep Deltaproteobacteria and detected nifH transcripts closely related to those of cultured diazotrophic Deltaproteobacteria. However, nifH expression was methane-dependent. 15N 2 incorporation was not observed in single DSS cells, but was detected in single DSB cells. Interestingly, 15N 2 incorporation in single DSB cells was methane-dependent, raising the possibility that DSB cells acquired reduced 15N products from diazotrophic ANME while spatially coupled, and then subsequently dissociated. In conclusion, with this combined data set we address several outstanding questions in methane seep microbial ecosystems and highlight the benefit of measuring microbial activity in the context of spatial associations.« less

Image-based modeling of radiation-induced foci

NASA Astrophysics Data System (ADS)

Costes, Sylvain; Cucinotta, Francis A.; Ponomarev, Artem; Barcellos-Hoff, Mary Helen; Chen, James; Chou, William; Gascard, Philippe

Several proteins involved in the response to DNA double strand breaks (DSB) form microscopically visible nuclear domains, or foci, after exposure to ionizing radiation. Radiation-induced foci (RIF) are believed to be located where DNA damage occurs. To test this assumption, we used Monte Carlo simulations to predict the spatial distribution of DSB in human nuclei exposed to high or low-LET radiation. We then compared these predictions to the distribution patterns of three DNA damage sensing proteins, i.e. 53BP1, phosphorylated ATM and γH2AX in human mammary epithelial. The probability to induce DSB can be derived from DNA fragment data measured experimentally by pulsed-field gel electrophoresis. We first used this probability in Monte Carlo simulations to predict DSB locations in synthetic nuclei geometrically described by a complete set of human chromosomes, taking into account microscope optics from real experiments. Simulations showed a very good agreement for high-LET, predicting 0.7 foci/µm along the path of a 1 GeV/amu Fe particle against measurement of 0.69 to 0.82 foci/µm for various RIF 5 min following exposure (LET 150 keV/µm). On the other hand, discrepancies were shown in foci frequency for low-LET, with measurements 20One drawback using a theoretical model for the nucleus is that it assumes a simplistic and static pattern for DNA densities. However DNA damage pattern is highly correlated to DNA density pattern (i.e. the more DNA, the more likely to have a break). Therefore, we generalized our Monte Carlo approach to real microscope images, assuming pixel intensity of DAPI in the nucleus was directly proportional to the amount of DNA in that pixel. With such approach we could predict DNA damage pattern in real images on a per nucleus basis. Since energy is randomly deposited along high-LET particle paths, RIF along these paths should also be randomly distributed. As expected, simulations produced DNA-weighted random (Poisson) distributions. In contrast, the distributions of RIF obtained as early as 5 min after exposure to high LET (1 GeV/amu Fe) were non-random. This deviation from the expected DNA-weighted random pattern was further characterized by "relative DNA image measurements". This novel imaging approach showed that RIF were located preferentially at the interface between high and low DNA density regions, and were more frequent than predicted in regions with lower DNA density. The same preferential nuclear location was also measured for RIF induced by 1 Gy of low-LET radiation. This deviation from random behavior was evident only 5 min after irradiation for phosphorylated ATM RIF, while γH2AX and 53BP1 RIF showed pronounced deviations up to 30 min after exposure. These data suggest that RIF within a few minutes following exposure to radiation cluster into open regions of the nucleus (i.e. euchromatin). It is possible that DNA lesions are collected in these nuclear sub-domains for more efficient repair. If so, this would imply that DSB are actively transported within the nucleus, a phenomenon that has not yet been considered in modeling DNA misrepair following exposure to radiation. These results are thus critical for more accurate risk models of radiation and we are actively working on characterizing further RIF movement in human nuclei using live cell imaging.

Generalized time-dependent model of radiation-induced chromosomal aberrations in normal and repair-deficient human cells.

PubMed

Ponomarev, Artem L; George, Kerry; Cucinotta, Francis A

2014-03-01

We have developed a model that can simulate the yield of radiation-induced chromosomal aberrations (CAs) and unrejoined chromosome breaks in normal and repair-deficient cells. The model predicts the kinetics of chromosomal aberration formation after exposure in the G₀/G₁ phase of the cell cycle to either low- or high-LET radiation. A previously formulated model based on a stochastic Monte Carlo approach was updated to consider the time dependence of DNA double-strand break (DSB) repair (proper or improper), and different cell types were assigned different kinetics of DSB repair. The distribution of the DSB free ends was derived from a mechanistic model that takes into account the structure of chromatin and DSB clustering from high-LET radiation. The kinetics of chromosomal aberration formation were derived from experimental data on DSB repair kinetics in normal and repair-deficient cell lines. We assessed different types of chromosomal aberrations with the focus on simple and complex exchanges, and predicted the DSB rejoining kinetics and misrepair probabilities for different cell types. The results identify major cell-dependent factors, such as a greater yield of chromosome misrepair in ataxia telangiectasia (AT) cells and slower rejoining in Nijmegen (NBS) cells relative to the wild-type. The model's predictions suggest that two mechanisms could exist for the inefficiency of DSB repair in AT and NBS cells, one that depends on the overall speed of joining (either proper or improper) of DNA broken ends, and another that depends on geometric factors, such as the Euclidian distance between DNA broken ends, which influences the relative frequency of misrepair.

Non-random distribution of DNA double-strand breaks induced by particle irradiation

NASA Technical Reports Server (NTRS)

Lobrich, M.; Cooper, P. K.; Rydberg, B.; Chatterjee, A. (Principal Investigator)

1996-01-01

Induction of DNA double-strand breaks (dsbs) in mammalian cells is dependent on the spatial distribution of energy deposition from the ionizing radiation. For high LET particle radiations the primary ionization sites occur in a correlated manner along the track of the particles, while for X-rays these sites are much more randomly distributed throughout the volume of the cell. It can therefore be expected that the distribution of dsbs linearly along the DNA molecule also varies with the type of radiation and the ionization density. Using pulsed-field gel and conventional gel techniques, we measured the size distribution of DNA molecules from irradiated human fibroblasts in the total range of 0.1 kbp-10 Mbp for X-rays and high LET particles (N ions, 97 keV/microns and Fe ions, 150 keV/microns). On a mega base pair scale we applied conventional pulsed-field gel electrophoresis techniques such as measurement of the fraction of DNA released from the well (FAR) and measurement of breakage within a specific NotI restriction fragment (hybridization assay). The induction rate for widely spaced breaks was found to decrease with LET. However, when the entire distribution of radiation-induced fragments was analysed, we detected an excess of fragments with sizes below about 200 kbp for the particles compared with X-irradiation. X-rays are thus more effective than high LET radiations in producing large DNA fragments but less effective in the production of smaller fragments. We determined the total induction rate of dsbs for the three radiations based on a quantitative analysis of all the measured radiation-induced fragments and found that the high LET particles were more efficient than X-rays at inducing dsbs, indicating an increasing total efficiency with LET. Conventional assays that are based only on the measurement of large fragments are therefore misleading when determining total dsb induction rates of high LET particles. The possible biological significance of this non-randomness for dsb induction is discussed.

Targeting PRMT5 as a Novel Radiosensitization Approach for Primary and Recurrent Prostate Cancer Treatment

DTIC Science & Technology

2016-10-01

Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author(s) and should not...with the expression of the androgen receptor ( AR ). Mechanistically PRMT5 epigenetically activates transcription of AR . Second, we have discovered two...DNA double-strand break (DSB) via homologous recombination and non-homologous end joining. Given that AR , DSB repair and NED all contribute to

The spatial regulation of meiotic recombination hotspots: are all DSB hotspots crossover hotspots?

PubMed

Serrentino, Maria-Elisabetta; Borde, Valérie

2012-07-15

A key step for the success of meiosis is programmed homologous recombination, during which crossovers, or exchange of chromosome arms, take place. Crossovers increase genetic diversity but their main function is to ensure accurate chromosome segregation. Defects in crossover number and position produce aneuploidies that represent the main cause of miscarriages and chromosomal abnormalities such as Down's syndrome. Recombination is initiated by the formation of programmed double strand breaks (DSBs), which occur preferentially at places called DSB hotspots. Among all DSBs generated, only a small fraction is repaired by crossover, the other being repaired by other homologous recombination pathways. Crossover maps have been generated in a number of organisms, defining crossover hotspots. With the availability of genome-wide maps of DSBs as well as the ability to measure genetically the repair outcome at several hotspots, it is becoming more and more clear that not all DSB hotspots behave the same for crossover formation, suggesting that chromosomal features distinguish different types of hotspots. Copyright © 2012. Published by Elsevier Inc.

The Distribution of Chromosomal Aberrations in Human Cells Predicted by a Generalized Time-Dependent Model of Radiation-Induced Formation of Aberrations

NASA Technical Reports Server (NTRS)

Ponomarev, Artem L.; George, K.; Cucinotta, F. A.

2011-01-01

New experimental data show how chromosomal aberrations for low- and high-LET radiation are dependent on DSB repair deficiencies in wild-type, AT and NBS cells. We simulated the development of chromosomal aberrations in these cells lines in a stochastic track-structure-dependent model, in which different cells have different kinetics of DSB repair. We updated a previously formulated model of chromosomal aberrations, which was based on a stochastic Monte Carlo approach, to consider the time-dependence of DSB rejoining. The previous version of the model had an assumption that all DSBs would rejoin, and therefore we called it a time-independent model. The chromosomal-aberrations model takes into account the DNA and track structure for low- and high-LET radiations, and provides an explanation and prediction of the statistics of rare and more complex aberrations. We compared the program-simulated kinetics of DSB rejoining to the experimentally-derived bimodal exponential curves of the DSB kinetics. We scored the formation of translocations, dicentrics, acentric and centric rings, deletions, and inversions. The fraction of DSBs participating in aberrations was studied in relation to the rejoining time. Comparisons of simulated dose dependence for simple aberrations to the experimental dose-dependence for HF19, AT and NBS cells will be made.

Relationship between radiation-induced aberrations in individual chromosomes and their DNA content: effects of interaction distance

NASA Technical Reports Server (NTRS)

Wu, H.; Durante, M.; Lucas, J. N.

2001-01-01

PURPOSE: To study the effect of the interaction distance on the frequency of inter- and intrachromosome exchanges in individual chromosomes with respect to their DNA content. Assumptions: Chromosome exchanges are formed by misrejoining of two DNA double-strand breaks (DSB) induced within an interaction distance, d. It is assumed that chromosomes in G(0)/G(1) phase of the cell cycle occupy a spherical domain in a cell nucleus, with no spatial overlap between individual chromosome domains. RESULTS: Formulae are derived for the probability of formation of inter-, as well as intra-, chromosome exchanges relating to the DNA content of the chromosome for a given interaction distance. For interaction distances <1 microm, the relative frequency of interchromosome exchanges predicted by the present model is similar to that by Cigarran et al. (1998) based on the assumption that the probability of interchromosome exchanges is proportional to the "surface area" of the chromosome territory. The "surface area" assumption is shown to be a limiting case of d-->0 in the present model. The present model also predicts that the probability of intrachromosome exchanges occurring in individual chromosomes is proportional to their DNA content with correction terms. CONCLUSION: When the interaction distance is small, the "surface area" distribution for chromosome participation in interchromosome exchanges has been expected. However, the present model shows that for the interaction distance as large as 1 microm, the predicted probability of interchromosome exchange formation is still close to the surface area distribution. Therefore, this distribution does not necessarily rule out the formation of complex chromosomal aberrations by long-range misrejoining of DSB.

The α-proteobacteria Wolbachia pipientis protein disulfide machinery has a regulatory mechanism absent in γ-proteobacteria.

PubMed

Walden, Patricia M; Halili, Maria A; Archbold, Julia K; Lindahl, Fredrik; Fairlie, David P; Inaba, Kenji; Martin, Jennifer L

2013-01-01

The α-proteobacterium Wolbachia pipientis infects more than 65% of insect species worldwide and manipulates the host reproductive machinery to enable its own survival. It can live in mutualistic relationships with hosts that cause human disease, including mosquitoes that carry the Dengue virus. Like many other bacteria, Wolbachia contains disulfide bond forming (Dsb) proteins that introduce disulfide bonds into secreted effector proteins. The genome of the Wolbachia strain wMel encodes two DsbA-like proteins sharing just 21% sequence identity to each other, α-DsbA1 and α-DsbA2, and an integral membrane protein, α-DsbB. α-DsbA1 and α-DsbA2 both have a Cys-X-X-Cys active site that, by analogy with Escherichia coli DsbA, would need to be oxidized to the disulfide form to serve as a disulfide bond donor toward substrate proteins. Here we show that the integral membrane protein α-DsbB oxidizes α-DsbA1, but not α-DsbA2. The interaction between α-DsbA1 and α-DsbB is very specific, involving four essential cysteines located in the two periplasmic loops of α-DsbB. In the electron flow cascade, oxidation of α-DsbA1 by α-DsbB is initiated by an oxidizing quinone cofactor that interacts with the cysteine pair in the first periplasmic loop. Oxidizing power is transferred to the second cysteine pair, which directly interacts with α-DsbA1. This reaction is inhibited by a non-catalytic disulfide present in α-DsbA1, conserved in other α-proteobacterial DsbAs but not in γ-proteobacterial DsbAs. This is the first characterization of the integral membrane protein α-DsbB from Wolbachia and reveals that the non-catalytic cysteines of α-DsbA1 regulate the redox relay system in cooperation with α-DsbB.

Description of the topographical changes associated to the different stages of the DsbA catalytic cycle

PubMed Central

Vinci, Floriana; Couprie, Joël; Pucci, Piero; Quéméneur, Eric; Moutiez, Mireille

2002-01-01

This paper provides a description of the surface topography of DsbA, the bacterial disulfide-bond forming enzyme, in the different phases of its catalytic cycle. Three representative states, that is, oxidized and reduced protein and a covalent complex mimicking the DsbA-substrate disulfide intermediate, have been investigated by a combination of limited proteolysis experiments and mass spectrometry methodologies. Protease-accessible sites are largely distributed in the oxidized form with a small predominance inside the thioredoxin domain. Proteolysis occurs even in secondary structure elements, revealing a significant mobility of the protein. Many cleavage sites disappear in the reduced form and most of the remaining ones appear with strongly reduced kinetics. The protein within the complex shows an intermediate behavior. This variation of flexibility in DsbA is probably the determining factor for the course of its catalytic cycle. In particular, the great mobility of the oxidized protein might facilitate the accommodation of its various substrates, whereas the increasing rigidity from the complexed to the reduced form could help the release of oxidized products. The formation of the complex between PID peptide and DsbA does not significantly protect the enzyme against proteolysis, reinforcing the results previously obtained by calorimetry concerning the weakness of their interaction. The few cleavage sites observed, however, are in favor of the presence of the peptide in the binding site postulated from crystallographic studies. As for the peptide itself, the proteolytic pattern and the protection effect exerted by DsbA could be explained by a preferential orientation within the binding site. PMID:12070313

DNA-incorporated 125I induces more than one double-strand break per decay in mammalian cells.

PubMed

Elmroth, Kecke; Stenerlöw, Bo

2005-04-01

The Auger-electron emitter 125I releases cascades of 20 electrons per decay that deposit a great amount of local energy, and for DNA-incorporated 125I, approximately one DNA double-strand break (DSB) is produced close to the decay site. To investigate the potential of 125I to induce additional DSBs within adjacent chromatin structures in mammalian cells, we applied DNA fragment-size analysis based on pulsed-field gel electrophoresis (PFGE) of hamster V79-379A cells exposed to DNA-incorporated 125IdU. After accumulation of decays at -70 degrees C in the presence of 10% DMSO, there was a non-random distribution of DNA fragments with an excess of fragments <0.5 Mbp and the measured yield was 1.6 DSBs/decay. However, since these experiments were performed under high scavenging conditions (DMSO) that reduce indirect effects, the yield in cells exposed to 125IdU under physiological conditions would most likely be even higher. In contrast, using a conventional low-resolution assay without measurement of smaller DNA fragments, the yield was close to one DSB/decay. We conclude that a large fraction of the DSBs induced by DNA-incorporated 125I are nonrandomly distributed and that significantly more than one DSB/decay is induced in an intact cell. Thus, in addition to DSBs produced close to the decay site, DSBs may also be induced within neighboring chromatin fibers, releasing smaller DNA fragments that are not detected by conventional DSB assays.

DNA fragmentation induced by Fe ions in human cells: shielding influence on spatially correlated damage

NASA Technical Reports Server (NTRS)

Antonelli, F.; Belli, M.; Campa, A.; Chatterjee, A.; Dini, V.; Esposito, G.; Rydberg, B.; Simone, G.; Tabocchini, M. A.

2004-01-01

Outside the magnetic field of the Earth, high energy heavy ions constitute a relevant part of the biologically significant dose to astronauts during the very long travels through space. The typical pattern of energy deposition in the matter by heavy ions on the microscopic scale is believed to produce spatially correlated damage in the DNA which is critical for radiobiological effects. We have investigated the influence of a lucite shielding on the initial production of very small DNA fragments in human fibroblasts irradiated with 1 GeV/u iron (Fe) ions. We also used gamma rays as reference radiation. Our results show: (1) a lower effect per incident ion when the shielding is used; (2) an higher DNA Double Strand Breaks (DSB) induction by Fe ions than by gamma rays in the size range 1-23 kbp; (3) a non-random DNA DSB induction by Fe ions. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

Functional and bioinformatics analysis of two Campylobacter jejuni homologs of the thiol-disulfide oxidoreductase, DsbA.

PubMed

Grabowska, Anna D; Wywiał, Ewa; Dunin-Horkawicz, Stanislaw; Łasica, Anna M; Wösten, Marc M S M; Nagy-Staroń, Anna; Godlewska, Renata; Bocian-Ostrzycka, Katarzyna; Pieńkowska, Katarzyna; Łaniewski, Paweł; Bujnicki, Janusz M; van Putten, Jos P M; Jagusztyn-Krynicka, E Katarzyna

2014-01-01

Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world. Phylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon. Our results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether the data presented here constitute the considerable insight to the Epsilonproteobacterial Dsb systems, which have been poorly understood so far.

Characterization of new DsbB-like thiol-oxidoreductases of Campylobacter jejuni and Helicobacter pylori and classification of the DsbB family based on phylogenomic, structural and functional criteria.

PubMed

Raczko, Anna M; Bujnicki, Janusz M; Pawlowski, Marcin; Godlewska, Renata; Lewandowska, Magdalena; Jagusztyn-Krynicka, Elzbieta K

2005-01-01

In Gram-negative bacterial cells, disulfide bond formation occurs in the oxidative environment of the periplasm and is catalysed by Dsb (disulfide bond) proteins found in the periplasm and in the inner membrane. In this report the identification of a new subfamily of disulfide oxidoreductases encoded by a gene denoted dsbI, and functional characterization of DsbI proteins from Campylobacter jejuni and Helicobacter pylori, as well as DsbB from C. jejuni, are described. The N-terminal domain of DsbI is related to DsbB proteins and comprises five predicted transmembrane segments, while the C-terminal domain is predicted to locate to the periplasm and to fold into a beta-propeller structure. The dsbI gene is co-transcribed with a small ORF designated dba (dsbI-accessory). Based on a series of deletion and complementation experiments it is proposed that DsbB can complement the lack of DsbI but not the converse. In the presence of DsbB, the activity of DsbI was undetectable, hence it probably acts only on a subset of possible substrates of DsbB. To reconstruct the principal events in the evolution of DsbB and DsbI proteins, sequences of all their homologues identifiable in databases were analysed. In the course of this study, previously undetected variations on the common thiol-oxidoreductase theme were identified, such as development of an additional transmembrane helix and loss or migration of the second pair of Cys residues between two distinct periplasmic loops. In conjunction with the experimental characterization of two members of the DsbI lineage, this analysis has resulted in the first comprehensive classification of the DsbB/DsbI family based on structural, functional and evolutionary criteria.

Campylobacter protein oxidation influences epithelial cell invasion or intracellular survival as well as intestinal tract colonization in chickens.

PubMed

Lasica, A M; Wyszynska, A; Szymanek, K; Majewski, P; Jagusztyn-Krynicka, E K

2010-01-01

The Dsb family of redox proteins catalyzes disulfide bond formation and isomerization. Since mutations in dsb genes change the conformation and stability of many extracytoplasmic proteins, and since many virulence factors of pathogenic bacteria are extracytoplasmic, inactivation of dsb genes often results in pathogen attenuation. This study investigated the role of 2 membrane-bound oxidoreductases, DsbB and DsbI, in the Campylobacter jejuni oxidative Dsb pathway. Campylobacter mutants, lacking DsbB or DsbI or both, were constructed by allelic replacement and used in the human intestinal epithelial T84 cell line for the gentamicin protection assay (invasion assay) and chicken colonization experiments. In C. coli strain 23/1, the inactivation of the dsbB or dsbI gene separately did not significantly affect the colonization process. However, simultaneous disruption of both membrane-bound oxidoreductase genes significantly decreased the strain’s ability to colonize chicken intestines. Moreover, C. jejuni strain 81-176 with mutated dsbB or dsbI genes showed reduced invasion/intracellular survival abilities. No cells of the double mutants (dsbB⁻ dsbI⁻) of C. jejuni 81-176 were recovered from human cells after 3 h of invasion.

An extracellular disulfide bond forming protein (DsbF) from Mycobacterium tuberculosis: Structural, biochemical and gene expression analysis

PubMed Central

Chim, Nicholas; Riley, Robert; The, Juliana; Im, Soyeon; Segelke, Brent; Lekin, Tim; Yu, Minmin; Hung, Li Wei; Terwilliger, Tom; Whitelegge, Julian P.; Goulding, Celia W.

2010-01-01

Disulfide bond forming (Dsb) proteins ensure correct folding and disulfide bond formation of secreted proteins. Previously, we showed that Mycobacterium tuberculosis DsbE (Mtb DsbE, Rv2878c) aids in vitro oxidative folding of proteins. Here we present structural, biochemical and gene expression analyses of another putative Mtb secreted disulfide bond isomerase protein homologous to Mtb DsbE, Mtb DsbF (Rv1677). The X-ray crystal structure of Mtb DsbF reveals a conserved thioredoxin fold although the active-site cysteines may be modeled in both oxidized and reduced forms, in contrast to the solely reduced form in Mtb DsbE. Furthermore, the shorter loop region in Mtb DsbF results in a more solvent-exposed active site. Biochemical analyses show that, similar to Mtb DsbE, Mtb DsbF can oxidatively refold reduced, unfolded hirudin and has a comparable pKa for the active-site solvent-exposed cysteine. However, contrary to Mtb DsbE, the Mtb DsbF redox potential is more oxidizing and its reduced state is more stable. From computational genomics analysis of the M. tuberculosis genome, we identified a potential Mtb DsbF interaction partner, Rv1676, a predicted peroxiredoxin. Complex formation is supported by protein co-expression studies and inferred by gene expression profiles, whereby Mtb DsbF and Rv1676 are upregulated under similar environments. Additionally, comparison of Mtb DsbF and Mtb DsbE gene expression data indicate anticorrelated gene expression patterns, suggesting that these two proteins and their functionally linked partners constitute analogous pathways that may function under different conditions. PMID:20060836

Preliminary crystallographic data of the three homologues of the thiol–disulfide oxidoreductase DsbA in Neisseria meningitidis

PubMed Central

Lafaye, Céline; Iwena, Thomas; Ferrer, Jean-Luc; Kroll, J. Simon; Griat, Mickael; Serre, Laurence

2008-01-01

Bacterial virulence depends on the correct folding of surface-exposed proteins, a process that is catalyzed by the thiol-disulfide oxidoreductase DsbA, which facilitates the synthesis of disulfide bonds in Gram-negative bacteria. Uniquely among bacteria, the Neisseria meningitidis genome possesses three genes encoding active DsbAs: DsbA1, DsbA2 and DsbA3. DsbA1 and DsbA2 have been characterized as lipoproteins involved in natural competence and in host-interactive biology, while the function of DsbA3 remains unknown. In an attempt to shed light on the reason for this multiplicity of dsbA genes, the three enzymes from N. meningitidis have been purified and crystallized in the presence of high concentrations of ammonium sulfate. The best crystals were obtained using DsbA1 and DsbA3; they belong to the orthorhombic and tetragonal systems and diffract to 1.5 and 2.7 Å resolution, respectively. PMID:18259062

Functional and Bioinformatics Analysis of Two Campylobacter jejuni Homologs of the Thiol-Disulfide Oxidoreductase, DsbA

PubMed Central

Grabowska, Anna D.; Wywiał, Ewa; Dunin-Horkawicz, Stanislaw; Łasica, Anna M.; Wösten, Marc M. S. M.; Nagy-Staroń, Anna; Godlewska, Renata; Bocian-Ostrzycka, Katarzyna; Pieńkowska, Katarzyna; Łaniewski, Paweł; Bujnicki, Janusz M.; van Putten, Jos P. M.; Jagusztyn-Krynicka, E. Katarzyna

2014-01-01

Background Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world. Methods and Results Phylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon. Conclusions Our results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether the data presented here constitute the considerable insight to the Epsilonproteobacterial Dsb systems, which have been poorly understood so far. PMID:25181355

Crystal Structure of the Dithiol Oxidase DsbA Enzyme from Proteus Mirabilis Bound Non-covalently to an Active Site Peptide Ligand

PubMed Central

Kurth, Fabian; Duprez, Wilko; Premkumar, Lakshmanane; Schembri, Mark A.; Fairlie, David P.; Martin, Jennifer L.

2014-01-01

The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery. PMID:24831013

Real-time Monitoring of Intermediates Reveals the Reaction Pathway in the Thiol-Disulfide Exchange between Disulfide Bond Formation Protein A (DsbA) and B (DsbB) on a Membrane-immobilized Quartz Crystal Microbalance (QCM) System*

PubMed Central

Yazawa, Kenjiro; Furusawa, Hiroyuki; Okahata, Yoshio

2013-01-01

Disulfide bond formation protein B (DsbBS-S,S-S) is an inner membrane protein in Escherichia coli that has two disulfide bonds (S-S, S-S) that play a role in oxidization of a pair of cysteine residues (SH, SH) in disulfide bond formation protein A (DsbASH,SH). The oxidized DsbAS-S, with one disulfide bond (S-S), can oxidize proteins with SH groups for maturation of a folding preprotein. Here, we have described the transient kinetics of the oxidation reaction between DsbASH,SH and DsbBS-S,S-S. We immobilized DsbBS-S,S-S embedded in lipid bilayers on the surface of a 27-MHz quartz crystal microbalance (QCM) device to detect both formation and degradation of the reaction intermediate (DsbA-DsbB), formed via intermolecular disulfide bonds, as a mass change in real time. The obtained kinetic parameters (intermediate formation, reverse, and oxidation rate constants (kf, kr, and kcat, respectively) indicated that the two pairs of cysteine residues in DsbBS-S,S-S were more important for the stability of the DsbA-DsbB intermediate than ubiquinone, an electron acceptor for DsbBS-S,S-S. Our data suggested that the reaction pathway of almost all DsbASH,SH oxidation processes would proceed through this stable intermediate, avoiding the requirement for ubiquinone. PMID:24145032

The contribution of thermally labile sugar lesions to DNA double-strand break formation in cells grown in the presence of BrdU.

PubMed

Li, Fanghua; Cheng, Yanlei; Iliakis, George

2015-04-01

Radiosensitization by bromodeoxyuridine (BrdU) is commonly attributed to an increase in the yield of double-strand breaks (DSB) in the DNA and an associated decrease in the reparability of these lesions. Radiation chemistry provides a mechanism for the increased yield of DSB through the generation, after bromine loss, of a highly reactive uracilyl radical that attacks the sugar moiety of the nucleotide to produce a single-strand break (SSB). The effects underpinning DSB repair inhibition remain, in contrast, incompletely characterized. A possible source of reduced reparability is a change in the nature or complexity of the DSB in BrdU-substituted DNA. Recent studies show that DSB-complexity or DSB-nature may also be affected by the presence within the cluster of thermally labile sugar lesions (TLSL) that break the DNA backbone only if they chemically evolve to SSB, a process thought to occur within the first hour post-irradiation. Since BrdU radiosensitization might be associated with increased yields and reduced reparability of DSB, we investigated whether BrdU underpins these effects by shifting the balance in the generation of TLSL. We employed asymmetric-field-inversion gel electrophoresis (AFIGE), a pulsed-field gel electrophoresis (PFGE) method to quantitate DSB in a battery of five cells lines grown in the presence of different concentrations of BrdU. We measured specifically the yields of promptly forming DSB (prDSB) using low temperature lysis protocols, and the yields of total DSB (tDSB = prDSB + tlDSB; tlDSB form after evolution to SSB of TLSL) using high temperature lysis protocols. We report that incorporation of BrdU generates similar increases in the formation of tlDSB and prDSB, but variations are noted among the different cell lines tested. The similar increase in the yields of tlDSB and prDSB in BrdU substituted DNA showed that shifts in the yields of these forms of lesions could not be invoked to explain BrdU radiosensitization.

Induction of single- and double-strand breaks in plasmid DNA by monoenergetic alpha-particles with energies below the Bragg-maximum.

PubMed

Scholz, V; Weidner, J; Köhnlein, W; Frekers, D; Wörtche, H J

1997-01-01

The yield of single-strand breaks (ssb) and double-strand breaks (dsb) produced by alpha-particles at the end of their track in DNA-films was determined experimentally. Helium nuclei were accelerated to 600 keV in the 400 kV ion accelerator and scattered at a carbon target. The elastically scattered alpha-particles with energies of 344 keV and 485 keV were used to irradiate supercircular plasmid DNA in vacuo. For the dosimetry of the alpha-particles a surface barrier detector was used and the energy distribution of the alpha-particles determined. The energy loss of the particles in the DNA-layer was calculated. DNA samples were separated into the three conformational isomers using agarose gel electrophoresis. After fluorochromation the number of ssb and dsb per plasmid DNA molecule was established from the band intensities assuming the validity of Poisson statistics. Linear dose effect correlations were found for ssb and dsb per plasmid molecule. In the case of 344 keV-alpha-particles the yield of dsb was (8.6 +/- 0.9) x 10(-11) breaks/Gy x dalton. The ratio of ssb/dsb was 0.5 +/- 0.2. This is at least a factor of six larger than the ratio found in experiments with higher energy alpha-particles and from model calculations. Similar experiments with protons yielded a relative biological effectiveness (rbe) value of 2.8 for the induction of double-strand breaks by track end alpha-particles.

Targeted induction of meiotic double-strand breaks reveals chromosomal domain-dependent regulation of Spo11 and interactions among potential sites of meiotic recombination

PubMed Central

Fukuda, Tomoyuki; Kugou, Kazuto; Sasanuma, Hiroyuki; Shibata, Takehiko

2008-01-01

Meiotic recombination is initiated by programmed DNA double-strand break (DSB) formation mediated by Spo11. DSBs occur with frequency in chromosomal regions called hot domains but are seldom seen in cold domains. To obtain insights into the determinants of the distribution of meiotic DSBs, we examined the effects of inducing targeted DSBs during yeast meiosis using a UAS-directed form of Spo11 (Gal4BD-Spo11) and a meiosis-specific endonuclease, VDE (PI-SceI). Gal4BD-Spo11 cleaved its target sequence (UAS) integrated in hot domains but rarely in cold domains. However, Gal4BD-Spo11 did bind to UAS and VDE efficiently cleaved its recognition sequence in either context, suggesting that a cold domain is not a region of inaccessible or uncleavable chromosome structure. Importantly, self-association of Spo11 occurred at UAS in a hot domain but not in a cold domain, raising the possibility that Spo11 remains in an inactive intermediate state in cold domains. Integration of UAS adjacent to known DSB hotspots allowed us to detect competitive interactions among hotspots for activation. Moreover, the presence of VDE-introduced DSB repressed proximal hotspot activity, implicating DSBs themselves in interactions among hotspots. Thus, potential sites for Spo11-mediated DSB are subject to domain-specific and local competitive regulations during and after DSB formation. PMID:18096626

Comparison of the biological effects of {sup 18}F at different intracellular levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kashino, Genro, E-mail: kashino@oita-u.ac.jp; Hayashi, Kazutaka; Douhara, Kazumasa

Highlights: • We estimated the inductions of DNA DSB in cell treated with {sup 18}F-FDG. • We found that inductions of DNA DSB are dependent on accumulation of {sup 18}F in cell. • Accumulation of {sup 18}F in cell may be indispensable for risk estimation of PET. - Abstract: We herein examined the biological effects of cells treated with {sup 18}F labeled drugs for positron emission tomography (PET). The relationship between the intracellular distribution of {sup 18}F and levels of damaged DNA has yet to be clarified in detail. We used culture cells (Chinese Hamster Ovary cells) treated with twomore » types of {sup 18}F labeled drugs, fluorodeoxyglucose (FDG) and fluorine ion (HF). FDG efficiently accumulated in cells, whereas HF did not. To examine the induction of DNA double strand breaks (DSB), we measured the number of foci for 53BP1 that formed at the site of DNA DSB. The results revealed that although radioactivity levels were the same, the induction of 53BP1 foci was stronger in cells treated with {sup 18}F-FDG than in those treated with {sup 18}F-HF. The clonogenic survival of cells was significantly lower with {sup 18}F-FDG than with {sup 18}F-HF. We concluded that the efficient accumulation of {sup 18}F in cells led to stronger biological effects due to more severe cellular lethality via the induction of DNA DSB.« less

Crystal structure of the dithiol oxidase DsbA enzyme from proteus mirabilis bound non-covalently to an active site peptide ligand.

PubMed

Kurth, Fabian; Duprez, Wilko; Premkumar, Lakshmanane; Schembri, Mark A; Fairlie, David P; Martin, Jennifer L

2014-07-11

The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of DSB-1, a Protein Required for Initiation of Meiotic Recombination in Caenorhabditis elegans, Illuminates a Crossover Assurance Checkpoint

PubMed Central

Stamper, Ericca L.; Rodenbusch, Stacia E.; Rosu, Simona; Ahringer, Julie; Villeneuve, Anne M.; Dernburg, Abby F.

2013-01-01

Meiotic recombination, an essential aspect of sexual reproduction, is initiated by programmed DNA double-strand breaks (DSBs). DSBs are catalyzed by the widely-conserved Spo11 enzyme; however, the activity of Spo11 is regulated by additional factors that are poorly conserved through evolution. To expand our understanding of meiotic regulation, we have characterized a novel gene, dsb-1, that is specifically required for meiotic DSB formation in the nematode Caenorhabditis elegans. DSB-1 localizes to chromosomes during early meiotic prophase, coincident with the timing of DSB formation. DSB-1 also promotes normal protein levels and chromosome localization of DSB-2, a paralogous protein that plays a related role in initiating recombination. Mutations that disrupt crossover formation result in prolonged DSB-1 association with chromosomes, suggesting that nuclei may remain in a DSB-permissive state. Extended DSB-1 localization is seen even in mutants with defects in early recombination steps, including spo-11, suggesting that the absence of crossover precursors triggers the extension. Strikingly, failure to form a crossover precursor on a single chromosome pair is sufficient to extend the localization of DSB-1 on all chromosomes in the same nucleus. Based on these observations we propose a model for crossover assurance that acts through DSB-1 to maintain a DSB-permissive state until all chromosome pairs acquire crossover precursors. This work identifies a novel component of the DSB machinery in C. elegans, and sheds light on an important pathway that regulates DSB formation for crossover assurance. PMID:23990794

A pleîotropic acid phosphatase-deficient mutant of Escherichia coli shows premature termination in the dsbA gene. Use of dsbA::phoA fusions to localize a structurally important domain in DsbA.

PubMed

Belin, P; Quéméneur, E; Boquet, P L

1994-01-01

A one-step mutant of Escherichia coli K-12 lacking both glucose-1-phosphatase (Agp) and pH 2.5 acid phosphatase (AppA) activities in the periplasmic space was isolated. The mutation which mapped close to chlB, at 87 min on the E. coli linkage map, also caused the loss of alkaline phosphatase (PhoA) activity, even when this activity was expressed from TnphoA fusions to genes encoding periplasmic or membrane proteins. A DNA fragment that complements the mutation was cloned and shown to carry the dsbA gene, which encodes a periplasmic disulphide bond-forming factor. The mutant had an ochre triplet in dsbA, truncating the protein at amino acid 70. Introduction of TnphoA fusions into a plasmid-borne dsbA gene resulted in DsbA-PhoA hybrid proteins that were all exported to the periplasmic space in both dsbA+ and dsbA strains. They belong to three different classes, depending on the length of the DsbA fragment fused to PhoA. When PhoA was fused to an amino-terminal DsbA heptapeptide, the protein was only seen in the periplasm of a dsbA+ strain, as in the case of wild-type PhoA. Hybrid proteins missing up to 29 amino acids at the carboxy-terminus of DsbA were stable and retained both the DsbA and PhoA activities. Those with shorter DsbA fragments that still carried the -Cys-Pro-His-Cys- motif were rapidly degraded (no DsbA activity). The presence is discussed of a structural domain lying around amino acid 170 of DsbA and which is probably essential for its folding into a proteolytic-resistant and enzymatically active form.

Biophysical modelling of early and delayed radiation damage at chromosome level

NASA Astrophysics Data System (ADS)

Andreev, S.; Eidelman, Y.

Exposure by ionising radiation increases cancer risk in human population Cancer is thought to originate from an altered expression of certain number of specific genes It is now widely recognised that chromosome aberrations CA are involved in stable change in expression of genes by gain or loss of their functions Thus CA can contribute to initiation or progression of cancer Therefore understanding mechanisms of CA formation in the course of cancer development might be valuable tool for quantification and prognosis of different stages of radiation carcinogenesis Early CA are defined as aberrations induced in first post-irradiation mitotic cycle The present work describes the original biophysical technique for early CA modelling It includes the following simulation steps the ionising particle track structure the structural organisation of all chromosomes in G 0 G 1 cell nucleus spatial distribution of radiation induced DNA double-strand breaks dsb within chromosomes dsb rejoining and misrejoining modelling cell cycle taking into account mitotic delay which results in complex time dependence of aberrant cells in first mitosis The results on prediction of dose-response curves for simple and complex CA measured in cells undergoing first division cycle are presented in comparison with recent experimental data There is increasing evidence that CA are also observed in descendents of irradiated cells many generations after direct DNA damage These delayed CA or chromosome instability CI are thought to be a manifestation of genome

The Active Structure of the Greater Dead Sea Basin

NASA Astrophysics Data System (ADS)

Shamir, G.

2002-12-01

The Greater Dead Sea Basin (GDSB) is a 220km long depression situated along the southern section of the Dead Sea Transform (DST), between two structurally and gravitationally elevated points, Wadi Malih in the north and Paran fault zone in the south. In its center is the Dead Sea basin 'sensu strictu' (DSB), which has been described since the 1970s as a pull-apart basin at a left step-over along the DST. However, several observations, or their lack thereof, contradict this scheme, e.g. (i) It is not supported by recent seismological and geomorphic data; (ii) It does not explain the fault pattern and mixed sinistral and dextral offset along the DSB western boundary; (iii) It does not simply explain the presence of intense deformation outside the presumed fault step zone; (iv) It is inconsistent with the orientation of seismically active faults within the Dead Sea and Jericho Valley; (v) The length of the DSB exceeds the total offset along the Dead Sea Transform, while its subsidence is about the age of the DST. In this study, newly acquired and analyzed data (high resolution seismic reflection and earthquake relocation and fault plane solutions) has been integrated with previously published data (structural mapping, fracture orientation distribution, Bouguer anomaly maps, sinkhole distribution, geomorphic lineaments). The results show that the GDSB is dominated by two active fault systems, one trending NNE and showing normal-dextral motion, the other trending NW. These systems are identified by earthquake activity, seismic reflection observations, alignment of recent sinkholes, and distribution of Bouguer anomaly gradients. As a result, the intra-basin structure is of a series of rectangular blocks. The dextral slip component along NNE trending faults, the mixed sense of lateral offset along the western boundary of the DSB and temporal change in fracture orientation in the Jericho Valley suggest that the intra-basin blocks have rotated counterclockwise since the Pleistocene. The overall sinistral motion between the Arabian and Israel-Sinai plates along the GDSB may thus be accommodated by the postulated, internally rotating shear zone. Then, the subsidence of the DSB may possibly be explained if the rate of the resulting internal E-W shortening is greater than the rate of plate convergence.

Clustering and interpretation of local earthquake tomography models in the southern Dead Sea basin

NASA Astrophysics Data System (ADS)

Bauer, Klaus; Braeuer, Benjamin

2016-04-01

The Dead Sea transform (DST) marks the boundary between the Arabian and the African plates. Ongoing left-lateral relative plate motion and strike-slip deformation started in the Early Miocene (20 MA) and produced a total shift of 107 km until presence. The Dead Sea basin (DSB) located in the central part of the DST is one of the largest pull-apart basins in the world. It was formed from step-over of different fault strands at a major segment boundary of the transform fault system. The basin development was accompanied by deposition of clastics and evaporites and subsequent salt diapirism. Ongoing deformation within the basin and activity of the boundary faults are indicated by increased seismicity. The internal architecture of the DSB and the crustal structure around the DST were subject of several large scientific projects carried out since 2000. Here we report on a local earthquake tomography study from the southern DSB. In 2006-2008, a dense seismic network consisting of 65 stations was operated for 18 months in the southern part of the DSB and surrounding regions. Altogether 530 well-constrained seismic events with 13,970 P- and 12,760 S-wave arrival times were used for a travel time inversion for Vp, Vp/Vs velocity structure and seismicity distribution. The work flow included 1D inversion, 2.5D and 3D tomography, and resolution analysis. We demonstrate a possible strategy how several tomographic models such as Vp, Vs and Vp/Vs can be integrated for a combined lithological interpretation. We analyzed the tomographic models derived by 2.5D inversion using neural network clustering techniques. The method allows us to identify major lithologies by their petrophysical signatures. Remapping the clusters into the subsurface reveals the distribution of basin sediments, prebasin sedimentary rocks, and crystalline basement. The DSB shows an asymmetric structure with thickness variation from 5 km in the west to 13 km in the east. Most importantly, a well-defined body under the eastern part of the basin down to 18 km depth was identified by the algorithm. Considering its geometry and petrophysical signature, this unit is interpreted as prebasin sediments and not as crystalline basement. The seismicity distribution supports our results, where events are concentrated along boundaries of the basin and the deep prebasin sedimentary body.

DNA damage on nano- and micrometer scales impacts dicentric induction: computer modelling of ion microbeam experiments

NASA Astrophysics Data System (ADS)

Friedland, Werner; Kundrat, Pavel; Schmitt, Elke

2016-07-01

Detailed understanding of the enhanced relative biological effectiveness (RBE) of ions, in particular at high linear energy transfer (LET) values, is needed to fully explore the radiation risk of manned space missions. It is generally accepted that the enhanced RBE of high-LET particles results from the DNA lesion patterns, in particular DNA double-strand breaks (DSB), due to the spatial clustering of energy deposits around their trajectories. In conventional experiments on biological effects of radiation types of diverse quality, however, clustering of energy deposition events on nanometer scale that is relevant for the induction and local complexity of DSB is inherently interlinked with regional (sub-)micrometer-scale DSB clustering along the particle tracks. Due to this limitation, the role of both (nano- and micrometer) scales on the induction of diverse biological endpoints cannot be frankly separated. To address this issue in a unique way, experiments at the ion microbeam SNAKE [1] and corresponding track-structure based model calculations of DSB induction and subsequent repair with the biophysical code PARTRAC [2] have been performed. In the experiments, hybrid human-hamster A_{L} cells were irradiated with 20 MeV (2.6 keV/μm) protons, 45 MeV (60 keV/μm) lithium ions or 55 MeV (310 keV/μm) carbon ions. The ions were either quasi-homogeneously distributed or focused to 0.5 x 1 μm^{2} spots on regular matrix patterns of 5.4 μm, 7.6 μm and 10.6 μm grid size, with pre-defined particle numbers per spot so as to deposit a mean dose of 1.7 Gy for all irradiation patterns. As expected, the induction of dicentrics by homogeneous irradiation increased with LET: lithium and carbon ions induced about two- and four-fold higher yields of dicentrics than protons. The induction of dicentrics is, however, affected by µm-scale, too: focusing 20 lithium ions or 451 protons per spot on a 10.6 μm grid induced two or three times more dicentrics, respectively, than a quasi-homogenous irradiation with these particles [3]. PARTRAC calculations of initial DNA damage showed that the sub-micrometer beam focusing of the ions in these experiments affects neither DSB yields nor local DSB complexity, but considerably enhances the formation of DSB fragments of 10 - 1000 kbp size [4], corresponding to DSB pairs in about 100 - 500 nm distance. Thus, the substantial impact of ion focusing on dicentric induction points out that nanoscale DNA damage clustering can explain only partly the increased RBE of high LET radiation regarding dicentric induction. The measured trends for dicentric induction as a function of grid size (or particle number per spot) were largely reproduced by the calculated induction of total chromosomal aberrations, whereas the calculation of dicentrics yielded apparent discrepancies, such as an overestimation of the focusing effect for protons and of the yield for quasi-homogeneous lithium ions [3]. Since this incongruity was found to be rather robust against model parameter variations, a more basic review of the chromosomal aberration model with in-depth testing of several hypotheses on the origin of misrejoining events of DNA ends has been started considering the reported experimental findings. The results of ongoing parameter studies will be presented at the meeting. Acknowledgement. This work was supported by the German Federal Ministry of Education and Research (Project 'LET-Verbund', Funding no. 02NUK031C). References [1] Schmid et al. 2012 Phys. Med. Biol. 57, 5889-5907 [2] Friedland et al. 2011 Mutat. Res. 711, 28-40 [3] Schmid et al. 2015 Mutat. Res. 793, 30-40 [4] Friedland et al. 2015 Radiat. Prot. Dosim. 166, 34-37

Plasma cholesterol reduction by defatted soy ontjom (fermented with Neurospora intermedia) in rats fed a cholesterol-free diet.

PubMed

Matsuo, M

2000-02-01

To popularize defatted soy ontjom (DSB-ontjom, soy product fermented with Neurospora intermedia) as a new food, I examined the plasma cholesterol-reducing effects of DSB-ontjom and DSB in rats fed cholesterol-free diets and compared the efficiencies of these effects. DSB-ontjom greatly reduced the plasma cholesterol level and increased fecal steroid excretion as compared to DSB. DSB-ontjom was rich in pepsin-resistant protein having a high bile acid binding capacity and was abundant in isoflavone-aglycones, especially daizein. The dietary fiber (DF) of DSB-ontjom stimulated the production of short-chain fatty acids (SCFAs) by intestinal microflora. The effect of DSB-ontjom on plasma cholesterol reduction was attributed to the collaborative effects of pepsin-resistant-protein, isoflavone-aglycones and SCFA-producing DF in DSB-ontjom.

Campylobacter jejuni dsb gene expression is regulated by iron in a Fur-dependent manner and by a translational coupling mechanism

PubMed Central

2011-01-01

Background Many bacterial extracytoplasmic proteins are stabilized by intramolecular disulfide bridges that are formed post-translationally between their cysteine residues. This protein modification plays an important role in bacterial pathogenesis, and is facilitated by the Dsb (disulfide bond) family of the redox proteins. These proteins function in two parallel pathways in the periplasmic space: an oxidation pathway and an isomerization pathway. The Dsb oxidative pathway in Campylobacter jejuni is more complex than the one in the laboratory E. coli K-12 strain. Results In the C. jejuni 81-176 genome, the dsb genes of the oxidative pathway are arranged in three transcriptional units: dsbA2-dsbB-astA, dsbA1 and dba-dsbI. Their transcription responds to an environmental stimulus - iron availability - and is regulated in a Fur-dependent manner. Fur involvement in dsb gene regulation was proven by a reporter gene study in a C. jejuni wild type strain and its isogenic fur mutant. An electrophoretic mobility shift assay (EMSA) confirmed that analyzed genes are members of the Fur regulon but each of them is regulated by a disparate mechanism, and both the iron-free and the iron-complexed Fur are able to bind in vitro to the C. jejuni promoter regions. This study led to identification of a new iron- and Fur-regulated promoter that drives dsbA1 gene expression in an indirect way. Moreover, the present work documents that synthesis of DsbI oxidoreductase is controlled by the mechanism of translational coupling. The importance of a secondary dba-dsbI mRNA structure for dsbI mRNA translation was verified by estimating individual dsbI gene expression from its own promoter. Conclusions The present work shows that iron concentration is a significant factor in dsb gene transcription. These results support the concept that iron concentration - also through its influence on dsb gene expression - might control the abundance of extracytoplasmic proteins during different stages of infection. Our work further shows that synthesis of the DsbI membrane oxidoreductase is controlled by a translational coupling mechanism. The dba expression is not only essential for the translation of the downstream dsbI gene, but also Dba protein that is produced might regulate the activity and/or stability of DsbI. PMID:21787430

Campylobacter jejuni dsb gene expression is regulated by iron in a Fur-dependent manner and by a translational coupling mechanism.

PubMed

Grabowska, Anna D; Wandel, Michał P; Łasica, Anna M; Nesteruk, Monika; Roszczenko, Paula; Wyszyńska, Agnieszka; Godlewska, Renata; Jagusztyn-Krynicka, Elzbieta K

2011-07-25

Many bacterial extracytoplasmic proteins are stabilized by intramolecular disulfide bridges that are formed post-translationally between their cysteine residues. This protein modification plays an important role in bacterial pathogenesis, and is facilitated by the Dsb (disulfide bond) family of the redox proteins. These proteins function in two parallel pathways in the periplasmic space: an oxidation pathway and an isomerization pathway. The Dsb oxidative pathway in Campylobacter jejuni is more complex than the one in the laboratory E. coli K-12 strain. In the C. jejuni 81-176 genome, the dsb genes of the oxidative pathway are arranged in three transcriptional units: dsbA2-dsbB-astA, dsbA1 and dba-dsbI. Their transcription responds to an environmental stimulus - iron availability - and is regulated in a Fur-dependent manner. Fur involvement in dsb gene regulation was proven by a reporter gene study in a C. jejuni wild type strain and its isogenic fur mutant. An electrophoretic mobility shift assay (EMSA) confirmed that analyzed genes are members of the Fur regulon but each of them is regulated by a disparate mechanism, and both the iron-free and the iron-complexed Fur are able to bind in vitro to the C. jejuni promoter regions. This study led to identification of a new iron- and Fur-regulated promoter that drives dsbA1 gene expression in an indirect way. Moreover, the present work documents that synthesis of DsbI oxidoreductase is controlled by the mechanism of translational coupling. The importance of a secondary dba-dsbI mRNA structure for dsbI mRNA translation was verified by estimating individual dsbI gene expression from its own promoter. The present work shows that iron concentration is a significant factor in dsb gene transcription. These results support the concept that iron concentration - also through its influence on dsb gene expression - might control the abundance of extracytoplasmic proteins during different stages of infection. Our work further shows that synthesis of the DsbI membrane oxidoreductase is controlled by a translational coupling mechanism. The dba expression is not only essential for the translation of the downstream dsbI gene, but also Dba protein that is produced might regulate the activity and/or stability of DsbI.

Expression and crystallization of SeDsbA, SeDsbL and SeSrgA from Salmonella enterica serovar Typhimurium.

PubMed

Jarrott, R; Shouldice, S R; Guncar, G; Totsika, M; Schembri, M A; Heras, B

2010-05-01

Pathogens require protein-folding enzymes to produce functional virulence determinants. These foldases include the Dsb family of proteins, which catalyze oxidative folding in bacteria. Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K-12 and these mechanisms have been extrapolated to other organisms. However, recent research indicates that the K-12 complement of Dsb proteins is not common to all bacteria. Importantly, many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence. To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins, Salmonella enterica serovar Typhimurium has been focused on. This Gram-negative bacterium contains three DsbA proteins: SeDsbA, SeDsbL and SeSrgA. Here, the expression, purification, crystallization and preliminary diffraction analysis of these three proteins are reported. SeDsbA, SeDsbL and SeSrgA crystals diffracted to resolution limits of 1.55, 1.57 and 2.6 A and belonged to space groups P2(1), P2(1)2(1)2 and C2, respectively.

Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain

PubMed Central

Bocian-Ostrzycka, Katarzyna M.; Łasica, Anna M.; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J.; Drabik, Karolina; Dobosz, Aneta M.; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K.

2015-01-01

Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation – periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity. PMID:26500620

Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain.

PubMed

Bocian-Ostrzycka, Katarzyna M; Łasica, Anna M; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J; Drabik, Karolina; Dobosz, Aneta M; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K

2015-01-01

Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation - periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.

Budding Yeast ATM/ATR Control Meiotic Double-Strand Break (DSB) Levels by Down-Regulating Rec114, an Essential Component of the DSB-machinery

PubMed Central

Carballo, Jesús A.; Panizza, Silvia; Serrentino, Maria Elisabetta; Johnson, Anthony L.; Geymonat, Marco; Borde, Valérie; Klein, Franz; Cha, Rita S.

2013-01-01

An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or “DSB homeostasis”, might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks. PMID:23825959

SU-E-J-274: Responses of Medulloblastoma Cells to Radiation Dosimetric Parameters in Intensity-Modulated Radiation Therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, J; Molecular Imaging Program at Stanford, Stanford, CA; Bio-X Program, Stanford, CA

2015-06-15

Purpose: To evaluate radiation responses of the medulloblastoma cell line Daoy in intensity-modulated radiation therapy (IMRT), quantitative variations to variable radiation dosimetic parameters were tracked by bioluminescent images (BLIs). Methods: The luciferase and green fluorescent protein positive Daoy cells were cultured on dishes. The medulloblastoma cells irradiated to different dose rate, interval of fractionated doses, field margin and misalignment, and dose uniformity in IMRT were monitored using bioluminescent images. The cultured cells were placed into a dedicated acrylic phantom to deliver intensity-modulated fluences and calculate accurate predicted dose distribution. The radiation with dose rate from 0.5 Gy/min to 15 Gy/minmore » was irradiated by adjusting monitor unit per minute and source-to-surface distances. The intervals of fractionated dose delivery were changed considering the repair time of double strand breaks (DSB) revealed by straining of gamma-H2AX.The effect of non-uniform doses on the cells were visualized by registering dose distributions and BLIs. The viability according to dosimetric parameters was correlated with bioluminescent intensities for cross-check of radiation responses. Results: The DSB and cell responses due to the first fractionated dose delivery significantly affected final tumor control rather than other parameters. The missing tumor volumes due to the smaller field margin than the tumor periphery or field misalignment caused relapse of cell responses on BLIs. The dose rate and gradient had effect on initial responses but could not bring out the distinguishable killing effect on cancer cells. Conclusion: Visualized and quantified bioluminescent images were useful to correlate the dose distributions with spatial radiation effects on cells. This would derive the effective combination of dose delivery parameters and fractionation. Radiation responses in particular IMRT configuration could be reflected to image based-dose re-optimization.« less

Mechanisms and Consequences of Double-strand DNA Break Formation in Chromatin

PubMed Central

Cannan, Wendy J.; Pederson, David S.

2016-01-01

All organisms suffer double-strand breaks (DSBs) in their DNA as a result of exposure to ionizing radiation. DSBs can also form when replication forks encounter DNA lesions or repair intermediates. The processing and repair of DSBs can lead to mutations, loss of heterozygosity, and chromosome rearrangements that result in cell death or cancer. The most common pathway used to repair DSBs in metazoans (non-homologous DNA end joining) is more commonly mutagenic than the alternative pathway (homologous recombination mediated repair). Thus, factors that influence the choice of pathways used DSB repair can affect an individual’s mutation burden and risk of cancer. This review describes radiological, chemical and biological mechanisms that generate DSBs, and discusses the impact of such variables as DSB etiology, cell type, cell cycle, and chromatin structure on the yield, distribution, and processing of DSBs. The final section focuses on nucleosome-specific mechanisms that influence DSB production, and the possible relationship between higher order chromosome coiling and chromosome shattering (chromothripsis). PMID:26040249

Structure of the Acinetobacter baumannii Dithiol Oxidase DsbA Bound to Elongation Factor EF-Tu Reveals a Novel Protein Interaction Site

PubMed Central

Premkumar, Lakshmanane; Kurth, Fabian; Duprez, Wilko; Grøftehauge, Morten K.; King, Gordon J.; Halili, Maria A.; Heras, Begoña; Martin, Jennifer L.

2014-01-01

The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA. PMID:24860094

Structure of the Acinetobacter baumannii dithiol oxidase DsbA bound to elongation factor EF-Tu reveals a novel protein interaction site.

PubMed

Premkumar, Lakshmanane; Kurth, Fabian; Duprez, Wilko; Grøftehauge, Morten K; King, Gordon J; Halili, Maria A; Heras, Begoña; Martin, Jennifer L

2014-07-18

The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Rv2969c, essential for optimal growth in Mycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Premkumar, Lakshmanane, E-mail: p.lakshmanane@imb.uq.edu.au; Heras, Begoña; Duprez, Wilko

The gene product of M. tuberculosis Rv2969c is shown to be a disulfide oxidase enzyme that has a canonical DsbA-like fold with novel structural and functional characteristics. The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins in Escherichia coli (Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB reoxidizes DsbA to maintain it in the active form. In Mycobacterium tuberculosis (Mtb), no DsbB homologue is encoded but a functionally similarmore » but structurally divergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c diverges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited substrate-binding specificity.« less

'Diffuse skin browning' in 1-MCP-treated apples: etiology and systems of control.

PubMed

Larrigaudière, Christian; Vilaplana, Rosa; Recasens, Inmaculada; Soria, Yolanda; Dupille, Eve

2010-11-01

'Diffuse skin browning' (DSB) is a physiological disorder that affects Golden Delicious apples treated with 1-methylcyclopropene (1-MCP). Although a very high incidence is found, very little is known about the etiology of this disorder. This study aims to provide an understanding of the causes of this disorder and prevent it. A very high incidence of DSB was found in 1-MCP-treated apples independent of the location of the orchard. Similar to superficial scald, harvest maturity determines the DSB incidence, with the more mature fruit being less sensitive. The 1-MCP dose (156 nL L(-1) or 625 nL L(-1)) and the temperature at which the 1-MCP treatment was applied (0.5 or 20 °C) did not affect the incidence of DSB. Diphenylamine (DPA) treatment did not prevent DSB, contrary to superficial scald. Additionally, controlled atmosphere storage only partially reduced the incidence of DSB, whereas progressive cooling strategies completely inhibited DSB occurrence. A direct correlation was found between the sensitivity of the Golden Delicious clone to russeting and its sensitivity to develop DSB during storage. Our results indicated that DSB and superficial scald are two different disorders involving different oxidative processes. DSB can be prevented by progressive cooling and selection of russeting-resistant clones. 2010 Society of Chemical Industry

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro.

PubMed

Santos, Clelton A; Toledo, Marcelo A S; Trivella, Daniela B B; Beloti, Lilian L; Schneider, Dilaine R S; Saraiva, Antonio M; Crucello, Aline; Azzoni, Adriano R; Souza, Alessandra A; Aparicio, Ricardo; Souza, Anete P

2012-10-01

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. © 2012 The Authors Journal compilation © 2012 FEBS.

Radiation-induced total-deletion mutations in the human hprt gene: a biophysical model based on random walk interphase chromatin geometry

NASA Technical Reports Server (NTRS)

Wu, H.; Sachs, R. K.; Yang, T. C.

1998-01-01

PURPOSE: To develop a biophysical model that explains the sizes of radiation-induced hprt deletions. METHODS: Key assumptions: (1) Deletions are produced by two DSB that are closer than an interaction distance at the time of DSB induction; (2) Interphase chromatin is modelled by a biphasic random walk distribution; and (3) Misrejoining of DSB from two separate tracks dominates at low-LET and misrejoining of DSB from a single track dominates at high-LET. RESULTS: The size spectra for radiation-induced total deletions of the hprt gene are calculated. Comparing with the results of Yamada and coworkers for gamma-irradiated human fibroblasts the study finds that an interaction distance of 0.75 microm will fit both the absolute frequency and the size spectrum of the total deletions. It is also shown that high-LET radiations produce, relatively, more total deletions of sizes below 0.5 Mb. The model predicts an essential gene to be located between 2 and 3 Mb from the hprt locus towards the centromere. Using the same assumptions and parameters as for evaluating mutation frequencies, a frequency of intra-arm chromosome deletions is calculated that is in agreement with experimental data. CONCLUSIONS: Radiation-induced total-deletion mutations of the human hprt gene and intrachange chromosome aberrations share a common mechanism for their induction.

Structural and biochemical characterization of Xylella fastidiosa DsbA family members: new insights into the enzyme-substrate interaction.

PubMed

Rinaldi, Fábio C; Meza, Andréia N; Guimarães, Beatriz G

2009-04-21

Disulfide oxidoreductase DsbA catalyzes disulfide bond formation in proteins secreted to the periplasm and has been related to the folding process of virulence factors in many organisms. It is among the most oxidizing of the thioredoxin-like proteins, and DsbA redox power is understood in terms of the electrostatic interactions involving the active site motif CPHC. The plant pathogen Xylella fastidiosa has two chromosomal genes encoding two oxidoreductases belonging to the DsbA family, and in one of them, the canonical motif CPHC is replaced by CPAC. Biochemical assays showed that both X. fastidiosa homologues have similar redox properties and the determination of the crystal structure of XfDsbA revealed substitutions in the active site of X. fastidiosa enzymes, which are proposed to compensate for the lack of the conserved histidine in XfDsbA2. In addition, electron density maps showed a ligand bound to the XfDsbA active site, allowing the characterization of the enzyme interaction with an 8-mer peptide. Finally, surface analysis of XfDsbA and XfDsbA2 suggests that X. fastidiosa enzymes may have different substrate specificities.

A disulfide-bond A oxidoreductase-like protein (DsbA-L) regulates adiponectin multimerization

PubMed Central

Liu, Meilian; Zhou, Lijun; Xu, Aimin; Lam, Karen S. L.; Wetzel, Michael D.; Xiang, Ruihua; Zhang, Jingjing; Xin, Xiaoban; Dong, Lily Q.; Liu, Feng

2008-01-01

Impairments in adiponectin multimerization lead to defects in adiponectin secretion and function and are associated with diabetes, yet the underlying mechanisms remain largely unknown. We have identified an adiponectin-interacting protein, previously named GST-kappa, by yeast 2-hybrid screening. The adiponectin-interacting protein contains 2 thioredoxin domains and has very little sequence similarity to other GST isoforms. However, this protein shares high sequence and secondary structure homology to bacterial disulfide-bond A oxidoreductase (DsbA) and is thus renamed DsbA-like protein (DsbA-L). DsbA-L is highly expressed in adipose tissue, and its expression level is negatively correlated with obesity in mice and humans. DsbA-L expression in 3T3-L1 adipocytes is stimulated by the insulin sensitizer rosiglitazone and inhibited by the inflammatory cytokine TNFα. Overexpression of DsbA-L promoted adiponectin multimerization while suppressing DsbA-L expression by RNAi markedly and selectively reduced adiponectin levels and secretion in 3T3-L1 adipocytes. Our results identify DsbA-L as a key regulator for adiponectin biosynthesis and uncover a potential new target for developing therapeutic drugs for the treatment of insulin resistance and its associated metabolic disorders. PMID:19011089

Structural and Biochemical Characterization of Xylella fastidiosa DsbA Family Members: New insightsinto the Enzyme-Substrate Interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rinaldi, F.; Meza, A; Gulmarges, B

2009-01-01

Disulfide oxidoreductase DsbA catalyzes disulfide bond formation in proteins secreted to the periplasm and has been related to the folding process of virulence factors in many organisms. It is among the most oxidizing of the thioredoxin-like proteins, and DsbA redox power is understood in terms of the electrostatic interactions involving the active site motif CPHC. The plant pathogen Xylella fastidiosa has two chromosomal genes encoding two oxidoreductases belonging to the DsbA family, and in one of them, the canonical motif CPHC is replaced by CPAC. Biochemical assays showed that both X. fastidiosa homologues have similar redox properties and the determinationmore » of the crystal structure of XfDsbA revealed substitutions in the active site of X. fastidiosa enzymes, which are proposed to compensate for the lack of the conserved histidine in XfDsbA2. In addition, electron density maps showed a ligand bound to the XfDsbA active site, allowing the characterization of the enzyme interaction with an 8-mer peptide. Finally, surface analysis of XfDsbA and XfDsbA2 suggests that X. fastidiosa enzymes may have different substrate specificities.« less

Association of treatment and outcomes of doctor-shopping behavior in patients with hepatocellular carcinoma

PubMed Central

Hsieh, Cheng-I; Chung, Kuo-Piao; Yang, Ming-Chin; Li, Tsai-Chung

2013-01-01

Background A variety of unfulfilled needs may trigger doctor-shopping behavior (DSB) in patients. In oncology, treatment results usually cause patients the most concern. This study investigated the association of DSB with active treatments received by patients with hepatocellular carcinoma (HCC) and outcomes. Methods With approval from the institutional review board, all patients registered in the cancer database of a teaching hospital and diagnosed as having HCC by self-referral from outside hospitals or by in-house diagnosis were retrospectively identified. Patient data were then reviewed and analyzed via electronic medical records. Results Hepatitis B carriers were significantly more likely than noncarriers to show first-time DSB. Recurrent disease was less likely to result in DSB than predicted. Patients from outside hospitals not receiving upfront first treatment after diagnosis were significantly more likely to show more frequent DSB than those receiving it. Male patients eligible for salvage treatment were less likely to have frequent occurrences of DSB than their female counterparts. Receiving first salvage treatment was not associated with more frequent DSB. Treatment recommendations offered in the study hospital did not influence patients’ decisions to leave or stay. Only elderly patients (>70 years) were less likely to show DSB. Conclusion DSB can occur throughout the entire course of treatment for HCC for a variety of reasons. Active treatments, disease status, and patient characteristics all exerted an influence on DSB. PMID:23874090

Association of treatment and outcomes of doctor-shopping behavior in patients with hepatocellular carcinoma.

PubMed

Hsieh, Cheng-I; Chung, Kuo-Piao; Yang, Ming-Chin; Li, Tsai-Chung

2013-01-01

A variety of unfulfilled needs may trigger doctor-shopping behavior (DSB) in patients. In oncology, treatment results usually cause patients the most concern. This study investigated the association of DSB with active treatments received by patients with hepatocellular carcinoma (HCC) and outcomes. With approval from the institutional review board, all patients registered in the cancer database of a teaching hospital and diagnosed as having HCC by self-referral from outside hospitals or by in-house diagnosis were retrospectively identified. Patient data were then reviewed and analyzed via electronic medical records. Hepatitis B carriers were significantly more likely than noncarriers to show first-time DSB. Recurrent disease was less likely to result in DSB than predicted. Patients from outside hospitals not receiving upfront first treatment after diagnosis were significantly more likely to show more frequent DSB than those receiving it. Male patients eligible for salvage treatment were less likely to have frequent occurrences of DSB than their female counterparts. Receiving first salvage treatment was not associated with more frequent DSB. Treatment recommendations offered in the study hospital did not influence patients' decisions to leave or stay. Only elderly patients (>70 years) were less likely to show DSB. DSB can occur throughout the entire course of treatment for HCC for a variety of reasons. Active treatments, disease status, and patient characteristics all exerted an influence on DSB.

Diversity of the Epsilonproteobacteria Dsb (disulfide bond) systems

PubMed Central

Bocian-Ostrzycka, Katarzyna M.; Grzeszczuk, Magdalena J.; Dziewit, Lukasz; Jagusztyn-Krynicka, Elżbieta K.

2015-01-01

The bacterial proteins of the Dsb family—important components of the post-translational protein modification system—catalyze the formation of disulfide bridges, a process that is crucial for protein structure stabilization and activity. Dsb systems play an essential role in the assembly of many virulence factors. Recent rapid advances in global analysis of bacteria have thrown light on the enormous diversity among bacterial Dsb systems. While the Escherichia coli disulfide bond-forming system is quite well understood, the mechanisms of action of Dsb systems in other bacteria, including members of class Epsilonproteobacteria that contain pathogenic and non-pathogenic bacteria colonizing extremely diverse ecological niches, are poorly characterized. Here we present a review of current knowledge on Epsilonproteobacteria Dsb systems. We have focused on the Dsb systems of Campylobacter spp. and Helicobacter spp. because our knowledge about Dsb proteins of Wolinella and Arcobacter spp. is still scarce and comes mainly from bioinformatic studies. Helicobacter pylori is a common human pathogen that colonizes the gastric epithelium of humans with severe consequences. Campylobacter spp. is a leading cause of zoonotic enteric bacterial infections in most developed and developing nations. We focus on various aspects of the diversity of the Dsb systems and their influence on pathogenicity, particularly because Dsb proteins are considered as potential targets for a new class of anti-virulence drugs to treat human infections by Campylobacter or Helicobacter spp. PMID:26106374

A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

DOE PAGES

Janssen, Aniek; Breuer, Gregory A.; Brinkman, Eva K.; ...

2016-07-15

Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here,more » we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.« less

A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Janssen, Aniek; Breuer, Gregory A.; Brinkman, Eva K.

Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here,more » we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.« less

A Monte-Carlo Model for the Formation of Radiation-induced Chromosomal Aberrations

NASA Technical Reports Server (NTRS)

Ponomarev, Artem L.; Cornforth, Michael N.; Loucas, Brad D.; Cucinotta, Francis A.

2009-01-01

Purpose: To simulate radiation-induced chromosome aberrations in mammalian cells (e.g., rings, translocations, and dicentrics) and to calculate their frequency distributions following exposure to DNA double strand breaks (DSBs) produced by high-LET ions. Methods: The interphase genome was assumed to be comprised of a collection of 2 kbp rigid-block monomers following the random-walk geometry. Additional details for the modeling of chromosomal structure, such as chromosomal domains and chromosomal loops, were included. A radial energy profile for heavy ion tracks was used to simulate the high-LET pattern of induced DSBs. The induced DSB pattern depended on the ion charge and kinetic energy, but always corresponded to the DSB yield of 25 DSBs/cell/Gy. The sum of all energy contributions from Poisson-distributed particle tracks was taken to account for all possible one-track and multi-track effects. The relevant output of the model was DNA fragments produced by DSBs. The DSBs, or breakpoints, were defined by (x, y, z, l) positions, where x, y, z were the Euclidian coordinates of a DSB, and where l was the relative position along the genome. Results: The code was used to carry out Monte Carlo simulations for DSB rejoinings at low doses. The resulting fragments were analyzed to estimate the frequencies of specific types of chromosomal aberrations. Histograms for relative frequencies of chromosomal aberrations and P.D.F.s (probability density functions) of a given aberration type were produced. The relative frequency of dicentrics to rings was compared to empirical data to calibrate rejoining probabilities. Of particular interest was the predicted distribution of ring sizes, irrespective of their frequencies relative to other aberrations. Simulated ring sizes were . 4 kbp, which are far too small to be observed experimentally (i.e., by microscopy) but which, nevertheless, are conjectured to exist. Other aberrations, for example, inversions, translocations, as well as multi-centrics were also recorded. Conclusion: High-LET DNA damage affects the frequencies of chromosomal aberrations. The ratio of rings to dicentrics is correct for the genomic size cut-offs corresponding to available experimental data. The present work predicts a relative abundance of small rings following irradiation by heavy ions.

Dissecting the machinery that introduces disulfide bonds in Pseudomonas aeruginosa.

PubMed

Arts, Isabelle S; Ball, Geneviève; Leverrier, Pauline; Garvis, Steven; Nicolaes, Valérie; Vertommen, Didier; Ize, Bérengère; Tamu Dufe, Veronica; Messens, Joris; Voulhoux, Romé; Collet, Jean-François

2013-12-10

Disulfide bond formation is required for the folding of many bacterial virulence factors. However, whereas the Escherichia coli disulfide bond-forming system is well characterized, not much is known on the pathways that oxidatively fold proteins in pathogenic bacteria. Here, we report the detailed unraveling of the pathway that introduces disulfide bonds in the periplasm of the human pathogen Pseudomonas aeruginosa. The genome of P. aeruginosa uniquely encodes two DsbA proteins (P. aeruginosa DsbA1 [PaDsbA1] and PaDsbA2) and two DsbB proteins (PaDsbB1 and PaDsbB2). We found that PaDsbA1, the primary donor of disulfide bonds to secreted proteins, is maintained oxidized in vivo by both PaDsbB1 and PaDsbB2. In vitro reconstitution of the pathway confirms that both PaDsbB1 and PaDsbB2 shuttle electrons from PaDsbA1 to membrane-bound quinones. Accordingly, deletion of both P. aeruginosa dsbB1 (PadsbB1) and PadsbB2 is required to prevent the folding of several P. aeruginosa virulence factors and to lead to a significant decrease in pathogenicity. Using a high-throughput proteomic approach, we also analyzed the impact of PadsbA1 deletion on the global periplasmic proteome of P. aeruginosa, which allowed us to identify more than 20 new potential substrates of this major oxidoreductase. Finally, we report the biochemical and structural characterization of PaDsbA2, a highly oxidizing oxidoreductase, which seems to be expressed under specific conditions. By fully dissecting the machinery that introduces disulfide bonds in P. aeruginosa, our work opens the way to the design of novel antibacterial molecules able to disarm this pathogen by preventing the proper assembly of its arsenal of virulence factors. The human pathogen Pseudomonas aeruginosa causes life-threatening infections in immunodepressed and cystic fibrosis patients. The emergence of P. aeruginosa strains resistant to all of the available antibacterial agents calls for the urgent development of new antibiotics active against this bacterium. The pathogenic power of P. aeruginosa is mediated by an arsenal of extracellular virulence factors, most of which are stabilized by disulfide bonds. Thus, targeting the machinery that introduces disulfide bonds appears to be a promising strategy to combat P. aeruginosa. Here, we unraveled the oxidative protein folding system of P. aeruginosa in full detail. The system uniquely consists of two membrane proteins that generate disulfide bonds de novo to deliver them to P. aeruginosa DsbA1 (PaDsbA1), a soluble oxidoreductase. PaDsbA1 in turn donates disulfide bonds to secreted proteins, including virulence factors. Disruption of the disulfide bond formation machinery dramatically decreases P. aeruginosa virulence, confirming that disulfide formation systems are valid targets for the design of antimicrobial drugs.

Spatially sculpted laser scissors for study of DNA damage and repair

NASA Astrophysics Data System (ADS)

Stephens, Jared; Mohanty, Samarendra K.; Genc, Suzanne; Kong, Xiangduo; Yokomori, Kyoko; Berns, Michael W.

2009-09-01

We present a simple and efficient method for controlled linear induction of DNA damage in live cells. By passing a pulsed laser beam through a cylindrical lens prior to expansion, an elongated elliptical beam profile is created with the ability to expose controlled linear patterns while keeping the beam and the sample stationary. The length and orientation of the beam at the sample plane were reliably controlled by an adjustable aperture and rotation of the cylindrical lens, respectively. Localized immunostaining by the DNA double strand break (DSB) markers phosphorylated H2AX (γH2AX) and Nbs1 in the nuclei of HeLa cells exposed to the ``line scissors'' was shown via confocal imaging. The line scissors method proved more efficient than the scanning mirror and scanning stage methods at induction of DNA DSB damage with the added benefit of having a greater potential for high throughput applications.

Virulence of the Melioidosis Pathogen Burkholderia pseudomallei Requires the Oxidoreductase Membrane Protein DsbB

PubMed Central

2018-01-01

ABSTRACT The naturally antibiotic-resistant bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a disease with stubbornly high mortality and a complex, protracted treatment regimen. The worldwide incidence of melioidosis is likely grossly underreported, though it is known to be highly endemic in northern Australia and Southeast Asia. Bacterial disulfide bond (DSB) proteins catalyze the oxidative folding and isomerization of disulfide bonds in substrate proteins. In the present study, we demonstrate that B. pseudomallei membrane protein disulfide bond protein B (BpsDsbB) forms a functional redox relay with the previously characterized virulence mediator B. pseudomallei disulfide bond protein A (BpsDsbA). Genomic analysis of diverse B. pseudomallei clinical isolates demonstrated that dsbB is a highly conserved core gene. Critically, we show that DsbB is required for virulence in B. pseudomallei. A panel of B. pseudomallei dsbB deletion strains (K96243, 576, MSHR2511, MSHR0305b, and MSHR5858) were phenotypically diverse according to the results of in vitro assays that assess hallmarks of virulence. Irrespective of their in vitro virulence phenotypes, two deletion strains were attenuated in a BALB/c mouse model of infection. A crystal structure of a DsbB-derived peptide complexed with BpsDsbA provides the first molecular characterization of their interaction. This work contributes to our broader understanding of DSB redox biology and will support the design of antimicrobial drugs active against this important family of bacterial virulence targets. PMID:29440370

Radiosensitization of tumour cell lines by the polyphenol Gossypol results from depressed double-strand break repair and not from enhanced apoptosis.

PubMed

Kasten-Pisula, Ulla; Windhorst, Sabine; Dahm-Daphi, Jochen; Mayr, Georg; Dikomey, Ekkehard

2007-06-01

New drugs are needed to increase the efficiency of radiotherapy in order to improve the therapeutic outcome of tumour patients. In this respect, the polyphenol Gossypol might be of interest, because of its effect on apoptosis and DNA repair, which is either mediated directly or indirectly via the inositol phosphate metabolism. It was investigated, whether these effects result in enhanced radiosensitivity of tumour cells. Tumour cell lines investigated: A549, FaDu, H1299, MCF7 and Du145. Cell cycle distribution was determined by FACS analysis, apoptosis was measured by DAPI staining and caspase3/7 activity. Double-strand breaks (DSB) were investigated via gammaH2AX-foci and cell survival by colony formation assay. The level of inositol phosphates was determined by HPLC, protein expression by Western blot. In A549 cells, Gossypol at concentrations 1microM strongly affects proliferation with only a modest arrest in the G1-phase, but with no increase in the fraction of apoptotic cells or the number of additional DSB. Additional DSB were only seen in FaDu cells, where Gossypol (2microM) was extremely toxic with a plating efficiency <0.002. When combined with irradiation, incubation with Gossypol (1-2microM) was found to result in an enhanced radiosensitivity with, however, a substantial variation. While there was a strong radiosensitization for FaDu and Du145 cells, there was an intermediate response for A549 cells, but almost no effect for H1299 and MCF7 cells. This sensitization was not caused from an elevated rate of apoptosis, but primarily resulted from reduced DSB repair capacity. The reduction in DSB repair could be ascribed neither to changes in the level of repair proteins relevant for non-homologous end-joining (Ku70, Ku80, DNA-PKcs) nor to changes in the level of higher phosphorylated inositols, whereby the latter were even found to be enhanced by Gossypol. For some tumour cell lines treatment with low concentrations of Gossypol can be used to inhibit DSB repair capacity and with that to increase the cellular radiosensitivity.

Monte-Carlo Simulation of Radiation Track Structure and Calculation of Dose Deposition in Nanovolumes

NASA Technical Reports Server (NTRS)

Plante, I.; Cucinotta, F. A.

2010-01-01

INTRODUCTION: The radiation track structure is of crucial importance to understand radiation damage to molecules and subsequent biological effects. Of a particular importance in radiobiology is the induction of double-strand breaks (DSBs) by ionizing radiation, which are caused by clusters of lesions in DNA, and oxidative damage to cellular constituents leading to aberrant signaling cascades. DSB can be visualized within cell nuclei with gamma-H2AX experiments. MATERIAL AND METHODS: In DSB induction models, the DSB probability is usually calculated by the local dose obtained from a radial dose profile of HZE tracks. In this work, the local dose imparted by HZE ions is calculated directly from the 3D Monte-Carlo simulation code RITRACKS. A cubic volume of 5 micron edge (Figure 1) is irradiated by a (Fe26+)-56 ion of 1 GeV/amu (LET approx.150 keV/micron) and by a fluence of 450 H+ ions, 300 MeV/amu (LET approx. 0.3 keV/micron). In both cases, the dose deposited in the volume is approx.1 Gy. The dose is then calculated into each 3D pixels (voxels) of 20 nm edge and visualized in 3D. RESULTS AND DISCUSSION: The dose is deposited uniformly in the volume by the H+ ions. The voxels which receive a high dose (orange) corresponds to electron track ends. The dose is deposited differently by the 56Fe26+ ion. Very high dose (red) is deposited in voxels with direct ion traversal. Voxels with electron track ends (orange) are also found distributed around the path of the track. In both cases, the appearance of the dose distribution looks very similar to DSBs seen in gammaH2AX experiments, particularly when the visualization threshold is applied. CONCLUSION: The refinement of the dose calculation to the nanometer scale has revealed important differences in the energy deposition between high- and low-LET ions. Voxels of very high dose are only found in the path of high-LET ions. Interestingly, experiments have shown that DSB induced by high-LET radiation are more difficult to repair. Therefore, this new approach may be useful to understand the nature of DSB and oxidative damage induced by ionizing radiation.

Virtual Screening of Peptide and Peptidomimetic Fragments Targeted to Inhibit Bacterial Dithiol Oxidase DsbA.

PubMed

Duprez, Wilko; Bachu, Prabhakar; Stoermer, Martin J; Tay, Stephanie; McMahon, Róisín M; Fairlie, David P; Martin, Jennifer L

2015-01-01

Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB--or peptides--complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.

DsbA Plays a Critical and Multifaceted Role in the Production of Secreted Virulence Factors by the Phytopathogen Erwinia carotovora subsp. atroseptica*S⃞

PubMed Central

Coulthurst, Sarah J.; Lilley, Kathryn S.; Hedley, Peter E.; Liu, Hui; Toth, Ian K.; Salmond, George P. C.

2008-01-01

Erwinia carotovora subsp. atroseptica is an enterobacterial phytopathogen causing economically significant soft rot disease. Pathogenesis is mediated by multiple secreted virulence factors, many of which are secreted by the type II (Out) secretion system. DsbA catalyzes the introduction of disulfide bonds into periplasmic and secreted proteins. In this study, the extracellular proteome (secretome) of wild type E. carotovora subsp. atroseptica SCRI1043, and dsbA and out mutants, was analyzed by spectral counting mass spectrometry. This revealed that dsbA inactivation had a huge impact on the secretome and identified diverse DsbA- and Out-dependent secreted proteins, representing known, predicted, and novel candidate virulence factors. Further characterization of the dsbA mutant showed that secreted enzyme activities, motility, production of the quorumsensing signal, and virulence were absent or substantially reduced. The impact of DsbA on secreted virulence factor production was mediated at multiple levels, including impacting on the Out secretion system and the virulence gene regulatory network. Transcriptome analyses revealed that the abundance of a broad, but defined, set of transcripts, including many virulence factors, was altered in the dsbA mutant, identifying a new virulence regulon responsive to extracytoplasmic conditions. In conclusion, DsbA plays a crucial, multifaceted role in the pathogenesis of E. carotovora subsp. atroseptica. PMID:18562317

Editing disulphide bonds: error correction using redox currencies.

PubMed

Ito, Koreaki

2010-01-01

The disulphide bond-introducing enzyme of bacteria, DsbA, sometimes oxidizes non-native cysteine pairs. DsbC should rearrange the resulting incorrect disulphide bonds into those with correct connectivity. DsbA and DsbC receive oxidizing and reducing equivalents, respectively, from respective redox components (quinones and NADPH) of the cell. Two mechanisms of disulphide bond rearrangement have been proposed. In the redox-neutral 'shuffling' mechanism, the nucleophilic cysteine in the DsbC active site forms a mixed disulphide with a substrate and induces disulphide shuffling within the substrate part of the enzyme-substrate complex, followed by resolution into a reduced enzyme and a disulphide-rearranged substrate. In the 'reduction-oxidation' mechanism, DsbC reduces those substrates with wrong disulphides so that DsbA can oxidize them again. In this issue of Molecular Microbiology, Berkmen and his collaborators show that a disulphide reductase, TrxP, from an anaerobic bacterium can substitute for DsbC in Escherichia coli. They propose that the reduction-oxidation mechanism of disulphide rearrangement can indeed operate in vivo. An implication of this work is that correcting errors in disulphide bonds can be coupled to cellular metabolism and is conceptually similar to the proofreading processes observed with numerous synthesis and maturation reactions of biological macromolecules.

Relative contribution of homologous recombination and non-homologous end-joining to DNA double-strand break repair after oxidative stress in Saccharomyces cerevisiae.

PubMed

Letavayová, Lucia; Marková, Eva; Hermanská, Katarína; Vlcková, Viera; Vlasáková, Danusa; Chovanec, Miroslav; Brozmanová, Jela

2006-05-10

Oxidative damage to DNA seems to be an important factor in developing many human diseases including cancer. It involves base and sugar damage, base-free sites, DNA-protein cross-links and DNA single-strand (SSB) and double-strand (DSB) breaks. Oxidative DSB can be formed in various ways such as their direct induction by the drug or their generation either through attempted and aborted repair of primary DNA lesions or through DNA replication-dependent conversion of SSB. In general, two main pathways are responsible for repairing DSB, homologous recombination (HR) and non-homologous end-joining (NHEJ), with both of them being potential candidates for the repair of oxidative DSB. We have examined relative contribution of HR and NHEJ to cellular response after oxidative stress in Saccharomyces cerevisiae. Therefore, cell survival, mutagenesis and DSB induction and repair in the rad52, yku70 and rad52 yku70 mutants after hydrogen peroxide (H(2)O(2)), menadione (MD) or bleomycin (BLM) exposure were compared to those obtained for the corresponding wild type. We show that MD exposure does not lead to observable DSB induction in yeast, suggesting that the toxic effects of this agent are mediated by other types of DNA damage. Although H(2)O(2) treatment generates some DSB, their yield is relatively low and hence DSB may only partially be responsible for toxicity of H(2)O(2), particularly at high doses of the agent. On the other hand, the basis of the BLM toxicity resides primarily in DSB induction. Both HR and NHEJ act on BLM-induced DSB, although their relative participation in the process is not equal. Based on our results we suggest that the complexity and/or the quality of the BLM-induced DSB might represent an obstacle for the NHEJ pathway.

Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 or PARP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stenerlöw, Bo; Karlsson, Karin H.; Radulescu, Irina

2008-04-29

Ionizing radiation induces a variety of different DNA lesions: in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have previously shown that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites (HLS) within clustered lesions, thus reflecting an artifact of preparation ofmore » genomic DNA at elevated temperature. To further characterize the influence of HLS on DSB induction and repair, four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for bi-phasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements the fraction of fast rejoining decreased to less than 50% of the total. However, neither the half-times of the fast (t{sub 1/2} = 7-8 min) or slow (t{sub 1/2} = 2.5 h) DSB rejoining were changed significantly. At t=0 the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSB/cell/Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all tested cells, including M059K cells treated with wortmannin or DNA-PKcs defect M059J cells. Furthermore, cells lacking XRCC1 or Poly(ADP-ribose) polymerase-1 (PARP-1) rejoined both total DSBs and heat-released DSBs similar to normal cells. In summary, the presence of heat-labile sites have a substantial impact on DSB induction yields and DSB rejoining rates measured by pulsed-field gel electrophoresis, and HLS repair is independent of DNA-PKcs, XRCC1 and PARP.« less

Kinetics of DSB rejoining and formation of simple chromosome exchange aberrations

NASA Technical Reports Server (NTRS)

Cucinotta, F. A.; Nikjoo, H.; O'Neill, P.; Goodhead, D. T.

2000-01-01

PURPOSE: To investigate the role of kinetics in the processing of DNA double strand breaks (DSB), and the formation of simple chromosome exchange aberrations following X-ray exposures to mammalian cells based on an enzymatic approach. METHODS: Using computer simulations based on a biochemical approach, rate-equations that describe the processing of DSB through the formation of a DNA-enzyme complex were formulated. A second model that allows for competition between two processing pathways was also formulated. The formation of simple exchange aberrations was modelled as misrepair during the recombination of single DSB with undamaged DNA. Non-linear coupled differential equations corresponding to biochemical pathways were solved numerically by fitting to experimental data. RESULTS: When mediated by a DSB repair enzyme complex, the processing of single DSB showed a complex behaviour that gives the appearance of fast and slow components of rejoining. This is due to the time-delay caused by the action time of enzymes in biomolecular reactions. It is shown that the kinetic- and dose-responses of simple chromosome exchange aberrations are well described by a recombination model of DSB interacting with undamaged DNA when aberration formation increases with linear dose-dependence. Competition between two or more recombination processes is shown to lead to the formation of simple exchange aberrations with a dose-dependence similar to that of a linear quadratic model. CONCLUSIONS: Using a minimal number of assumptions, the kinetics and dose response observed experimentally for DSB rejoining and the formation of simple chromosome exchange aberrations are shown to be consistent with kinetic models based on enzymatic reaction approaches. A non-linear dose response for simple exchange aberrations is possible in a model of recombination of DNA containing a DSB with undamaged DNA when two or more pathways compete for DSB repair.

Fat-Specific DsbA-L Overexpression Promotes Adiponectin Multimerization and Protects Mice From Diet-Induced Obesity and Insulin Resistance

PubMed Central

Liu, Meilian; Xiang, Ruihua; Wilk, Sarah Ann; Zhang, Ning; Sloane, Lauren B.; Azarnoush, Kian; Zhou, Lijun; Chen, Hongzhi; Xiang, Guangda; Walter, Christi A.; Austad, Steven N.; Musi, Nicolas; DeFronzo, Ralph A.; Asmis, Reto; Scherer, Philipp E.; Dong, Lily Q.; Liu, Feng

2012-01-01

The antidiabetic and antiatherosclerotic effects of adiponectin make it a desirable drug target for the treatment of metabolic and cardiovascular diseases. However, the adiponectin-based drug development approach turns out to be difficult due to extremely high serum levels of this adipokine. On the other hand, a significant correlation between adiponectin multimerization and its insulin-sensitizing effects has been demonstrated, suggesting a promising alternative therapeutic strategy. Here we show that transgenic mice overexpressing disulfide bond A oxidoreductase-like protein in fat (fDsbA-L) exhibited increased levels of total and the high-molecular-weight form of adiponectin compared with wild-type (WT) littermates. The fDsbA-L mice also displayed resistance to diet-induced obesity, insulin resistance, and hepatic steatosis compared with WT control mice. The protective effects of DsbA-L overexpression on diet-induced insulin resistance, but not increased body weight and fat cell size, were significantly decreased in adiponectin-deficient fDsbA-L mice (fDsbA-L/Ad−/−). In addition, the fDsbA-L/Ad−/− mice displayed greater activity and energy expenditure compared with adiponectin knockout mice under a high-fat diet. Taken together, our results demonstrate that DsbA-L protects mice from diet-induced obesity and insulin resistance through adiponectin-dependent and independent mechanisms. In addition, upregulation of DsbA-L could be an effective therapeutic approach for the treatment of obesity and its associated metabolic disorders. PMID:22807031

The active structure of the Dead Sea depression

NASA Astrophysics Data System (ADS)

Shamir, G.

2003-04-01

The ~220km long gravitational and structural Dead Sea Depression (DSD), situated along the southern section of the Dead Sea Transform (DST), is centered by the Dead Sea basin sensu strictu (DSB), which has been described since the 1960?s as a pull-apart basin over a presumed left-hand fault step. However, several observations, or their lack thereof, question this scheme, e.g. (i) It is not supported by recent seismological and geomorphic data; (ii) It does not explain the fault pattern and mixed sinistral and dextral offset along the DSB western boundary; (iii) It does not simply explain the presence of intense deformation outside the presumed fault step zone; (iv) It is inconsistent with the orientation of seismically active faults within the Dead Sea and Jericho Valley; (v); It is apparently inconsistent with the symmetrical structure of the DSD; (vi) The length of the DSB exceeds the total offset along the Dead Sea Transform, while its subsidence is about the age of the DST. Integration of newly acquired and analyzed data (high resolution and petroleum seismic reflection data, earthquake relocation and fault plane solutions) with previously published data (structural mapping, fracture orientation distribution, Bouguer anomaly maps, sinkhole distribution, geomorphic lineaments) now shows that the active upper crustal manifestation of the DSD is a broad shear zone dominated by internal fault systems oriented NNE and NNW. These fault systems are identified by earthquake activity, seismic reflection observations, alignment of recent sinkholes, and distribution of Bouguer anomaly gradients. Motion on the NNE system is normal-dextral, suggesting that counterclockwise rotation may have taken place within the shear zone. The overall sinistral motion between the Arabian and Israel-Sinai plates along the DSD is thus accommodated by distributed shear across the N-S extending DSD. The three-dimensionality of this motion at the DSD may be related to the rate of convergence between the two plates.

Super-resolution imaging identifies PARP1 and the Ku complex acting as DNA double-strand break sensors

PubMed Central

Yang, Guang; Liu, Chao; Chen, Shih-Hsun; Kassab, Muzaffer A; Hoff, J Damon; Yu, Xiaochun

2018-01-01

Abstract DNA double-strand breaks (DSBs) are fatal DNA lesions and activate a rapid DNA damage response. However, the earliest stage of DSB sensing remains elusive. Here, we report that PARP1 and the Ku70/80 complex localize to DNA lesions considerably earlier than other DSB sensors. Using super-resolved fluorescent particle tracking, we further examine the relocation kinetics of PARP1 and the Ku70/80 complex to a single DSB, and find that PARP1 and the Ku70/80 complex are recruited to the DSB almost at the same time. Notably, only the Ku70/80 complex occupies the DSB exclusively in the G1 phase; whereas PARP1 competes with the Ku70/80 complex at the DSB in the S/G2 phase. Moreover, in the S/G2 phase, PARP1 removes the Ku70/80 complex through its enzymatic activity, which is further confirmed by in vitro DSB-binding assays. Taken together, our results reveal PARP1 and the Ku70/80 complex as critical DSB sensors, and suggest that PARP1 may function as an important regulator of the Ku70/80 complex at the DSBs in the S/G2 phase. PMID:29447383

Mechanisms underlying radiosensitivity : iIvestigations in xrs-5, an X-ray sensitive hamster cell line

NASA Astrophysics Data System (ADS)

Johnston, Peter James

The damage caused to cells by ionising radiation is believed to center on damage to the DNA. In particular, the induction of DNA double strand breaks (DSB) have been implicated in biological end-points such as cell killing and the formation of chromosomal aberrations. The xrs-5 cell line is a mutant Chinese hamster ovary fibroblast (CHO-K1) mutant which exhibits sensitivity to ionising radiation and a number of other DNA damaging agents. This mutation, postulated to involve the hamster homologue of the human XRCC5 gene, is believed to be involved in the repair of the DSB. In addition, there are constitutive differences between the wild type and xrs cells involving the structure and function of the nucleus and higher order chromatin structures. The aims of this thesis were to study further the xrs-5 cell line and its response to DNA damage and to investigate the possible link between chromatin structure and DSB repair. By the examination of the response of xrs-5 cells to a number of DNA damaging agents and potential modulators of this response using the cytokinesis block micronucleus assay [Fenech and Morley, 1985] a possible cell cycle defect was identified in addition to elevated levels of chromosomal damage. Xrs-5 cells appeared to be partially defective in the cell cycle checkpoints involving the passage from G2 phase to mitosis. By the use of a modified neutral filter elution procedure variations in the repair of DSB were observed between xrs-5 and CHO. Conventional neutral filter elution requires harsh lysis conditions to remove higher order chromatin structures which interfere with the elution of DNA containing DSB. By lysing cells with non-ionic detergent in the presence of 2 M NaC1, histone depleted structures which retain the higher order nuclear matrix organisation, including chromatin loops, can be produced. Elution from these structures will only occur if two or more DSB lie within a single looped domain delineated by points of attachment to the nuclear matrix. Repair experiments indicate that in CHO cells repair of DSB in loops containing multiple DSB are repaired with "slow" kinetics (t1/2 = 5 hrs) whilst DSB occurring in loops containing single DSB are repaired with "fast" kinetics (t1/2 " 10 min). Xrs- 5 cells are incapable of repairing these multiply damaged loops. This work indicates that the spatial orientation of DSB in higher order structures of chromatin are a possible factor in the repair of these lesions. By construction of a mathematical model of the process of elution from chromatin loops it was possible to postulate the size of the loops to approximate to 2.5-3 Mbp. Further evidence of a potential structural defect in the chromatin of xrs-5 cells was provided by examination of the polypeptide composition and DNA binding activity of nuclear extracts. The affinity of extracted proteins for double-stranded calf-thymus DNA was measured in nuclear extracts of xrs-5 and CHO cells. There was an alteration in the DNA binding activity of salt extractable proteins from xrs-5 as measured by a filter binding assay. By the use of SDS-PAGE and the technique of South-Western blotting, it was possible to identify the approximate molecular weights of these DNA binding proteins. Differences were found in DNA binding between proteins from CHO and xrs-5 extracts of both non-irradiated and irradiated cells. Two proteins with apparent molecular weights of 32.2 and 31.8 kDa exhibited a lower DNA binding activity in xrs-5 than proteins of similar extracts from CHO. The amount of the 32.2 kDa protein was less in the xrs-5 extracts than in CHO extracts, as measured by Coomassie blue staining. The two proteins have not yet been identified but comprise a major DNA binding activity in CHO extracts obtained by detergent-free extraction procedures. This work provides circumstantial evidence that suggests these two polypeptides may form part of the histone H1 family.

Crystallization and preliminary diffraction analysis of a DsbA homologue from Wolbachia pipientis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurz, M.; Iturbe-Ormaetxe, I.; Jarrott, R.

2008-02-01

The first crystallization of a W. pipientis protein, α-DsbA1, was achieved using hanging-drop and sitting-drop vapour diffusion. α-DsbA1 is one of two DsbA homologues encoded by the Gram-negative α-proteobacterium Wolbachia pipientis, an endosymbiont that can behave as a reproductive parasite in insects and as a mutualist in medically important filarial nematodes. The α-DsbA1 protein is thought to be important for the folding and secretion of Wolbachia proteins involved in the induction of reproductive distortions. Crystals of native and SeMet α-DsbA1 were grown by vapour diffusion and belong to the monoclinic space group C2, with unit-cell parameters a = 71.4, bmore » = 49.5, c = 69.3 Å, β = 107.0° and one molecule in the asymmetric unit (44% solvent content). X-ray data were recorded from native crystals to a resolution of 2.01 Å using a copper anode and data from SeMet α-DsbA1 crystals were recorded to 2.45 Å resolution using a chromium anode.« less

The Mechanism of Gene Targeting in Human Somatic Cells

PubMed Central

Kan, Yinan; Ruis, Brian; Lin, Sherry; Hendrickson, Eric A.

2014-01-01

Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB) repair known as homologous recombination (HR). The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells. PMID:24699519

The effect of deep and slow breathing on pain perception, autonomic activity, and mood processing--an experimental study.

PubMed

Busch, Volker; Magerl, Walter; Kern, Uwe; Haas, Joachim; Hajak, Göran; Eichhammer, Peter

2012-02-01

Deep and slow breathing (DSB) techniques, as a component of various relaxation techniques, have been reported as complementary approaches in the treatment of chronic pain syndromes, but the relevance of relaxation for alleviating pain during a breathing intervention was not evaluated so far. In order to disentangle the effects of relaxation and respiration, we investigated two different DSB techniques at the same respiration rates and depths on pain perception, autonomic activity, and mood in 16 healthy subjects. In the attentive DSB intervention, subjects were asked to breathe guided by a respiratory feedback task requiring a high degree of concentration and constant attention. In the relaxing DSB intervention, the subjects relaxed during the breathing training. The skin conductance levels, indicating sympathetic tone, were measured during the breathing maneuvers. Thermal detection and pain thresholds for cold and hot stimuli and profile of mood states were examined before and after the breathing sessions. The mean detection and pain thresholds showed a significant increase resulting from the relaxing DSB, whereas no significant changes of these thresholds were found associated with the attentive DSB. The mean skin conductance levels indicating sympathetic activity decreased significantly during the relaxing DSB intervention but not during the attentive DSB. Both breathing interventions showed similar reductions in negative feelings (tension, anger, and depression). Our results suggest that the way of breathing decisively influences autonomic and pain processing, thereby identifying DSB in concert with relaxation as the essential feature in the modulation of sympathetic arousal and pain perception. Wiley Periodicals, Inc.

Comparison of DSB effects of the beta particles of iodine-131 and 6 MV X-ray at a dose of 2 Gy in the presence of 2-Methoxyestradiol, IUdR, and TPT in glioblastoma spheroids

NASA Astrophysics Data System (ADS)

Neshasteh-Riz, Ali; Eyvazzadeh, Nazila; Koosha, Fereshteh; Cheraghi, Susan

2017-02-01

Glioblastoma is one of the lethal brain tumors and one of the resistant tumors against radiotherapy. Multiple treatment methods and different types of radiation and Radiosensitizers drugs have been combined to optimize the efficacy of radiotherapy. Radiosensitizers are employed to reinforce tumor cell killing and have much fewer effects on the normal tissue. Inducing DNA double strand break in tumoral cells is a major goal of radiation sensitivity. In this study, the level of DNA double strand break in glioblastoma spheroids irradiated by 2 Gy beta particles of iodine-131 and 6 MV X-rays in the presence of 2-Methoxyestradiol (2ME2), iodo-deoxy-uridine (IUdR) and Topotecan (TPT) was measured using the PicoGreen method. Spheroids of the U87MG cell line were cultured to reach a 300 μm diameter. In the phase one of the study, the spheroids were treated in four groups individually, including 2 Gy of iodine-131, TPT+iodine-131, IUdR+iodine-131, IUdR+2ME2+iodine-131. In the next phase, the cells were treated with 2 Gy of 6 MV X-ray, TPT+6 MV X-ray, IUdR+6 MV X-ray, TPT+IUdR+6 MV X-ray. DSB lesions were measured by the Pico Green assay. The amount of DSB lesions in groups irradiated with iodine-131 individually was greater than the group irradiated with 6 MV X-ray (p<0.05). DNA double strand breaks became more significant in combination with TPT. However, the amount of DSBs in the two independent groups of TPT+IUdR+2ME2+iodine-131 and TPT+IUdR+2ME2+6 MV X-ray was approximately in the same range (P>0.05). The level of DNA double strand breaks in cells irradiated with Iodine-131 was higher than cells irradiated with 6 MV X-ray at the same dose and Topotecan had a positive effect on inducing the damage. The role of 2ME2+IUdR in increasing the damage caused by beta particles of iodine-131 was not significant. Iodine-131 could lead to major DSB damage than 6 MV X-ray at the same dose due to its cross fire effect and spatial distribution of energy in different angels. This study showed that a combination of chemotherapy and iodine-131 had better efficacy than radiotherapy with 6 MV X-ray in the treatment of glioblastoma.

The formation of double-strand breaks at multiply damaged sites is driven by the kinetics of excision/incision at base damage in eukaryotic cells

PubMed Central

Kozmin, Stanislav G.; Sedletska, Yuliya; Reynaud-Angelin, Anne; Gasparutto, Didier; Sage, Evelyne

2009-01-01

It has been stipulated that repair of clustered DNA lesions may be compromised, possibly leading to the formation of double-strand breaks (DSB) and, thus, to deleterious events. Using a variety of model multiply damaged sites (MDS), we investigated parameters that govern the formation of DSB during the processing of MDS. Duplexes carrying MDS were inserted into replicative or integrative vectors, and used to transform yeast Saccharomyces cerevisiae. Formation of DSB was assessed by a relevant plasmid survival assay. Kinetics of excision/incision and DSB formation at MDS was explored using yeast cell extracts. We show that MDS composed of two uracils or abasic sites, were rapidly incised and readily converted into DSB in yeast cells. In marked contrast, none of the MDS carrying opposed oG and hU separated by 3–8 bp gave rise to DSB, despite the fact that some of them contained preexisting single-strand break (a 1-nt gap). Interestingly, the absence of DSB formation in this case correlated with slow excision/incision rates of lesions. We propose that the kinetics of the initial repair steps at MDS is a major parameter that direct towards the conversion of MDS into DSB. Data provides clues to the biological consequences of MDS in eukaryotic cells. PMID:19174565

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites

PubMed Central

Kato, Takamitsu A.; Suzuki, Takehiro; Dohmae, Naoshi; Takizawa, Kazuya; Nakazawa, Yuka; Genet, Matthew D.; Saotome, Mika; Hama, Michio; Nakajima, Nakako Izumi; Hazawa, Masaharu; Tomita, Masanori; Koike, Manabu; Noshiro, Katsuko; Tomiyama, Kenichi; Obara, Chizuka; Gotoh, Takaya; Ui, Ayako; Fujimori, Akira; Nakayama, Fumiaki; Sugasawa, Kaoru; Okayasu, Ryuichi; Tajima, Katsushi

2018-01-01

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism. PMID:29590107

Fusion of SpCas9 to E. coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells.

PubMed

Lin, Lin; Petersen, Trine Skov; Jensen, Kristopher Torp; Bolund, Lars; Kühn, Ralf; Luo, Yonglun

2017-04-10

Mammalian cells repair double-strand DNA breaks (DSB) by a range of different pathways following DSB induction by the engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9. While CRISPR-Cas9 thus enables predesigned modifications of the genome, applications of CRISPR-Cas9-mediated genome-editing are frequently hampered by the unpredictable and varying pathways for DSB repair in mammalian cells. Here we present a strategy of fusing Cas9 to recombinant proteins for fine-tuning of the DSB repair preferences in mammalian cells. By fusing Streptococcus Pyogenes Cas9 (SpCas9) to the recombinant protein A (Rec A, NP_417179.1) from Escherichia coli, we create a recombinant Cas9 protein (rSpCas9) which enhances the generation of indel mutations at DSB sites in mammalian cells, increases the frequency of DSB repair by homology-directed single-strand annealing (SSA), and represses homology-directed gene conversion by approximately 33%. Our study thus proves for the first time that fusing SpCas9 to recombinant proteins can influence the balance between DSB repair pathways in mammalian cells. This approach may form the basis for further investigations of the applications of recombinant Cas9 proteins to fine-tuning DSB repair pathways in eukaryotic cells. Copyright © 2017 Elsevier B.V. All rights reserved.

On the mutagenicity of homologous recombination and double-strand break repair in bacteriophage.

PubMed

Shcherbakov, Victor P; Plugina, Lidiya; Shcherbakova, Tamara; Sizova, Svetlana; Kudryashova, Elena

2011-01-02

The double-strand break (DSB) repair via homologous recombination is generally construed as a high-fidelity process. However, some molecular genetic observations show that the recombination and the recombinational DSB repair may be mutagenic and even highly mutagenic. Here we developed an effective and precise method for studying the fidelity of DSB repair in vivo by combining DSBs produced site-specifically by the SegC endonuclease with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. The method is based on the comparison of the rate of reversion of rII mutation in the presence and in the absence of a DSB repair event initiated in the proximity of the mutation. We observed that DSB repair may moderately (up to 6-fold) increase the apparent reversion frequency, the effect of being dependent on the mutation structure. We also studied the effect of the T4 recombinase deficiency (amber mutation in the uvsX gene) on the fidelity of DSB repair. We observed that DSBs are still repaired via homologous recombination in the uvsX mutants, and the apparent fidelity of this repair is higher than that seen in the wild-type background. The mutator effect of the DSB repair may look unexpected given that most of the normal DNA synthesis in bacteriophage T4 is performed via a recombination-dependent replication (RDR) pathway, which is thought to be indistinguishable from DSB repair. There are three possible explanations for the observed mutagenicity of DSB repair: (1) the origin-dependent (early) DNA replication may be more accurate than the RDR; (2) the step of replication initiation may be more mutagenic than the process of elongation; and (3) the apparent mutagenicity may just reflect some non-randomness in the pool of replicating DNA, i.e., preferential replication of the sequences already involved in replication. We discuss the DSB repair pathway in the absence of UvsX recombinase. Copyright © 2010 Elsevier B.V. All rights reserved.

Sulfur Denitrosylation by an Engineered Trx-like DsbG Enzyme Identifies Nucleophilic Cysteine Hydrogen Bonds as Key Functional Determinant.

PubMed

Lafaye, Céline; Van Molle, Inge; Tamu Dufe, Veronica; Wahni, Khadija; Boudier, Ariane; Leroy, Pierre; Collet, Jean-François; Messens, Joris

2016-07-15

Exposure of bacteria to NO results in the nitrosylation of cysteine thiols in proteins and low molecular weight thiols such as GSH. The cells possess enzymatic systems that catalyze the denitrosylation of these modified sulfurs. An important player in these systems is thioredoxin (Trx), a ubiquitous, cytoplasmic oxidoreductase that can denitrosylate proteins in vivo and S-nitrosoglutathione (GSNO) in vitro However, a periplasmic or extracellular denitrosylase has not been identified, raising the question of how extracytoplasmic proteins are repaired after nitrosative damage. In this study, we tested whether DsbG and DsbC, two Trx family proteins that function in reducing pathways in the Escherichia coli periplasm, also possess denitrosylating activity. Both DsbG and DsbC are poorly reactive toward GSNO. Moreover, DsbG is unable to denitrosylate its specific substrate protein, YbiS. Remarkably, by borrowing the CGPC active site of E. coli Trx-1 in combination with a T200M point mutation, we transformed DsbG into an enzyme highly reactive toward GSNO and YbiS. The pKa of the nucleophilic cysteine, as well as the redox and thermodynamic properties of the engineered DsbG are dramatically changed and become similar to those of E. coli Trx-1. X-ray structural insights suggest that this results from a loss of two direct hydrogen bonds to the nucleophilic cysteine sulfur in the DsbG mutant. Our results highlight the plasticity of the Trx structural fold and reveal that the subtle change of the number of hydrogen bonds in the active site of Trx-like proteins is the key factor that thermodynamically controls reactivity toward nitrosylated compounds. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis.

PubMed

Liu, Mingming; Ba, Zhaoqing; Costa-Nunes, Pedro; Wei, Wei; Li, Lanxia; Kong, Fansi; Li, Yan; Chai, Jijie; Pontes, Olga; Qi, Yijun

2017-03-01

Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Arabidopsis thaliana Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA binding ARGONAUTE2 leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from single-stranded DNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair. © 2017 American Society of Plant Biologists. All rights reserved.

Delayed repair of radiation induced clustered DNA damage: Friend or foe?

PubMed Central

Eccles, Laura J.; O’Neill, Peter; Lomax, Martine E.

2011-01-01

A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with associated base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will concentrate on the experimental findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addition certain non-DSB clustered damaged sites are processed within the cell to form additional DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a “friend”, leading to cell killing in tumour cells or as a “foe”, resulting in the formation of mutations and genetic instability in normal tissue. PMID:21130102

Study on doctor shopping behavior: insight from patients with upper respiratory tract infection in Taiwan.

PubMed

Wang, Ming-Jye; Lin, Shu-Ping

2010-01-01

Based on the actual medical records of ambulatory care visits, this study analyzed patients' healthcare seeking behavior and doctor shopping behavior (DSB), and investigated the underlying factors and the impact on the depletion of the healthcare resources for health policy makers to build a better health delivery system. Among a cohort comprised of 200,000 patients randomly chosen from the National Health Insurance Research Database of Taiwan in 2004, only the patients seeking ambulatory care visits for upper respiratory tract infection (URI) were analyzed. Among the 45,951 URI patients, 2875 of them exhibited DSB (prevalence 6.3%). The DSB showed a reverse U-shaped relationship with the patient age (the highest DSB in age 18-34 years). The episodes of the URI had a negative impact on the DSB. The odds ratios of gender and the frequency of consultation versus DSB were 1.10 and 4.72, respectively, and the depletion of days of medication and repeat prescription increased with doctor shopping. Health education to raise DSB awareness is necessary, especially for female's age 18-34 years. Implementing a proper referral system with efficient data exchange, setting up control parameters in the IC cards, and strengthening the integrated care plan could reduce the unnecessary waste of the healthcare resources.

Sent packing: protein engineering generates a new crystal form of Pseudomonas aeruginosa DsbA1 with increased catalytic surface accessibility

DOE Office of Scientific and Technical Information (OSTI.GOV)

McMahon, Roisin M., E-mail: r.mcmahon1@uq.edu.au; Coinçon, Mathieu; Tay, Stephanie

The crystal structure of a P. aeruginosa DsbA1 variant is more suitable for fragment-based lead discovery efforts to identify inhibitors of this antimicrobial drug target. In the reported structures the active site of the protein can simultaneously bind multiple ligands introduced in the crystallization solution or via soaking. Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discoverymore » approach to PaDsbA1 inhibitor development, the identification of a crystal form of PaDsbA1 that was more suitable for fragment-soaking experiments was sought. A previously identified crystallization condition for this protein was unsuitable, as in this crystal form of PaDsbA1 the active-site surface loops are engaged in the crystal packing, occluding access to the target site. A single residue involved in crystal-packing interactions was substituted with an amino acid commonly found at this position in closely related enzymes, and this variant was successfully used to generate a new crystal form of PaDsbA1 in which the active-site surface is more accessible for soaking experiments. The PaDsbA1 variant displays identical redox character and in vitro activity to wild-type PaDsbA1 and is structurally highly similar. Two crystal structures of the PaDsbA1 variant were determined in complex with small molecules bound to the protein active site. These small molecules (MES, glycerol and ethylene glycol) were derived from the crystallization or cryoprotectant solutions and provide a proof of principle that the reported crystal form will be amenable to co-crystallization and soaking with small molecules designed to target the protein active-site surface.« less

Analysis of Biological Features Associated with Meiotic Recombination Hot and Cold Spots in Saccharomyces cerevisiae

PubMed Central

Hansen, Loren; Kim, Nak-Kyeong; Mariño-Ramírez, Leonardo; Landsman, David

2011-01-01

Meiotic recombination is not distributed uniformly throughout the genome. There are regions of high and low recombination rates called hot and cold spots, respectively. The recombination rate parallels the frequency of DNA double-strand breaks (DSBs) that initiate meiotic recombination. The aim is to identify biological features associated with DSB frequency. We constructed vectors representing various chromatin and sequence-based features for 1179 DSB hot spots and 1028 DSB cold spots. Using a feature selection approach, we have identified five features that distinguish hot from cold spots in Saccharomyces cerevisiae with high accuracy, namely the histone marks H3K4me3, H3K14ac, H3K36me3, and H3K79me3; and GC content. Previous studies have associated H3K4me3, H3K36me3, and GC content with areas of mitotic recombination. H3K14ac and H3K79me3 are novel predictions and thus represent good candidates for further experimental study. We also show nucleosome occupancy maps produced using next generation sequencing exhibit a bias at DSB hot spots and this bias is strong enough to obscure biologically relevant information. A computational approach using feature selection can productively be used to identify promising biological associations. H3K14ac and H3K79me3 are novel predictions of chromatin marks associated with meiotic DSBs. Next generation sequencing can exhibit a bias that is strong enough to lead to incorrect conclusions. Care must be taken when interpreting high throughput sequencing data where systematic biases have been documented. PMID:22242140

Human immunodeficiency virus type 1 resistance to the small molecule maturation inhibitor 3-O-(3',3'-dimethylsuccinyl)-betulinic acid is conferred by a variety of single amino acid substitutions at the CA-SP1 cleavage site in Gag.

PubMed

Zhou, Jing; Chen, Chin Ho; Aiken, Christopher

2006-12-01

The compound 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB) potently and specifically inhibits human immunodeficiency virus type 1 (HIV-1) replication by delaying the cleavage of the CA-SP1 junction in Gag, leading to impaired maturation of the viral core. In this study, we investigated HIV-1 resistance to DSB by analyzing HIV-1 mutants encoding a variety of individual amino acid substitutions in the CA-SP1 cleavage site. Three of the substitutions were lethal to HIV-1 replication owing to a deleterious effect on particle assembly. The remaining mutants exhibited a range of replication efficiencies; however, each mutant was capable of replicating in the presence of concentrations of DSB that effectively inhibited wild-type HIV-1. Mutations conferring resistance to DSB also led to impaired binding of the compound to immature HIV-1 virions and loss of DSB-mediated inhibition of cleavage of Gag. Surprisingly, two of the DSB-resistant mutants retained an intermediate ability to bind the compound, suggesting that binding of DSB to immature HIV-1 particles may not be sufficient for antiviral activity. Overall, our results indicate that Gag amino acids L363 and A364 are critical for inhibition of HIV-1 replication by DSB and suggest that these residues form key contacts with the drug in the context of the assembling HIV-1 particle. These results have implications for the design of and screening for novel inhibitors of HIV-1 maturation.

Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1

PubMed Central

Headey, Stephen J.; Vazirani, Mansha; Shouldice, Stephen R.; Coinçon, Mathieu; Tay, Stephanie; Morton, Craig J.; Simpson, Jamie S.; Martin, Jennifer L.

2017-01-01

At a time when the antibiotic drug discovery pipeline has stalled, antibiotic resistance is accelerating with catastrophic implications for our ability to treat bacterial infections. Globally we face the prospect of a future when common infections can once again kill. Anti-virulence approaches that target the capacity of the bacterium to cause disease rather than the growth or survival of the bacterium itself offer a tantalizing prospect of novel antimicrobials. They may also reduce the propensity to induce resistance by removing the strong selection pressure imparted by bactericidal or bacteriostatic agents. In the human pathogen Pseudomonas aeruginosa, disulfide bond protein A (PaDsbA1) plays a central role in the oxidative folding of virulence factors and is therefore an attractive target for the development of new anti-virulence antimicrobials. Using a fragment-based approach we have identified small molecules that bind to PaDsbA1. The fragment hits show selective binding to PaDsbA1 over the DsbA protein from Escherichia coli, suggesting that developing species-specific narrow-spectrum inhibitors of DsbA enzymes may be feasible. Structures of a co-complex of PaDsbA1 with the highest affinity fragment identified in the screen reveal that the fragment binds on the non-catalytic surface of the protein at a domain interface. This biophysical and structural data represent a starting point in the development of higher affinity compounds, which will be assessed for their potential as selective PaDsbA1 inhibitors. PMID:28346540

The double-stranded break-forming activity of plant SPO11s and a novel rice SPO11 revealed by a Drosophila bioassay

PubMed Central

2012-01-01

Background SPO11 is a key protein for promoting meiotic recombination, by generating chromatin locus- and timing-specific DNA double-strand breaks (DSBs). The DSB activity of SPO11 was shown by genetic analyses, but whether SPO11 exerts DSB-forming activity by itself is still an unanswered question. DSB formation by SPO11 has not been detected by biochemical means, probably because of a lack of proper protein-folding, posttranslational modifications, and/or specific SPO11-interacting proteins required for this activity. In addition, plants have multiple SPO11-homologues. Results To determine whether SPO11 can cleave DNA by itself, and to identify which plant SPO11 homologue cleaves DNA, we developed a Drosophila bioassay system that detects the DSB signals generated by a plant SPO11 homologue expressed ectopically. We cytologically and genetically demonstrated the DSB activities of Arabidopsis AtSPO11-1 and AtSPO11-2, which are required for meiosis, in the absence of other plant proteins. Using this bioassay, we further found that a novel SPO11-homologue, OsSPO11D, which has no counterpart in Arabidopsis, displays prominent DSB-forming activity. Quantitative analyses of the rice SPO11 transcripts revealed the specific increase in OsSPO11D mRNA in the anthers containing meiotic pollen mother cells. Conclusions The Drosophila bioassay system successfully demonstrated that some plant SPO11 orthologues have intrinsic DSB activities. Furthermore, we identified a novel SPO11 homologue, OsSPO11D, with robust DSB activity and a possible meiotic function. PMID:22248237

Helicobacter pylori protein oxidation influences the colonization process.

PubMed

Godlewska, Renata; Dzwonek, Artur; Mikuła, Michał; Ostrowski, Jerzy; Pawłowski, Marcin; Bujnicki, Janusz M; Jagusztyn-Krynicka, Elzbieta K

2006-08-01

Dsb proteins control the formation and rearrangement of disulfide bonds during the folding of membrane and exported proteins. Here we examined the role of DsbI protein in Helicobacter pylori pathogenesis and demonstrated that a dsbI mutant impaired in disulfide bond formation revealed a greatly reduced ability to colonize mice gastric mucosa.

Preparation and structural characterization of zwitterionic surfactant intercalated into NiZn-layered hydroxide salts

NASA Astrophysics Data System (ADS)

Liu, Jiexiang; Wang, Jianlong; Zhang, Xiaoguang; Fang, Binbin; Hu, Pan; Zhao, Xuyang

2015-10-01

Three zwitterionic surfactants, dodecyl dimethyl carboxylbetaine (DCB), dodecyl dimethyl sulfobetaine (DSB) and N-dodecyl-β-aminoprpionate (DAP), intercalated into NiZn-layered hydroxide salts (NZL-DCB, NZL-DSB and NZL-DAP) were synthesized by the coprecipitation method. The effect of surfactant content, pH, temperature and time of hydrothermal treatment on preparation was investigated and discussed. The NZL-DCB, NZL-DSB and NZL-DAP were characterized by powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), and thermogravimetry analysis and differential thermal analysis (TGA/DTA). The results showed that basal spacings of NZL-DCB, NZL-DSB and NZL-DAP were around 3.45, 3.68 and 3.94 nm, respectively. DCB, DSB and DAP probably form an overlapped bilayer in the gallery. TGA/DTA data indicated that NZL-DCB, NZL-DSB and NZL-DAP displayed three loss weight stages: loss of adsorbed and structural water, dehydroxylation of matrix and decomposition of nitrate ions, decomposition and combustion of surfactants. Furthermore, chemical analysis data, BET surface area and scanning electron microscopic (SEM) were also measured and analyzed.

DNA Repair Alterations in Children With Pediatric Malignancies: Novel Opportunities to Identify Patients at Risk for High-Grade Toxicities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruebe, Claudia E., E-mail: claudia.ruebe@uks.e; Fricke, Andreas; Schneider, Ruth

Purpose: To evaluate, in a pilot study, the phosphorylated H2AX ({gamma}H2AX) foci approach for identifying patients with double-strand break (DSB) repair deficiencies, who may overreact to DNA-damaging cancer therapy. Methods and Materials: The DSB repair capacity of children with solid cancers was analyzed compared with that of age-matched control children and correlated with treatment-related normal-tissue responses (n = 47). Double-strand break repair was investigated by counting {gamma}H2AX foci in blood lymphocytes at defined time points after irradiation of blood samples. Results: Whereas all healthy control children exhibited proficient DSB repair, 3 children with tumors revealed clearly impaired DSB repair capacities,more » and 2 of these repair-deficient children developed life-threatening or even lethal normal-tissue toxicities. The underlying mutations affecting regulatory factors involved in DNA repair pathways were identified. Moreover, significant differences in mean DSB repair capacity were observed between children with tumors and control children, suggesting that childhood cancer is based on genetic alterations affecting DSB repair function. Conclusions: Double-strand break repair alteration in children may predispose to cancer formation and may affect children's susceptibility to normal-tissue toxicities. Phosphorylated H2AX analysis of blood samples allows one to detect DSB repair deficiencies and thus enables identification of children at risk for high-grade toxicities.« less

Double-strand break repair-adox: Restoration of suppressed double-strand break repair during mitosis induces genomic instability

PubMed Central

Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

2014-01-01

Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G1 phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability. PMID:25287622

Identification of the ubiquinone-binding domain in the disulfide catalyst disulfide bond protein B.

PubMed

Xie, Tong; Yu, Linda; Bader, Martin W; Bardwell, James C A; Yu, Chang-An

2002-01-18

Disulfide bond (Dsb) formation is catalyzed in the periplasm of prokaryotes by the Dsb proteins. DsbB, a key enzyme in this process, generates disulfides de novo by using the oxidizing power of quinones. To explore the mechanism of this newly described enzymatic activity, we decided to study the ubiquinone-protein interaction and identify the ubiquinone-binding domain in DsbB by cross-linking to photoactivatable quinone analogues. When purified Escherichia coli DsbB was incubated with an azidoubiquinone derivative, 3-azido-2-methyl-5-[(3)H]methoxy-6-decyl-1,4-benzoquinone ([(3)H]azido-Q), and illuminated with long wavelength UV light, the decrease in enzymatic activity correlated with the amount of 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone (azido-Q) incorporated into the protein. One azido-Q-linked peptide with a retention time of 33.5 min was obtained by high performance liquid chromatography of the V8 digest of [(3)H]azido-Q-labeled DsbB. This peptide has a partial NH(2)-terminal amino acid sequence of NH(2)-HTMLQLY corresponding to residues 91-97. This sequence occurs in the second periplasmic domain of the inner membrane protein DsbB in a loop connecting transmembrane helices 3 and 4. We propose that the quinone-binding site is within or very near to this sequence.

Extrapolation of the dna fragment-size distribution after high-dose irradiation to predict effects at low doses

NASA Technical Reports Server (NTRS)

Ponomarev, A. L.; Cucinotta, F. A.; Sachs, R. K.; Brenner, D. J.; Peterson, L. E.

2001-01-01

The patterns of DSBs induced in the genome are different for sparsely and densely ionizing radiations: In the former case, the patterns are well described by a random-breakage model; in the latter, a more sophisticated tool is needed. We used a Monte Carlo algorithm with a random-walk geometry of chromatin, and a track structure defined by the radial distribution of energy deposition from an incident ion, to fit the PFGE data for fragment-size distribution after high-dose irradiation. These fits determined the unknown parameters of the model, enabling the extrapolation of data for high-dose irradiation to the low doses that are relevant for NASA space radiation research. The randomly-located-clusters formalism was used to speed the simulations. It was shown that only one adjustable parameter, Q, the track efficiency parameter, was necessary to predict DNA fragment sizes for wide ranges of doses. This parameter was determined for a variety of radiations and LETs and was used to predict the DSB patterns at the HPRT locus of the human X chromosome after low-dose irradiation. It was found that high-LET radiation would be more likely than low-LET radiation to induce additional DSBs within the HPRT gene if this gene already contained one DSB.

Individualization of radiotherapy in breast cancer patients: possible usefulness of a DNA damage assay to measure normal cell radiosensitivity.

PubMed

Ruiz de Almodóvar, José Mariano; Guirado, Damian; Isabel Núñez, María; López, Escarlata; Guerrero, Rosario; Valenzuela, María Teresa; Villalobos, Mercedes; del Moral, Rosario

2002-03-01

The purpose of this study was to determine whether the distribution of sensitivities in breast cancer patients, measured using a DNA damage assay on lymphocytes, is likely to provide sufficient discrimination to enable the reliable identification of patients with abnormal sensitivities. Radiosensitivity (x) was assessed in 226 samples of lymphocytes from unselected women with breast cancer and was quantified as the initial number of DNA double-strand breaks (dsb) induced per Gy and per DNA unit (200 Mbp). The existence of an inter-individual variation in the parameter (x) is described through the range (0.40-4.72 dsb/Gy/DNA unit) of values found, which have been fitted to the mathematical model defined by the log-normal distribution (mu = 0.42+/-0.03; sigma = 0.52+/-0.03; R(2)=0.9475). A total of 189 patients received radiotherapy after surgical treatment. Among them, we have detected 15 patients who developed severe skin reactions and we have compared their radiosensitivity values with the rest of patients treated. Our results suggest that DNA initial damage measured on lymphocytes offers an approach to predict the acute response of human normal tissues prior to radiotherapy. Values of x higher than 3.20 dsb/Gy/DNA unit theoretically should correspond to the highly radio-sensitive patients. Using the experimental results, we have calculated the strength of the test by means of the area under the receiver operator characteristic curves (A(Z)) to determine whether the radiosensitivity assay can discriminate between patients according to their radiation response. The value found (A(Z)=0.675+/-0.072) is indicative of a fair-poor discriminating capacity of the test to identify the patients with higher risk of developing a severe acute reaction during the radiotherapy treatment.

The sequence of the CA-SP1 junction accounts for the differential sensitivity of HIV-1 and SIV to the small molecule maturation inhibitor 3-O-{3',3'-dimethylsuccinyl}-betulinic acid.

PubMed

Zhou, Jing; Chen, Chin Ho; Aiken, Christopher

2004-06-29

Despite the effectiveness of currently available antiretroviral therapies in the treatment of HIV-1 infection, a continuing need exists for novel compounds that can be used in combination with existing drugs to slow the emergence of drug-resistant viruses. We previously reported that the small molecule 3-O-{3',3'-dimethylsuccinyl}-betulinic acid (DSB) specifically inhibits HIV-1 replication by delaying the processing of the CA-SP1 junction in Pr55Gag. By contrast, SIVmac239 replicates efficiently in the presence of high concentrations of DSB. To determine whether sequence differences in the CA-SP1 junction can fully account for the differential sensitivity of HIV-1 and SIV to DSB, we engineered mutations in this region of two viruses and tested their sensitivity to DSB in replication assays using activated human primary CD4+ T cells. Substitution of the P2 and P1 residues of HIV-1 by the corresponding amino acids of SIV resulted in strong resistance to DSB, but the mutant virus replicated with reduced efficiency. Conversely, replication of an SIV mutant containing three amino acid substitutions in the CA-SP1 cleavage site was highly sensitive to DSB, and the mutations resulted in delayed cleavage of the CA-SP1 junction in the presence of the drug. These results demonstrate that the CA-SP1 junction in Pr55Gag represents the primary viral target of DSB. They further suggest that the therapeutic application of DSB will be accompanied by emergence of mutant viruses that are highly resistant to the drug but which exhibit reduced fitness relative to wild type HIV-1.

DNA-damage foci to detect and characterize DNA repair alterations in children treated for pediatric malignancies.

PubMed

Schuler, Nadine; Palm, Jan; Kaiser, Mareike; Betten, Dominik; Furtwängler, Rhoikos; Rübe, Christian; Graf, Norbert; Rübe, Claudia E

2014-01-01

In children diagnosed with cancer, we evaluated the DNA damage foci approach to identify patients with double-strand break (DSB) repair deficiencies, who may overreact to DNA-damaging radio- and chemotherapy. In one patient with Fanconi anemia (FA) suffering relapsing squamous cell carcinomas of the oral cavity we also characterized the repair defect in biopsies of skin, mucosa and tumor. In children with histologically confirmed tumors or leukemias and healthy control-children DSB repair was investigated by counting γH2AX-, 53BP1- and pATM-foci in blood lymphocytes at defined time points after ex-vivo irradiation. This DSB repair capacity was correlated with treatment-related normal-tissue responses. For the FA patient the defective repair was also characterized in tissue biopsies by analyzing DNA damage response proteins by light and electron microscopy. Between tumor-children and healthy control-children we observed significant differences in mean DSB repair capacity, suggesting that childhood cancer is based on genetic alterations affecting DNA repair. Only 1 out of 4 patients with grade-4 normal-tissue toxicities revealed an impaired DSB repair capacity. The defective DNA repair in FA patient was verified in irradiated blood lymphocytes as well as in non-irradiated mucosa and skin biopsies leading to an excessive accumulation of heterochromatin-associated DSBs in rapidly cycling cells. Analyzing human tissues we show that DSB repair alterations predispose to cancer formation at younger ages and affect the susceptibility to normal-tissue toxicities. DNA damage foci analysis of blood and tissue samples allows one to detect and characterize DSB repair deficiencies and enables identification of patients at risk for high-grade toxicities. However, not all treatment-associated normal-tissue toxicities can be explained by DSB repair deficiencies.

Xrcc1-dependent and Ku-dependent DNA double-strand break repair kinetics in Arabidopsis plants.

PubMed

Charbonnel, Cyril; Gallego, Maria E; White, Charles I

2010-10-01

Double-strand breakage (DSB) of DNA involves loss of information on the two strands of the DNA fibre and thus cannot be repaired by simple copying of the complementary strand which is possible with single-strand DNA damage. Homologous recombination (HR) can precisely repair DSB using another copy of the genome as template and non-homologous recombination (NHR) permits repair of DSB with little or no dependence on DNA sequence homology. In addition to the well-characterised Ku-dependent non-homologous end-joining (NHEJ) pathway, much recent attention has been focused on Ku-independent NHR. The complex interrelationships and regulation of NHR pathways remain poorly understood, even more so in the case of plants, and we present here an analysis of Ku-dependent and Ku-independent repair of DSB in Arabidopsis thaliana. We have characterised an Arabidopsis xrcc1 mutant and developed quantitative analysis of the kinetics of appearance and loss of γ-H2AX foci as a tool to measure DSB repair in dividing root tip cells of γ-irradiated plants in vivo. This approach has permitted determination of DSB repair kinetics in planta following a short pulse of γ-irradiation, establishing the existence of a Ku-independent, Xrcc1-dependent DSB repair pathway. Furthermore, our data show a role for Ku80 during the first minutes post-irradiation and that Xrcc1 also plays such a role, but only in the absence of Ku. The importance of Xrcc1 is, however, clearly visible at later times in the presence of Ku, showing that alternative end-joining plays an important role in DSB repair even in the presence of active NHEJ. © 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.

Induction and repair of radiation-induced DNA double-strand breaks in human fibroblasts are not affected by terminal differentiation.

PubMed

Brammer, Ingo; Herskind, Carsten; Haase, Oliver; Rodemann, H Peter; Dikomey, Ekkehard

2004-02-03

It was studied for human skin fibroblasts, whether the induction or repair of DNA double-strand breaks (dsb) depend on the differentiation status. These studies were performed (a) with a fibroblast strain (HSF1) kept in progenitor state (mitotic fibroblasts, MF) or triggered to premature terminal differentiation (postmitotic fibrocytes, PMF) by exposure to mitomycin C or (b) with 20 fibroblast strains differing intrinsically in their differentiation status. The differentiation status was quantified by determining the fraction of postmitotic fibrocytes by light microscopy. DNA dsb were measured by constant-field gel electrophoresis, and the fraction of apoptotic cells by comet assay. MF and PMF cultures of HSF1 cells were irradiated with X-ray doses up to 160 Gy, and dsb were measured either immediately after irradiation or after a repair incubation of 4 or 24 h. There were a difference neither in the number of initial nor residual dsb. PMF cultures, however, showed a slightly higher number of dsb already present in non-irradiated cells, which was measured to result from a small fraction of 5% apoptotic cells. The 20 analysed fibroblast strains showed a substantial variation in the fraction of postmitotic fibrocytes (9-51%) as well as in the number of dsb remaining at 24 h after irradiation (1.9-4.9%), but there was no correlation between these two parameters. These data demonstrate that for fibroblasts the terminal differentiation has an effect neither on the induction nor the repair of radiation-induced dsb. This result indicates that the variation in dsb-repair capacity previously observed for fibroblast strains and which was considered to be the main cause for the variation in the cellular radiosensitivity, cannot be ascribed to differences in the differentiation status.

CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants.

PubMed

Shen, Hexi; Strunks, Gary D; Klemann, Bart J P M; Hooykaas, Paul J J; de Pater, Sylvia

2017-01-05

Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants. Copyright © 2017 Shen et al.

DNA DSB measurements and modelling approaches based on gamma-H2AX foci time evolution

NASA Astrophysics Data System (ADS)

Esposito, Giuseppe; Campa, Alessandro; Antonelli, Francesca; Mariotti, Luca; Belli, Mauro; Giardullo, Paola; Simone, Giustina; Antonella Tabocchini, Maria; Ottolenghi, Andrea

DNA double strand breaks (DSBs) induced by ionising radiation are considered the main dam-age related to the deleterious consequences in the cells. Unrepaired or mis-repaired DSBs can cause mutations or loss of chromosome regions which can eventually lead to cell death or neo-plastic transformation. Quantification of the number and complexity of DSBs induced by low doses of radiation remains a complex problem. About ten years ago Rogakou et al. proposed an immunofluorescent technique able to detect even a single DSB per cell. This approach is based on the serine 139 phosphorylation of many molecules (up to 2000) of histone H2AX (γg-H2AX) following the induction of a DSB in the DNA. DSB can be visualized as foci by immunofluores-cence by using phospho-specific antibodies, so that enumeration of foci can be used to measure DSB induction and processing. It is still not completely clear how γ-H2AX dephosphorylation takes place; however it has been related with DSB repair, in particular with the efficiency of DSB repair. In this work we analyse the H2AX phosphorylation-dephosphorylation kinetics after irradiation of primary human fibroblasts (AG1522 cell line) with radiation of differing quality, that is γ-rays and α-particles (125 keV/µm), with the aim of comparing the time evolution of γ-H2AX foci. Our results show that, after a dose of 0.5 Gy, both γ-rays and α-particles induce the maximum number of γ-H2AX foci within 30 minutes from irradiation, that this number depends on the radiation type and is consistent with the number of track traversal in α-irradiated nuclei, that the dephosphorylation kinetics are very different, being the α-induced foci rate of disappearence slower than that of γ-induced foci. In this work a modellistic approach to estimate the number of DSB induced by γ-rays detectable by using the γ-H2AX assay is presented. The competing processes of appearance and disappearance of visible foci will be modeled taking into account the phosphorylation and dephosphorylation phenomena. The effect of possible overlapping DSB induced at short distance (within 2 Mbp) will be also taken into account. Moreover, for charged particles (in particular for α-particles) the average number of DSB per focus is calculated.

Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment

PubMed Central

Schipler, Agnes; Mladenova, Veronika; Soni, Aashish; Nikolov, Vladimir; Saha, Janapriya; Mladenov, Emil; Iliakis, George

2016-01-01

Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect. PMID:27257076

Double-strand break repair-adox: Restoration of suppressed double-strand break repair during mitosis induces genomic instability.

PubMed

Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

2014-12-01

Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G1 phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Acetylated Histone H3K9 is associated with meiotic recombination hotspots, and plays a role in recombination redundantly with other factors including the H3K4 methylase Set1 in fission yeast

PubMed Central

Yamada, Shintaro; Ohta, Kunihiro; Yamada, Takatomi

2013-01-01

Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double-strand break (DSB) formation at hotspots, but the role and precise landscape of involved modifications remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is elevated, and H3K4me3 is not significantly enriched. Mutating H3K9 to non-acetylatable alanine mildly reduced levels of the DSB-inducing protein Rec12 (the fission yeast homologue of Spo11) and DSB at hotspots, indicating that H3K9ac may be involved in DSB formation by enhancing the interaction between Rec12 and hotspots. In addition, we found that the lack of the H3K4 methyltransferase Set1 generally increased Rec12 binding to chromatin but partially reduced DSB formation at some loci, suggesting that Set1 is also involved in DSB formation. These results suggest that meiotic DSB formation is redundantly regulated by multiple chromatin-related factors including H3K9ac and Set1 in fission yeast. PMID:23382177

A modified microdosimetric kinetic model for relative biological effectiveness calculation

NASA Astrophysics Data System (ADS)

Chen, Yizheng; Li, Junli; Li, Chunyan; Qiu, Rui; Wu, Zhen

2018-01-01

In the heavy ion therapy, not only the distribution of physical absorbed dose, but also the relative biological effectiveness (RBE) weighted dose needs to be taken into account. The microdosimetric kinetic model (MKM) can predict the RBE value of heavy ions with saturation-corrected dose-mean specific energy, which has been used in clinical treatment planning at the National Institute of Radiological Sciences. In the theoretical assumption of the MKM, the yield of the primary lesion is independent of the radiation quality, while the experimental data shows that DNA double strand break (DSB) yield, considered as the main primary lesion, depends on the LET of the particle. Besides, the β parameter of the MKM is constant with LET resulting from this assumption, which also differs from the experimental conclusion. In this study, a modified MKM was developed, named MMKM. Based on the experimental DSB yield of mammalian cells under the irradiation of ions with different LETs, a RBEDSB (RBE for the induction of DSB)-LET curve was fitted as the correction factor to modify the primary lesion yield in the MKM, and the variation of the primary lesion yield with LET is considered in the MMKM. Compared with the present the MKM, not only the α parameter of the MMKM for mono-energetic ions agree with the experimental data, but also the β parameter varies with LET and the variation trend of the experimental result can be reproduced on the whole. Then a spread-out Bragg peaks (SOBP) distribution of physical dose was simulated with Geant4 Monte Carlo code, and the biological and clinical dose distributions were calculated, under the irradiation of carbon ions. The results show that the distribution of clinical dose calculated with the MMKM is closed to the distribution with the MKM in the SOBP, while the discrepancy before and after the SOBP are both within 10%. Moreover, the MKM might overestimate the clinical dose at the distal end of the SOBP more than 5% because of its constant β value, while a minimal value of β is calculated with the MMKM at this position. Besides, the discrepancy of the averaged cell survival fraction in the SOBP calculated with the two models is more than 15% at the high dose level. The MMKM may provide a reference for the accurate calculation of the RBE value in heavy ion therapy.

The Role of Oxidoreductases in Determining the Function of the Neisserial Lipid A Phosphoethanolamine Transferase Required for Resistance to Polymyxin

PubMed Central

Piek, Susannah; Wang, Zhirui; Ganguly, Jhuma; Lakey, Adam M.; Bartley, Stephanie N.; Mowlaboccus, Shakeel; Anandan, Anandhi; Stubbs, Keith A.; Scanlon, Martin J.; Vrielink, Alice; Azadi, Parastoo; Carlson, Russell W.; Kahler, Charlene M.

2014-01-01

The decoration of the lipid A headgroups of the lipooligosaccharide (LOS) by the LOS phosphoethanolamine (PEA) transferase (LptA) in Neisseria spp. is central for resistance to polymyxin. The structure of the globular domain of LptA shows that the protein has five disulphide bonds, indicating that it is a potential substrate of the protein oxidation pathway in the bacterial periplasm. When neisserial LptA was expressed in Escherichia coli in the presence of the oxidoreductase, EcDsbA, polymyxin resistance increased 30-fold. LptA decorated one position of the E. coli lipid A headgroups with PEA. In the absence of the EcDsbA, LptA was degraded in E. coli. Neisseria spp. express three oxidoreductases, DsbA1, DsbA2 and DsbA3, each of which appear to donate disulphide bonds to different targets. Inactivation of each oxidoreductase in N. meningitidis enhanced sensitivity to polymyxin with combinatorial mutants displaying an additive increase in sensitivity to polymyxin, indicating that the oxidoreductases were required for multiple pathways leading to polymyxin resistance. Correlates were sought between polymyxin sensitivity, LptA stability or activity and the presence of each of the neisserial oxidoreductases. Only meningococcal mutants lacking DsbA3 had a measurable decrease in the amount of PEA decoration on lipid A headgroups implying that LptA stability was supported by the presence of DsbA3 but did not require DsbA1/2 even though these oxidoreductases could oxidise the protein. This is the first indication that DsbA3 acts as an oxidoreductase in vivo and that multiple oxidoreductases may be involved in oxidising the one target in N. meningitidis. In conclusion, LptA is stabilised by disulphide bonds within the protein. This effect was more pronounced when neisserial LptA was expressed in E. coli than in N. meningitidis and may reflect that other factors in the neisserial periplasm have a role in LptA stability. PMID:25215579

DNA double-strand-break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice.

PubMed

Schipler, Agnes; Iliakis, George

2013-09-01

Although the DNA double-strand break (DSB) is defined as a rupture in the double-stranded DNA molecule that can occur without chemical modification in any of the constituent building blocks, it is recognized that this form is restricted to enzyme-induced DSBs. DSBs generated by physical or chemical agents can include at the break site a spectrum of base alterations (lesions). The nature and number of such chemical alterations define the complexity of the DSB and are considered putative determinants for repair pathway choice and the probability that errors will occur during this processing. As the pathways engaged in DSB processing show distinct and frequently inherent propensities for errors, pathway choice also defines the error-levels cells opt to accept. Here, we present a classification of DSBs on the basis of increasing complexity and discuss how complexity may affect processing, as well as how it may cause lethal or carcinogenic processing errors. By critically analyzing the characteristics of DSB repair pathways, we suggest that all repair pathways can in principle remove lesions clustering at the DSB but are likely to fail when they encounter clusters of DSBs that cause a local form of chromothripsis. In the same framework, we also analyze the rational of DSB repair pathway choice.

Evaluation of DNA Single and Double Strand Breaks in Women with Cervical Neoplasia Based on Alkaline and Neutral Comet Assay Techniques

PubMed Central

Cortés-Gutiérrez, Elva I.; Hernández-Garza, Fernando; García-Pérez, Jorge O.; Dávila-Rodríguez, Martha I.; Aguado-Barrera, Miguel E.; Cerda-Flores, Ricardo M.

2012-01-01

A hospital-based unmatched case-control study was performed in order to determine the relation of DNA single (ssb) and double (dsb) strand breaks in women with and without cervical neoplasia. Cervical epithelial cells of 30 women: 10 with low grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 without cervical lesions were evaluated using alkaline and neutral comet assays. A significant increase in global DNA damage (ssb + dsb) and dsb was observed in patients with HG-SIL (48.90 ± 12.87 and 23.50 ± 13.91), patients with LG-SIL (33.60 ± 14.96 and 11.20 ± 5.71), and controls (21.70 ± 11.87 and 5.30 ± 5.38; resp.). Pearson correlation coefficient reveled a strong relation between the levels ssb and dsb (r2 = 0.99, P = 0.03, and r2 = 0.94, P = 0.16, resp.) and progression of neoplasia. The increase of dsb damage in patients with HG-SIL was confirmed by DNA breakage detection-FISH (DBD-FISH) on neutral comets. Our results argue in favor of a real genomic instability in women with cervical neoplasia, which was strengthened by our finding of a higher proportion of DNA dsb. PMID:23093842

N-Acetyl-L-cysteine protects thyroid cells against DNA damage induced by external and internal irradiation.

PubMed

Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji

2017-11-01

We evaluated the effect of the antioxidant N-acetyl-L-cysteine (NAC) on the levels of reactive oxygen species (ROS), DNA double strand breaks (DSB) and micronuclei (MN) induced by internal and external irradiation using a rat thyroid cell line PCCL3. In internal irradiation experiments, ROS and DSB levels increased immediately after 131 I addition and then gradually declined, resulting in very high levels of MN at 24 and 48 h. NAC administration both pre- and also post- 131 I addition suppressed ROS, DSB and MN. In external irradiation experiments with a low dose (0.5 Gy), ROS and DSB increased shortly and could be prevented by NAC administration pre-, but not post-irradiation. In contrast, external irradiation with a high dose (5 Gy) increased ROS and DSB in a bimodal way: ROS and DSB levels increased immediately after irradiation, quickly returned to the basal levels and gradually rose again after >24 h. The second phase was in parallel with an increase in 4-hydroxy-2-nonenal. The number of MN induced by the second wave of ROS/DSB elevations was much higher than that by the first peak. In this situation, NAC administered pre- and post-irradiation comparably suppressed MN induced by a delayed ROS elevation. In conclusion, a prolonged ROS increase during internal irradiation and a delayed ROS increase after external irradiation with a high dose caused serious DNA damage, which were efficiently prevented by NAC. Thus, NAC administration even both after internal or external irradiation prevents ROS increase and eventual DNA damage.

Doctor Shopping Behavior for Zolpidem Among Insomnia Patients in Taiwan: A Nationwide Population-Based Study.

PubMed

Lu, Tzu-Hsuan; Lee, Yen-Ying; Lee, Hsin-Chien; Lin, You-Meei

2015-07-01

Although zolpidem is listed as a controlled drug in Taiwan, patients' behavior has not been restricted and has led to the problem of doctor shopping behavior (DSB), leading to overutilization of medical resources and excess spending. The National Health Insurance Administration in Taiwan has instituted a new policy to regulate physicians' prescribing behavior and decrease DSB. This retrospective study aimed to analyze the DSB for zolpidem by insomnia patients and assess related factors. Data were extracted from the Longitudinal Health Insurance Database in Taiwan. Individuals with a diagnosis of insomnia who received more than one prescription of zolpidem in 2008 were followed for 24 mo. Doctor shopping was defined as ≥ 2 prescriptions by different doctors within ≥ 1 day overlapping in the duration of therapy. The percentage of zolpidem obtained through doctor shopping was used as an indicator of the DSB of each patient. Among the 6,947 insomnia patients who were prescribed zolpidem, 1,652 exhibited DSB (23.78%). The average dose of zolpidem dispensed for each patient during 24 mo was 244.21 daily defined doses. The doctor shopping indicator (DSI) was 0.20 (standard deviation, 0.23) among patients with DSB. Younger age, chronic diseases, high number of diseases, higher premium status, high socioeconomic status, and fewer people served per practicing physicians were all factors significantly related to doctor shopping behavior. Doctor shopping for zolpidem appears to be an important issue in Taiwan. Implementing a proper referral system with efficient data exchange by physician or pharmacist-led medication reconciliation process might reduce DSB. © 2015 Associated Professional Sleep Societies, LLC.

Microfabricated high-bandpass foucault aperture for electron microscopy

DOEpatents

Glaeser, Robert; Cambie, Rossana; Jin, Jian

2014-08-26

A variant of the Foucault (knife-edge) aperture is disclosed that is designed to provide single-sideband (SSB) contrast at low spatial frequencies but retain conventional double-sideband (DSB) contrast at high spatial frequencies in transmission electron microscopy. The aperture includes a plate with an inner open area, a support extending from the plate at an edge of the open area, a half-circle feature mounted on the support and located at the center of the aperture open area. The radius of the half-circle portion of reciprocal space that is blocked by the aperture can be varied to suit the needs of electron microscopy investigation. The aperture is fabricated from conductive material which is preferably non-oxidizing, such as gold, for example.

Genome-wide control of the distribution of meiotic recombination.

PubMed

Grey, Corinne; Baudat, Frédéric; de Massy, Bernard

2009-02-17

Meiotic recombination events are not randomly distributed in the genome but occur in specific regions called recombination hotspots. Hotspots are predicted to be preferred sites for the initiation of meiotic recombination and their positions and activities are regulated by yet-unknown controls. The activity of the Psmb9 hotspot on mouse Chromosome 17 (Chr 17) varies according to genetic background. It is active in strains carrying a recombinant Chr 17 where the proximal third is derived from Mus musculus molossinus. We have identified the genetic locus required for Psmb9 activity, named Dsbc1 for Double-strand break control 1, and mapped this locus within a 6.7-Mb region on Chr 17. Based on cytological analysis of meiotic DNA double-strand breaks (DSB) and crossovers (COs), we show that Dsbc1 influences DSB and CO, not only at Psmb9, but in several other regions of Chr 17. We further show that CO distribution is also influenced by Dsbc1 on Chrs 15 and 18. Finally, we provide direct molecular evidence for the regulation in trans mediated by Dsbc1, by showing that it controls the CO activity at the Hlx1 hotspot on Chr 1. We thus propose that Dsbc1 encodes for a trans-acting factor involved in the specification of initiation sites of meiotic recombination genome wide in mice.

Induction and Processing of the Radiation-Induced Gamma-H2AX Signal and Its Link to the Underlying Pattern of DSB: A Combined Experimental and Modelling Study

PubMed Central

Tommasino, Francesco; Friedrich, Thomas; Jakob, Burkhard; Meyer, Barbara; Durante, Marco; Scholz, Michael

2015-01-01

We present here an analysis of DSB induction and processing after irradiation with X-rays in an extended dose range based on the use of the γH2AX assay. The study was performed by quantitative flow cytometry measurements, since the use of foci counting would result in reasonable accuracy only in a limited dose range of a few Gy. The experimental data are complemented by a theoretical analysis based on the GLOBLE model. In fact, original aim of the study was to test GLOBLE predictions against new experimental data, in order to contribute to the validation of the model. Specifically, the γH2AX signal kinetics has been investigated up to 24 h after exposure to increasing photon doses between 2 and 500 Gy. The prolonged persistence of the signal at high doses strongly suggests dose dependence in DSB processing after low LET irradiation. Importantly, in the framework of our modelling analysis, this is related to a gradually increased fraction of DSB clustering at the micrometre scale. The parallel study of γH2AX dose response curves shows the onset of a pronounced saturation in two cell lines at a dose of about 20 Gy. This dose is much lower than expected according to model predictions based on the values usually adopted for the DSB induction yield (≈ 30 DSB/Gy) and for the γH2AX foci extension of approximately 2 Mbp around the DSB. We show and discuss how theoretical predictions and experimental findings can be in principle reconciled by combining an increased DSB induction yield with the assumption of a larger genomic extension for the single phosphorylated regions. As an alternative approach, we also considered in our model the possibility of a 3D spreading-mechanism of the H2AX phosphorylation around the induced DSB, and applied it to the analysis of both the aspects considered. Our results are found to be supportive for the basic assumptions on which GLOBLE is built. Apart from giving new insights into the H2AX phosphorylation process, experiments performed at high doses are of relevance in the context of radiation therapy, where hypo-fractionated schemes become increasingly popular. PMID:26067661

Doctor Shopping Behavior for Zolpidem Among Insomnia Patients in Taiwan: A Nationwide Population-Based Study

PubMed Central

Lu, Tzu-Hsuan; Lee, Yen-Ying; Lee, Hsin-Chien; Lin, You-Meei

2015-01-01

Objectives: Although zolpidem is listed as a controlled drug in Taiwan, patients' behavior has not been restricted and has led to the problem of doctor shopping behavior (DSB), leading to overutilization of medical resources and excess spending. The National Health Insurance Administration in Taiwan has instituted a new policy to regulate physicians' prescribing behavior and decrease DSB. This retrospective study aimed to analyze the DSB for zolpidem by insomnia patients and assess related factors. Design and Participants: Data were extracted from the Longitudinal Health Insurance Database in Taiwan. Individuals with a diagnosis of insomnia who received more than one prescription of zolpidem in 2008 were followed for 24 mo. Doctor shopping was defined as ≥ 2 prescriptions by different doctors within ≥ 1 day overlapping in the duration of therapy. The percentage of zolpidem obtained through doctor shopping was used as an indicator of the DSB of each patient. Results: Among the 6,947 insomnia patients who were prescribed zolpidem, 1,652 exhibited DSB (23.78%). The average dose of zolpidem dispensed for each patient during 24 mo was 244.21 daily defined doses. The doctor shopping indicator (DSI) was 0.20 (standard deviation, 0.23) among patients with DSB. Younger age, chronic diseases, high number of diseases, higher premium status, high socioeconomic status, and fewer people served per practicing physicians were all factors significantly related to doctor shopping behavior. Conclusion: Doctor shopping for zolpidem appears to be an important issue in Taiwan. Implementing a proper referral system with efficient data exchange by physician or pharmacist-led medication reconciliation process might reduce DSB. Citation: Lu TH, Lee YY, Lee HC, Lin YM. Doctor shopping behavior for zolpidem among insomnia patients in Taiwan: a nationwide population-based study. SLEEP 2015;38(7):1039–1044. PMID:25761979

The Impact of Individual In Vivo Repair of DNA Double-Strand Breaks on Oral Mucositis in Adjuvant Radiotherapy of Head-and-Neck Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fleckenstein, Jochen, E-mail: rajfle@uks.eu; Kuehne, Martin; Seegmueller, Katharina

2011-12-01

Purpose: To evaluate the impact of individual in vivo DNA double-strand break (DSB) repair capacity on the incidence of severe oral mucositis in patients with head-and-neck cancer undergoing adjuvant radiotherapy (RT) or radiochemotherapy (RCT). Patients and Methods: Thirty-one patients with resected head-and-neck cancer undergoing adjuvant RT or RCT were examined. Patients underwent RT of the primary tumor site and locoregional lymph nodes with a total dose of 60-66 Gy (single dose 2 Gy, five fractions per week). Chemotherapy consisted of two cycles of cisplatin and 5-fluorouracil. To assess DSB repair, {gamma}-H2AX foci in blood lymphocytes were quantified before and 0.5more » h, 2.5 h, 5 h, and 24 h after in vivo radiation exposure (the first fraction of RT). World Health Organization scores for oral mucositis were documented weekly and correlated with DSB repair. Results: Sixteen patients received RT alone; 15 patients received RCT. In patients who developed Grade {>=} 3 mucositis (n = 18) the amount of unrepaired DSBs 24 h after radiation exposure and DSB repair half-times did not differ significantly from patients with Grade {<=}2 mucositis (n = 13). Patients with a proportion of unrepaired DSBs after 24 h higher than the mean value + one standard deviation had an increased incidence of severe oral mucositis. Conclusions: Evaluation of in vivo DSB repair by determination of {gamma}-H2AX foci loss is feasible in clinical practice and allows identification of patients with impaired DSB repair. The incidence of oral mucositis is not closely correlated with DSB repair under the evaluated conditions.« less

Role of Double-Strand Break End-Tethering during Gene Conversion in Saccharomyces cerevisiae

PubMed Central

Haber, James E.

2016-01-01

Correct repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Whereas gene conversion (GC)-mediated repair is mostly error-free, repair by break-induced replication (BIR) is associated with non-reciprocal translocations and loss of heterozygosity. We have previously shown that a Recombination Execution Checkpoint (REC) mediates this competition by preventing the BIR pathway from acting on DSBs that can be repaired by GC. Here, we asked if the REC can also determine whether the ends that are engaged in a GC-compatible configuration belong to the same break, since repair involving ends from different breaks will produce potentially deleterious translocations. We report that the kinetics of repair are markedly delayed when the two DSB ends that participate in GC belong to different DSBs (termed Trans) compared to the case when both DSB ends come from the same break (Cis). However, repair in Trans still occurs by GC rather than BIR, and the overall efficiency of repair is comparable. Hence, the REC is not sensitive to the “origin” of the DSB ends. When the homologous ends for GC are in Trans, the delay in repair appears to reflect their tethering to sequences on the other side of the DSB that themselves recombine with other genomic locations with which they share sequence homology. These data support previous observations that the two ends of a DSB are usually tethered to each other and that this tethering facilitates both ends encountering the same donor sequence. We also found that the presence of homeologous/repetitive sequences in the vicinity of a DSB can distract the DSB end from finding its bona fide homologous donor, and that inhibition of GC by such homeologous sequences is markedly increased upon deleting Sgs1 but not Msh6. PMID:27074148

DNA double-strand break repair of blood lymphocytes and normal tissues analysed in a preclinical mouse model: implications for radiosensitivity testing.

PubMed

Rübe, Claudia E; Grudzenski, Saskia; Kühne, Martin; Dong, Xiaorong; Rief, Nicole; Löbrich, Markus; Rübe, Christian

2008-10-15

Radiotherapy is an effective cancer treatment, but a few patients suffer severe radiation toxicities in neighboring normal tissues. There is increasing evidence that the variable susceptibility to radiation toxicities is caused by the individual genetic predisposition, by subtle mutations, or polymorphisms in genes involved in cellular responses to ionizing radiation. Double-strand breaks (DSB) are the most deleterious form of radiation-induced DNA damage, and DSB repair deficiencies lead to pronounced radiosensitivity. Using a preclinical mouse model, the highly sensitive gammaH2AX-foci approach was tested to verify even subtle, genetically determined DSB repair deficiencies known to be associated with increased normal tissue radiosensitivity. By enumerating gammaH2AX-foci in blood lymphocytes and normal tissues (brain, lung, heart, and intestine), the induction and repair of DSBs after irradiation with therapeutic doses (0.1-2 Gy) was investigated in repair-proficient and repair-deficient mouse strains in vivo and blood samples irradiated ex vivo. gammaH2AX-foci analysis allowed to verify the different DSB repair deficiencies; even slight impairments caused by single polymorphisms were detected similarly in both blood lymphocytes and solid tissues, indicating that DSB repair measured in lymphocytes is valid for different and complex organs. Moreover, gammaH2AX-foci analysis of blood samples irradiated ex vivo was found to reflect repair kinetics measured in vivo and, thus, give reliable information about the individual DSB repair capacity. gammaH2AX analysis of blood and tissue samples allows to detect even minor genetically defined DSB repair deficiencies, affecting normal tissue radiosensitivity. Future studies will have to evaluate the clinical potential to identify patients more susceptible to radiation toxicities before radiotherapy.

Radiation-induced double-strand breaks in mammalian DNA: influence of temperature and DMSO.

PubMed

Elmroth, K; Nygren, J; Erkell, L J; Hultborn, R

2000-11-01

To investigate the effects of subphysiological irradiation temperature (2 28 degrees C) and the influence of the radical scavenger DMSO on the induction of double-strand breaks (DSB) in chromosomal DNA from a human breast cancer cell line (MCF-7) as well as in intact cells. The rejoining of DSB in cells irradiated at 2 degrees C or 37 degrees C was also investigated. Agarose plugs with [14C]thymidine labelled MCF-7 cells were lysed in EDTA-NLS-proteinase-K buffer. The plugs containing chromosomal DNA were irradiated with X-rays under different temperatures and scavenging conditions. Intact MCF-7 cells were irradiated in Petri dishes and plugs were made. The cells were then lysed in EDTA-NLS-proteinase-K buffer. The induction of DSB was studied by constant field gel electrophoresis and expressed as DSB/100/Mbp, calculated from the fraction of activity released into the gel. The induction of DSB in chromosomal DNA was reduced by a decrease in temperature. This protective effect of low temperature was inhibited when the DNA was irradiated in the presence of DMSO. No difference was found when intact cells were irradiated at different temperatures. However, the rapid phase of rejoining was slower in cells irradiated at 37 degrees C than at 2 degrees C. The induction of DSB in naked DNA was reduced by hypothermic irradiation. The temperature had no influence on the induction of DSB in the presence of a high concentration of DMSO, indicating that the temperature effect is mediated via the indirect effects of ionizing radiation. Results are difficult to interpret in intact cells. Rejoining during irradiation at the higher temperature may counteract an increased induction. The difference in rejoining may be interpreted in terms of qualitative differences between breaks induced at the two temperatures.

Biochemical Kinetics Model of DSB Repair and GammaH2AX FOCI by Non-homologous End Joining

NASA Technical Reports Server (NTRS)

Cucinotta, Francis, A.; Pluth, Janice M.; Anderson, Jennifer A.; Harper, Jane V.; O'Neill, Peter

2007-01-01

We developed a biochemical kinetics approach to describe the repair of double strand breaks (DSB) produced by low LET radiation by modeling molecular events associated with the mechanisms of non-homologous end-joining (NHEJ). A system of coupled non-linear ordinary differential equations describes the induction of DSB and activation pathways for major NHEJ components including Ku(sub 70/80), DNA-PK(sub cs), and the Ligase IV-XRCC4 hetero-dimer. The autophosphorylation of DNA-PK(sub cs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of DNA-PK(sub cs) regulation of repair was developed with the initial step allowing access of other NHEJ components to breaks, and a second step limiting access to Ligase IV-XRCC4. Our model assumes that the transition from the first to second-step depends on DSB complexity, with a much slower-rate for complex DSB. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field electrophoresis (PFGE), quantification of the induction of gamma-H2AX foci, and live cell imaging of the induction of Ku(sub 70/80). Predictions are made for the behaviors of NHEJ components at low doses and dose-rates, where a steady-state is found at dose-rates of 0.1 Gy/hr or lower.

Evidence for double-strand break mediated mitochondrial DNA replication in Saccharomyces cerevisiae

PubMed Central

Prasai, Kanchanjunga; Robinson, Lucy C.; Scott, Rona S.; Tatchell, Kelly

2017-01-01

Abstract The mechanism of mitochondrial DNA (mtDNA) replication in Saccharomyces cerevisiae is controversial. Evidence exists for double-strand break (DSB) mediated recombination-dependent replication at mitochondrial replication origin ori5 in hypersuppressive ρ− cells. However, it is not clear if this replication mode operates in ρ+ cells. To understand this, we targeted bacterial Ku (bKu), a DSB binding protein, to the mitochondria of ρ+ cells with the hypothesis that bKu would bind persistently to mtDNA DSBs, thereby preventing mtDNA replication or repair. Here, we show that mitochondrial-targeted bKu binds to ori5 and that inducible expression of bKu triggers petite formation preferentially in daughter cells. bKu expression also induces mtDNA depletion that eventually results in the formation of ρ0 cells. This data supports the idea that yeast mtDNA replication is initiated by a DSB and bKu inhibits mtDNA replication by binding to a DSB at ori5, preventing mtDNA segregation to daughter cells. Interestingly, we find that mitochondrial-targeted bKu does not decrease mtDNA content in human MCF7 cells. This finding is in agreement with the fact that human mtDNA replication, typically, is not initiated by a DSB. Therefore, this study provides evidence that DSB-mediated replication is the predominant form of mtDNA replication in ρ+ yeast cells. PMID:28549155

DNA double-strand–break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice

PubMed Central

Schipler, Agnes; Iliakis, George

2013-01-01

Although the DNA double-strand break (DSB) is defined as a rupture in the double-stranded DNA molecule that can occur without chemical modification in any of the constituent building blocks, it is recognized that this form is restricted to enzyme-induced DSBs. DSBs generated by physical or chemical agents can include at the break site a spectrum of base alterations (lesions). The nature and number of such chemical alterations define the complexity of the DSB and are considered putative determinants for repair pathway choice and the probability that errors will occur during this processing. As the pathways engaged in DSB processing show distinct and frequently inherent propensities for errors, pathway choice also defines the error-levels cells opt to accept. Here, we present a classification of DSBs on the basis of increasing complexity and discuss how complexity may affect processing, as well as how it may cause lethal or carcinogenic processing errors. By critically analyzing the characteristics of DSB repair pathways, we suggest that all repair pathways can in principle remove lesions clustering at the DSB but are likely to fail when they encounter clusters of DSBs that cause a local form of chromothripsis. In the same framework, we also analyze the rational of DSB repair pathway choice. PMID:23804754

Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-x(L)

NASA Technical Reports Server (NTRS)

Wiese, Claudia; Pierce, Andrew J.; Gauny, Stacey S.; Jasin, Maria; Kronenberg, Amy; Chatterjee, A. (Principal Investigator)

2002-01-01

Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in DSB repair in human cells. However, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We demonstrated previously that overexpression of BCL-2 or BCL-x(L) enhanced the frequency of X-ray-induced TK1 mutations, including loss of heterozygosity events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells and to determine whether ectopic expression of BCL-x(L) affects HDR. Using TK6-neo cells, we find that a single DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold, demonstrating efficient DSB repair by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3-4-fold more frequent in TK6 cells that stably overexpress the antiapoptotic protein BCL-X(L). Thus, HDR plays an important role in maintaining genomic integrity in human cells, and ectopic expression of BCL-x(L) enhances HDR of DSBs. This is the first study to highlight a function for BCL-x(L) in modulating DSB repair in human cells.

A double-strand break can trigger immunoglobulin gene conversion

PubMed Central

Bastianello, Giulia; Arakawa, Hiroshi

2017-01-01

All three B cell-specific activities of the immunoglobulin (Ig) gene re-modeling system—gene conversion, somatic hypermutation and class switch recombination—require activation-induced deaminase (AID). AID-induced DNA lesions must be further processed and dissected into different DNA recombination pathways. In order to characterize potential intermediates for Ig gene conversion, we inserted an I-SceI recognition site into the complementarity determining region 1 (CDR1) of the Ig light chain locus of the AID knockout DT40 cell line, and conditionally expressed I-SceI endonuclease. Here, we show that a double-strand break (DSB) in CDR1 is sufficient to trigger Ig gene conversion in the absence of AID. The pattern and pseudogene usage of DSB-induced gene conversion were comparable to those of AID-induced gene conversion; surprisingly, sometimes a single DSB induced multiple gene conversion events. These constitute direct evidence that a DSB in the V region can be an intermediate for gene conversion. The fate of the DNA lesion downstream of a DSB had more flexibility than that of AID, suggesting two alternative models: (i) DSBs during the physiological gene conversion are in the minority compared to single-strand breaks (SSBs), which are frequently generated following DNA deamination, or (ii) the physiological gene conversion is mediated by a tightly regulated DSB that is locally protected from non-homologous end joining (NHEJ) or other non-homologous DNA recombination machineries. PMID:27701075

MO-FG-CAMPUS-TeP3-02: Benchmarks of a Proton Relative Biological Effectiveness (RBE) Model for DNA Double Strand Break (DSB) Induction in the FLUKA, MCNP, TOPAS, and RayStation™ Treatment Planning System

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, R; Streitmatter, S; Traneus, E

2016-06-15

Purpose: Validate implementation of a published RBE model for DSB induction (RBEDSB) in several general purpose Monte Carlo (MC) code systems and the RayStation™ treatment planning system (TPS). For protons and other light ions, DSB induction is a critical initiating molecular event that correlates well with the RBE for cell survival. Methods: An efficient algorithm to incorporate information on proton and light ion RBEDSB from the independently tested Monte Carlo Damage Simulation (MCDS) has now been integrated into MCNP (Stewart et al. PMB 60, 8249–8274, 2015), FLUKA, TOPAS and a research build of the RayStation™ TPS. To cross-validate the RBEDSBmore » model implementation LET distributions, depth-dose and lateral (dose and RBEDSB) profiles for monodirectional monoenergetic (100 to 200 MeV) protons incident on a water phantom are compared. The effects of recoil and secondary ion production ({sub 2}H{sub +}, {sub 3}H{sub +}, {sub 3}He{sub 2+}, {sub 4}He{sub 2+}), spot size (3 and 10 mm), and transport physics on beam profiles and RBEDSB are examined. Results: Depth-dose and RBEDSB profiles among all of the MC models are in excellent agreement using a 1 mm distance criterion (width of a voxel). For a 100 MeV proton beam (10 mm spot), RBEDSB = 1.2 ± 0.03 (− 2–3%) at the tip of the Bragg peak and increases to 1.59 ± 0.3 two mm distal to the Bragg peak. RBEDSB tends to decrease as the kinetic energy of the incident proton increases. Conclusion: The model for proton RBEDSB has been accurately implemented into FLUKA, MCNP, TOPAS and the RayStation™TPS. The transport of secondary light ions (Z > 1) has a significant impact on RBEDSB, especially distal to the Bragg peak, although light ions have a small effect on (dosexRBEDSB) profiles. The ability to incorporate spatial variations in proton RBE within a TPS creates new opportunities to individualize treatment plans and increase the therapeutic ratio. Dr. Erik Traneus is employed full-time as a Research Scientist at RaySearch Laboratories. The research build of the RayStation used in the study was made available to the University of Washington free of charge. RaySearch Laboratories did not provide any monetary support for the reported studies.« less

Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

2009-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems.

DNA double-strand breaks in blood lymphocytes induced by two-day 99mTc-MIBI myocardial perfusion scintigraphy.

PubMed

Rief, Matthias; Hartmann, Lisa; Geisel, Dominik; Richter, Felicitas; Brenner, Winfried; Dewey, Marc

2018-07-01

To investigate DNA double-strand breaks (DSBs) in blood lymphocytes induced by two-day 99m Tc-MIBI myocardial perfusion scintigraphy (MPS) using y-H2AX immunofluorescence microscopy and to correlate the results with 99m Tc activity in blood samples. Eleven patients who underwent two-day MPS were included. DSB blood sampling was performed before and 5min, 1h and 24h after the first and second radiotracer injections. 99m Tc activity was measured in each blood sample. For immunofluorescence microscopy, distinct foci representing DSBs were quantified in lymphocytes after staining for the phosphorylated histone variant y-H2AX. The 99m Tc-MIBI activity measured on days one and two was similar (254±25 and 258±27 MBq; p=0.594). Compared with baseline DSB foci (0.09±0.05/cell), a significant increase was found at 5min (0.19±0.04/cell) and 1h (0.18±0.04/cell) after the first injection and at 5min and 1h after the second injection (0.21±0.03 and 0.19±0.04/cell, respectively; p=0.003 for both). At 24h after the first and second injections, the number of DSB foci had returned to baseline (0.06±0.02 and 0.12±0.05/cell, respectively). 99m Tc activity levels in peripheral blood samples correlated well with DSB counts (r=0.451). DSB counts reflect 99m Tc-MIBI activity after injection for two-day MPS, and might allow individual monitoring of biological effects of cardiac nuclear imaging. • Myocardial perfusion scintigraphy using 99m Tc induces time-dependent double-strand breaks (DSBs) • γ-H2AX immunofluorescence microscopy shows DSB as an early response to radiotracer injection • Activity measurements of 99m Tc correlate well with detected DSB • DSB foci induced by 99m Tc return to baseline 24h after radiotracer injection.

NF-κB regulates DNA double-strand break repair in conjunction with BRCA1-CtIP complexes.

PubMed

Volcic, Meta; Karl, Sabine; Baumann, Bernd; Salles, Daniela; Daniel, Peter; Fulda, Simone; Wiesmüller, Lisa

2012-01-01

NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP-BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP-BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation.

Recruitment of RecA homologs Dmc1p and Rad51p to the double-strand break repair site initiated by meiosis-specific endonuclease VDE (PI-SceI).

PubMed

Fukuda, Tomoyuki; Ohya, Yoshikazu

2006-02-01

During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination, being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of VDE-initiated homing for the study of meiotic recombination.

DNA Breaks and End Resection Measured Genome-wide by End Sequencing.

PubMed

Canela, Andres; Sridharan, Sriram; Sciascia, Nicholas; Tubbs, Anthony; Meltzer, Paul; Sleckman, Barry P; Nussenzweig, André

2016-09-01

DNA double-strand breaks (DSBs) arise during physiological transcription, DNA replication, and antigen receptor diversification. Mistargeting or misprocessing of DSBs can result in pathological structural variation and mutation. Here we describe a sensitive method (END-seq) to monitor DNA end resection and DSBs genome-wide at base-pair resolution in vivo. We utilized END-seq to determine the frequency and spectrum of restriction-enzyme-, zinc-finger-nuclease-, and RAG-induced DSBs. Beyond sequence preference, chromatin features dictate the repertoire of these genome-modifying enzymes. END-seq can detect at least one DSB per cell among 10,000 cells not harboring DSBs, and we estimate that up to one out of 60 cells contains off-target RAG cleavage. In addition to site-specific cleavage, we detect DSBs distributed over extended regions during immunoglobulin class-switch recombination. Thus, END-seq provides a snapshot of DNA ends genome-wide, which can be utilized for understanding genome-editing specificities and the influence of chromatin on DSB pathway choice. Published by Elsevier Inc.

A significant role of non-thermal equilibrated electrons in the formation of deleterious complex DNA damage.

PubMed

Kai, Takeshi; Yokoya, Akinari; Ukai, Masatoshi; Fujii, Kentaro; Toigawa, Tomohiro; Watanabe, Ritsuko

2018-01-24

Although most of the radiation damage to genomic DNA could be rendered harmless using repair enzymes in a living cell, a certain fraction of the damage is persistent resulting in serious genetic effects, such as mutation induction. In order to understand the mechanisms of the deleterious DNA damage formation in terms of its earliest physical stage at the radiation track end, dynamics of low energy electrons and their thermalization processes around DNA molecules were investigated using a dynamic Monte Carlo code. The primary incident (1 keV) electrons multiply collide within 1 nm (equivalent to three DNA-base-pairs, 3bp) and generate secondary electrons which show non-Gaussian and non-thermal equilibrium distributions within 300 fs. On the other hand, the secondary electrons are mainly distributed within approximately 10 nm from their parent cations although approximately 5% of the electrons are localized within 1 nm of the cations owing to the interaction of their Coulombic fields. The mean electron energy is 0.7 eV; however, more than 10% of the electrons fall into a much lower-energy region than 0.1 eV at 300 fs. These results indicate that pre-hydrated electrons are formed from the extremely decelerated electrons over a few nm from the cations. DNA damage sites comprising multiple nucleobase lesions or single strand breaks can therefore be formed by multiple collisions of these electrons within 3bp. This multiple damage site is hardly processed by base excision repair enzymes. However, pre-hydrated electrons can also be produced resulting in an additional base lesion (or a strand break) more than 3bp away from the multi-damage site. These damage sites may be finally converted into a double strand break (DSB) when base excision enzymes process the additional base lesions. This DSB includes another base lesion(s) at their termini, and may introduce miss-rejoining by DSB repair enzymes, and hence may result in biological effects such as mutation in surviving cells.

Development of Bone-Conducted Ultrasonic Hearing Aid for the Profoundly Deaf: Assessments of the Modulation Type with Regard to Intelligibility and Sound Quality

NASA Astrophysics Data System (ADS)

Nakagawa, Seiji; Fujiyuki, Chika; Kagomiya, Takayuki

2012-07-01

Bone-conducted ultrasound (BCU) is perceived even by the profoundly sensorineural deaf. A novel hearing aid using the perception of amplitude-modulated BCU (BCU hearing aid: BCUHA) has been developed; however, further improvements are needed, especially in terms of articulation and sound quality. In this study, the intelligibility and sound quality of BCU speech with several types of amplitude modulation [double-sideband with transmitted carrier (DSB-TC), double-sideband with suppressed carrier (DSB-SC), and transposed modulation] were evaluated. The results showed that DSB-TC and transposed speech were more intelligible than DSB-SC speech, and transposed speech was closer than the other types of BCU speech to air-conducted speech in terms of sound quality. These results provide useful information for further development of the BCUHA.

Involvement of Matrin 3 and SFPQ/NONO in the DNA damage response.

PubMed

Salton, Maayan; Lerenthal, Yaniv; Wang, Shih-Ya; Chen, David J; Shiloh, Yosef

2010-04-15

The DNA damage response (DDR) is a complex signaling network that is induced by DNA lesions and vigorously activated by double strand breaks (DSBs). The DSB response is mobilized by the nuclear protein kinase ATM, which phosphorylates key players in its various branches. SFPQ (PSF) and NONO (p54) are nuclear proteins that interact with each other and have diverse roles in nucleic acids metabolism. The SFPQ/NONO heterodimer was previously found to enhance DNA strand break rejoining in vitro. Our attention was drawn to these two proteins as they interact with the nuclear matrix protein Matrin 3 (MATR3), which we found to be a novel ATM target. We asked whether SFPQ and NONO too are involved in the DSB response. Proteins that function at the early phase of this response are often recruited to the damaged sites. We observed rapid recruitment of SFPQ/NONO to sites of DNA damage induced by laser microbeam. In MATR3 knockdown cells SFPQ/NONO retention at DNA damage sites was prolonged. SFPQ and MATR3 depletion led to abnormal accumulation of cells at the S-phase of the cell cycle following treatment with the radiomimetic chemical neocarzinostatin. Notably, proteins involved in DSB repair via nonhomologous end-joining co-immunoprecipitated with NONO; SFPQ depletion delayed DSB repair. Collectively the data suggest that SFPQ, NONO and MATR3 are involved in the early stage of the DSB response, setting the scene for DSB repair.

Evidence for double-strand break mediated mitochondrial DNA replication in Saccharomyces cerevisiae.

PubMed

Prasai, Kanchanjunga; Robinson, Lucy C; Scott, Rona S; Tatchell, Kelly; Harrison, Lynn

2017-07-27

The mechanism of mitochondrial DNA (mtDNA) replication in Saccharomyces cerevisiae is controversial. Evidence exists for double-strand break (DSB) mediated recombination-dependent replication at mitochondrial replication origin ori5 in hypersuppressive ρ- cells. However, it is not clear if this replication mode operates in ρ+ cells. To understand this, we targeted bacterial Ku (bKu), a DSB binding protein, to the mitochondria of ρ+ cells with the hypothesis that bKu would bind persistently to mtDNA DSBs, thereby preventing mtDNA replication or repair. Here, we show that mitochondrial-targeted bKu binds to ori5 and that inducible expression of bKu triggers petite formation preferentially in daughter cells. bKu expression also induces mtDNA depletion that eventually results in the formation of ρ0 cells. This data supports the idea that yeast mtDNA replication is initiated by a DSB and bKu inhibits mtDNA replication by binding to a DSB at ori5, preventing mtDNA segregation to daughter cells. Interestingly, we find that mitochondrial-targeted bKu does not decrease mtDNA content in human MCF7 cells. This finding is in agreement with the fact that human mtDNA replication, typically, is not initiated by a DSB. Therefore, this study provides evidence that DSB-mediated replication is the predominant form of mtDNA replication in ρ+ yeast cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Defense Science Board 2001 Summer Study on Defense Science and Technology

DTIC Science & Technology

2002-05-01

SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10 . SPONSOR/MONITOR’S ACRONYM(S) 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION...Today, it takes roughly 10 to 15 years to develop a safe drug for a specific purpose. The task force believes that it is possible for the United...performance in later screens. DSB Summer Study on Defense Science and Technology ___________________________________________ 10 The

The DESIRE Airborne gravity project in the Dead Sea Basin and 3D numerical gravity modeling

NASA Astrophysics Data System (ADS)

Choi, Sungchan; Götze, Hans-Jürgen; Meyer, Uwe; Desire-Group

2010-05-01

This geo-scientific research focuses on the geological setting of the Dead Sea Transform (DST) and the Dead Sea Basin (DSB) and its resulting pull-apart basins. Since the late 1970s, crustal scale geophysical experiments have been carried out in this region. However, the nature of the crust underlying the eastern and western shoulders of the DSB and underneath the DST itself is still a hotly debated topic among researchers. To address one of the central questions of plate tectonics - How do large transform systems work and what are their typical features? - An international geoscientific Dead Sea Integrated Research project (DESIRE) is being conducted by colleagues from Germany, Israel, Palestine, and Jordan. In order to provide a high resolution gravity database that support 3D numerical modeling and hence a more comprehensive understanding of the nature and segmentation of the DST, an airborne gravity survey as a part of the DESIRE project has been carried out from February to March 2007. The airborne gravity survey covered the DST from Elat/Aqaba in the South to the northern rim of the Dead Sea. The low speed and terrain-following helicopter gravity flights were performed to acquire the highest possible data quality. In total, 32 north-south profiles and 16 west-east profiles crossing the DST have been measured. Most of the profiles concentrated in areas that lacked terrestrial gravity data coverage, e. g. over the shoulders of the DSB. The airborne gravity data are merged with existing conventional (terrestrial) data sets to provide a seamless gravity map of the area of interest. The results of the 3D gravity modelling based the GPS analysis, magnetic field characters, seismic researches and analysis of earthquake data allow us to propose that (1) the DSB is divided into two tectonic blocks by the region between the Lisan peninsula and the southern margin of the northern DSB and (2) the tectonic system in the DSB is defined as a counter-clockwise rotating pull apart basin due to the ‘Riedel flaking', by which the northern DSB is rotated counter-clockwise from the region and the southern DSB to the opposite direction. The salt diapir below the Dead Sea is suspected to be migrated from the Lisan peninsula to present region by the rotation of the northern DSB, while the Sedom diapir is extended to the SE direction. The Almacik flake along the North Anatolian fault, Turkey is probably another example of such basin.

DsbA-L prevents obesity-induced inflammation and insulin resistance by suppressing the mtDNA release-activated cGAS-cGAMP-STING pathway

PubMed Central

Cervantes, Christopher; Liu, Juan; He, Sijia; Zhou, Haiyan; Zhang, Bilin; Cai, Huan; Yin, Dongqing; Hu, Derong; Li, Zhi; Chen, Hongzhi; Gao, Xiaoli; Wang, Fang; O’Connor, Jason C.; Xu, Yong; Liu, Meilian; Dong, Lily Q.

2017-01-01

Chronic inflammation in adipose tissue plays a key role in obesity-induced insulin resistance. However, the mechanisms underlying obesity-induced inflammation remain elusive. Here we show that obesity promotes mtDNA release into the cytosol, where it triggers inflammatory responses by activating the DNA-sensing cGAS-cGAMP-STING pathway. Fat-specific knockout of disulfide-bond A oxidoreductase-like protein (DsbA-L), a chaperone-like protein originally identified in the mitochondrial matrix, impaired mitochondrial function and promoted mtDNA release, leading to activation of the cGAS-cGAMP-STING pathway and inflammatory responses. Conversely, fat-specific overexpression of DsbA-L protected mice against high-fat diet-induced activation of the cGAS-cGAMP-STING pathway and inflammation. Taken together, we identify DsbA-L as a key molecule that maintains mitochondrial integrity. DsbA-L deficiency promotes inflammation and insulin resistance by activating the cGAS-cGAMP-STING pathway. Our study also reveals that, in addition to its well-characterized roles in innate immune surveillance, the cGAS-cGAMP-STING pathway plays an important role in mediating obesity-induced metabolic dysfunction. PMID:29087318

DsbA-L prevents obesity-induced inflammation and insulin resistance by suppressing the mtDNA release-activated cGAS-cGAMP-STING pathway.

PubMed

Bai, Juli; Cervantes, Christopher; Liu, Juan; He, Sijia; Zhou, Haiyan; Zhang, Bilin; Cai, Huan; Yin, Dongqing; Hu, Derong; Li, Zhi; Chen, Hongzhi; Gao, Xiaoli; Wang, Fang; O'Connor, Jason C; Xu, Yong; Liu, Meilian; Dong, Lily Q; Liu, Feng

2017-11-14

Chronic inflammation in adipose tissue plays a key role in obesity-induced insulin resistance. However, the mechanisms underlying obesity-induced inflammation remain elusive. Here we show that obesity promotes mtDNA release into the cytosol, where it triggers inflammatory responses by activating the DNA-sensing cGAS-cGAMP-STING pathway. Fat-specific knockout of disulfide-bond A oxidoreductase-like protein (DsbA-L), a chaperone-like protein originally identified in the mitochondrial matrix, impaired mitochondrial function and promoted mtDNA release, leading to activation of the cGAS-cGAMP-STING pathway and inflammatory responses. Conversely, fat-specific overexpression of DsbA-L protected mice against high-fat diet-induced activation of the cGAS-cGAMP-STING pathway and inflammation. Taken together, we identify DsbA-L as a key molecule that maintains mitochondrial integrity. DsbA-L deficiency promotes inflammation and insulin resistance by activating the cGAS-cGAMP-STING pathway. Our study also reveals that, in addition to its well-characterized roles in innate immune surveillance, the cGAS-cGAMP-STING pathway plays an important role in mediating obesity-induced metabolic dysfunction.

BRCA1 Mutation: A Predictive Marker for Radiation Therapy?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kan, Charlene; Zhang, Junran, E-mail: Junran.zhang@case.edu

2015-10-01

DNA repair, in particular, DNA double-strand break (DSB) repair, is essential for the survival of both normal and cancer cells. An elaborate repair mechanism has been developed in cells to efficiently repair the damaged DNA. The pathways predominately involved in DSB repair are homologous recombination and classic nonhomologous end-joining, although the alternative NHEJ pathway, a third DSB repair pathway, could also be important in certain contexts. The protein of BRCA1 encoded by the tumor suppressor gene BRCA1 regulates all DSB repair pathways. Given that DSBs represent the most biologically significant lesions induced by ionizing radiation and that impaired DSB repairmore » leads to radiation sensitivity, it has been expected that cancer patients with BRCA1 mutations should benefit from radiation therapy. However, the clinical data have been conflicting and inconclusive. We provide an overview about the current status of the data regarding BRCA1 deficiency and radiation therapy sensitivity in both experimental models and clinical investigations. In addition, we discuss a strategy to potentiate the effects of radiation therapy by poly(ADP-ribose) polymerase inhibitors, the pharmacologic drugs being investigated as monotherapy for the treatment of patients with BRCA1/2 mutations.« less

Systematic characterization of deubiquitylating enzymes for roles in maintaining genome integrity.

PubMed

Nishi, Ryotaro; Wijnhoven, Paul; le Sage, Carlos; Tjeertes, Jorrit; Galanty, Yaron; Forment, Josep V; Clague, Michael J; Urbé, Sylvie; Jackson, Stephen P

2014-10-01

DNA double-strand breaks (DSBs) are perhaps the most toxic of all DNA lesions, with defects in the DNA-damage response to DSBs being associated with various human diseases. Although it is known that DSB repair pathways are tightly regulated by ubiquitylation, we do not yet have a comprehensive understanding of how deubiquitylating enzymes (DUBs) function in DSB responses. Here, by carrying out a multidimensional screening strategy for human DUBs, we identify several with hitherto unknown links to DSB repair, the G2/M DNA-damage checkpoint and genome-integrity maintenance. Phylogenetic analyses reveal functional clustering within certain DUB subgroups, suggesting evolutionally conserved functions and/or related modes of action. Furthermore, we establish that the DUB UCHL5 regulates DSB resection and repair by homologous recombination through protecting its interactor, NFRKB, from degradation. Collectively, our findings extend the list of DUBs promoting the maintenance of genome integrity, and highlight their potential as therapeutic targets for cancer.

Inactivation, DNA double strand break induction and their rejoining in bacterial cells irradiated with heavy ions

NASA Technical Reports Server (NTRS)

Schaefer, M.; Zimmermann, H.; Schmitz, C.

1994-01-01

Besides inactivation one of the major interests in our experiments is to study the primary damage in the DNA double strand breaks (DSB) after heavy ion irradiation. These damages lead not only to cell death but also under repair activities to mutations. In further experiments we have investigated the inactivation with two different strains of Deinococcus radiodurans (R1, Rec 30) and the induction of DSB as well as the rejoining of DSB in stationary cells of E. coli (strain B/r) irradiated with radiations of different quality. In the latter case irradiations were done so that the cell survival was roughly at the same level. We measured the DSB using the pulse field gelelectrophoresis which allows to separate between intact (circular) and damaged (linear) DNA. The irradiated cells were transferred to NB medium and incubated for different times to allow rejoining.

Optical and tunneling microscopy and spectroscopy at the ultimate spatial limit

NASA Astrophysics Data System (ADS)

Chen, Chi

2009-12-01

The combination of optical detection system with a scanning tunneling microscope (STM) leads to the possibility of resolving radiative transition probability with the ultrahigh spatial resolution of STM in real space. This opens an innovative approach toward revealing the correlation between molecular structure, electronic characteristics, and optical properties. This thesis describes a series of experiments that manifests this correlation, including atomic silver chains and single porphine molecules. In atomic silver chains, the number and positions of the emission maxima in the photon images match the nodes in the dI/d V images of "particle-in-a-box" states. This surprising correlation between the emission maxima and nodes in the density of states is a manifestation of Fermi's golden rule in real space for radiative transitions, which provides an understanding of the mechanism of STM induced light emission. From single porphine molecules, orthogonal spatial contrast of two types of vibronic coupling is resolved by both photon spectroscopy and vibronic-mode-selected photon images. Intramolecular transitions from the two orthogonal LUMOs individually couple to different molecular normal modes. This is the first demonstration of the photon emission probability of a single molecule and its direct correlations with the molecular orbitals. This also provides the first real space experimental evidence to separate the tangled effects of molecular conformations and nano-environments on the inhomogeneity of molecular emission. DSB molecules are found to have two conformational isomers and one of them shows surface chirality. All these conformers and enantiomers can be switched to each other by electron injection. Different DSB conformers present distinct manipulation dynamics, which demonstrate how different conformations and their preferred adsorption geometries can have pronounced influence on the molecular mechanics on the surface. Overall, this thesis studies the very fundamental nature of single molecules and artificial nanostructures by integrating all kinds of important functions of STM: topography, spectroscopy, manipulation, and photon emission. Detailed correlations between the emission patterns and orbital structures are revealed by the ultimate spatial resolution of our "STM photon microscopy".

New small-molecule inhibitor class targeting human immunodeficiency virus type 1 virion maturation.

PubMed

Blair, Wade S; Cao, Joan; Fok-Seang, Juin; Griffin, Paul; Isaacson, Jason; Jackson, R Lynn; Murray, Edward; Patick, Amy K; Peng, Qinghai; Perros, Manos; Pickford, Chris; Wu, Hua; Butler, Scott L

2009-12-01

A new small-molecule inhibitor class that targets virion maturation was identified from a human immunodeficiency virus type 1 (HIV-1) antiviral screen. PF-46396, a representative molecule, exhibits antiviral activity against HIV-1 laboratory strains and clinical isolates in T-cell lines and peripheral blood mononuclear cells (PBMCs). PF-46396 specifically inhibits the processing of capsid (CA)/spacer peptide 1 (SP1) (p25), resulting in the accumulation of CA/SP1 (p25) precursor proteins and blocked maturation of the viral core particle. Viral variants resistant to PF-46396 contain a single amino acid substitution in HIV-1 CA sequences (CAI201V), distal to the CA/SP1 cleavage site in the primary structure, which we demonstrate is sufficient to confer significant resistance to PF-46396 and 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB), a previously described maturation inhibitor. Conversely, a single amino substitution in SP1 (SP1A1V), which was previously associated with DSB in vitro resistance, was sufficient to confer resistance to DSB and PF-46396. Further, the CAI201V substitution restored CA/SP1 processing in HIV-1-infected cells treated with PF-46396 or DSB. Our results demonstrate that PF-46396 acts through a mechanism that is similar to DSB to inhibit the maturation of HIV-1 virions. To our knowledge, PF-46396 represents the first small-molecule HIV-1 maturation inhibitor that is distinct in chemical class from betulinic acid-derived maturation inhibitors (e.g., DSB), demonstrating that molecules of diverse chemical classes can inhibit this mechanism.

Pseudosynapsis and Decreased Stringency of Meiotic Repair Pathway Choice on the Hemizygous Sex Chromosome of Caenorhabditis elegans Males

PubMed Central

Checchi, Paula M.; Lawrence, Katherine S.; Van, Mike V.; Larson, Braden J.; Engebrecht, JoAnne

2014-01-01

During meiosis, accurate chromosome segregation relies on homology to mediate chromosome pairing, synapsis, and crossover recombination. Crossovers are dependent upon formation and repair of double-strand breaks (DSBs) by homologous recombination (HR). In males of many species, sex chromosomes are largely hemizygous, yet DSBs are induced along nonhomologous regions. Here we analyzed the genetic requirements for meiotic DSB repair on the completely hemizygous X chromosome of Caenorhabditis elegans males. Our data reveal that the kinetics of DSB formation, chromosome pairing, and synapsis are tightly linked in the male germ line. Moreover, DSB induction on the X is concomitant with a brief period of pseudosynapsis that may allow X sister chromatids to masquerade as homologs. Consistent with this, neither meiotic kleisins nor the SMC-5/6 complex are essential for DSB repair on the X. Furthermore, early processing of X DSBs is dependent on the CtIP/Sae2 homolog COM-1, suggesting that as with paired chromosomes, HR is the preferred pathway. In contrast, the X chromosome is refractory to feedback mechanisms that ensure crossover formation on autosomes. Surprisingly, neither RAD-54 nor BRC-2 are essential for DSB repair on the X, suggesting that unlike autosomes, the X is competent for repair in the absence of HR. When both RAD-54 and the structure-specific nuclease XPF-1 are abrogated, X DSBs persist, suggesting that single-strand annealing is engaged in the absence of HR. Our findings indicate that alteration in sister chromatid interactions and flexibility in DSB repair pathway choice accommodate hemizygosity on sex chromosomes. PMID:24939994

Global analysis of double-strand break processing reveals in vivo properties of the helicase-nuclease complex AddAB

PubMed Central

Badrinarayanan, Anjana; Cisse, Ibrahim I.

2017-01-01

In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicase-nuclease complex such as AddAB. The activity of AddAB has been well-studied in vitro, with translocation speeds between 400–2000 bp/s on linear DNA suggesting that a large section of DNA around a break site is processed for repair. However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear. To examine the functions and mechanistic regulation of AddAB inside bacterial cells, we developed a next-generation sequencing-based approach to assay DNA processing after a site-specific DSB was introduced on the chromosome of Caulobacter crescentus. Using this assay we determined the in vivo rates of DSB processing by AddAB and found that putative chi sites attenuate processing in a RecA-dependent manner. This RecA-mediated regulation of AddAB prevents the excessive loss of DNA around a break site, limiting the effects of DSB processing on transcription. In sum, our results, taken together with prior studies, support a mechanism for regulating AddAB that couples two key events of DSB repair–the attenuation of DNA-end processing and the initiation of homology search by RecA–thereby helping to ensure that genomic integrity is maintained during DSB repair. PMID:28489851

Fumarase is involved in DNA double-strand break resection through a functional interaction with Sae2.

PubMed

Leshets, Michael; Ramamurthy, Dharanidharan; Lisby, Michael; Lehming, Norbert; Pines, Ophry

2018-06-01

One of the most severe forms of DNA damage is the double-strand break (DSB). Failure to properly repair the damage can cause mutation, gross chromosomal rearrangements and lead to the development of cancer. In eukaryotes, homologous recombination (HR) and non-homologous end joining (NHEJ) are the main DSB repair pathways. Fumarase is a mitochondrial enzyme which functions in the tricarboxylic acid cycle. Intriguingly, the enzyme can be readily detected in the cytosolic compartment of all organisms examined, and we have shown that cytosolic fumarase participates in the DNA damage response towards DSBs. In human cells, fumarase was shown to be involved in NHEJ, but it is still unclear whether fumarase is also important for the HR pathway. Here we show that the depletion of cytosolic fumarase in yeast prolongs the presence of Mre11 at the DSBs, and decreases the kinetics of repair by the HR pathway. Overexpression of Sae2 endonuclease reduced the DSB sensitivity of the cytosolic fumarase depleted yeast, suggesting that Sae2 and fumarase functionally interact. Our results also suggest that Sae2 and cytosolic fumarase physically interact in vivo. Sae2 has been shown to be important for the DSB resection process, which is essential for the repair of DSBs by the HR pathway. Depletion of cytosolic fumarase inhibited DSB resection, while the overexpression of cytosolic fumarase or Sae2 restored resection. Together with our finding that cytosolic fumarase depletion reduces Sae2 cellular amounts, our results suggest that cytosolic fumarase is important for the DSB resection process by regulating Sae2 levels.

Manganese supplementation increases adiponectin and lowers ICAM-1 and creatinine blood levels in Zucker type 2 diabetic rats, and downregulates ICAM-1 by upregulating adiponectin multimerization protein (DsbA-L) in endothelial cells.

PubMed

Burlet, Elodie; Jain, Sushil K

2017-05-01

Blood and tissue levels of manganese (Mn) are lower in type 2 diabetic and atherosclerosis patients compared with healthy subjects. Adiponectin has anti-diabetic and anti-atherogenic properties. Impairment in Disulfide bond A-like protein (DsbA-L) is associated with low adiponectin levels and diabetes. This study investigates the hypothesis that the beneficial effects of Mn supplementation are mediated by adiponectin and DsbA-L. At 6 weeks of age, Male Zucker diabetic fatty rats (ZDF) were randomly divided into two groups: diabetic controls and Mn-supplemented diabetic rats. Each rat was supplemented with Mn (D+Mn, 16 mg/kg BW) or water (placebo, D+P) daily for 7 weeks by oral gavage. For cell culture studies, Human Umbilical Vein Endothelial Cells (HUVEC) or 3T3L1 adipocytes were pretreated with Mn (0-10 µM MnCl 2 ) for 24 h, followed by high glucose (HG, 25 mM) or normal glucose (5 mM) exposure for another 24 h. Mn supplementation resulted in higher adiponectin (p = 0.01), and lower ICAM-1 (p = 0.04) and lower creatinine (p = 0.04) blood levels compared to those in control ZDF rats. Mn-supplemented rats also caused reduced oxidative stress (ROS) and NADPH oxidase, and higher DsbA-L expression in the liver (p = 0.03) of ZDF rats compared to those in livers of control rats; however, Fe levels in liver were lower but not significant (p = 0.08). Similarly, treatment with high glucose (25 mM) caused a decrease in DsbA-L, which was prevented by Mn supplementation in HUVEC and adipocytes. Mechanistic studies with DsbA-L siRNA showed that the beneficial effects of Mn supplementation on ROS, NOX4, and ICAM-1 expression were abolished in DsbA-L knock-down HUVEC. These studies demonstrate that DsbA-L-linked adiponectin mediates the beneficial effects observed with Mn supplementation and provides evidence for a novel mechanism by which Mn supplementation can increase adiponectin and reduce the biomarkers of endothelial dysfunction in diabetes.

A novel automatic quantification method for high-content screening analysis of DNA double strand-break response.

PubMed

Feng, Jingwen; Lin, Jie; Zhang, Pengquan; Yang, Songnan; Sa, Yu; Feng, Yuanming

2017-08-29

High-content screening is commonly used in studies of the DNA damage response. The double-strand break (DSB) is one of the most harmful types of DNA damage lesions. The conventional method used to quantify DSBs is γH2AX foci counting, which requires manual adjustment and preset parameters and is usually regarded as imprecise, time-consuming, poorly reproducible, and inaccurate. Therefore, a robust automatic alternative method is highly desired. In this manuscript, we present a new method for quantifying DSBs which involves automatic image cropping, automatic foci-segmentation and fluorescent intensity measurement. Furthermore, an additional function was added for standardizing the measurement of DSB response inhibition based on co-localization analysis. We tested the method with a well-known inhibitor of DSB response. The new method requires only one preset parameter, which effectively minimizes operator-dependent variations. Compared with conventional methods, the new method detected a higher percentage difference of foci formation between different cells, which can improve measurement accuracy. The effects of the inhibitor on DSB response were successfully quantified with the new method (p = 0.000). The advantages of this method in terms of reliability, automation and simplicity show its potential in quantitative fluorescence imaging studies and high-content screening for compounds and factors involved in DSB response.

Improving Fab' fragment retention in an autonucleolytic Escherichia coli strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA.

PubMed

Schofield, Desmond M; Sirka, Ernestas; Keshavarz-Moore, Eli; Ward, John M; Nesbeth, Darren N

2017-12-01

To reduce unwanted Fab' leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab' fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives. We attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab' grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab' coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab' leakage relative to the original autonucleolytic Fab' strain with OmpA-fused staphylococcal nuclease. We successfully rescued Fab' leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab' fragment to the surrounding growth media.

Middle Infrared Radiation Induces G2/M Cell Cycle Arrest in A549 Lung Cancer Cells

PubMed Central

Huang, Hsuan-Cheng; Tsai, Shang-Ru; Juan, Hsueh-Fen; Lee, Si-Chen

2013-01-01

There were studies investigating the effects of broadband infrared radiation (IR) on cancer cell, while the influences of middle-infrared radiation (MIR) are still unknown. In this study, a MIR emitter with emission wavelength band in the 3–5 µm region was developed to irradiate A549 lung adenocarcinoma cells. It was found that MIR exposure inhibited cell proliferation and induced morphological changes by altering the cellular distribution of cytoskeletal components. Using quantitative PCR, we found that MIR promoted the expression levels of ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related and Rad3-related), TP53 (tumor protein p53), p21 (CDKN1A, cyclin-dependent kinase inhibitor 1A) and GADD45 (growth arrest and DNA-damage inducible), but decreased the expression levels of cyclin B coding genes, CCNB1 and CCNB2, as well as CDK1 (Cyclin-dependent kinase 1). The reduction of protein expression levels of CDC25C, cyclin B1 and the phosphorylation of CDK1 at Thr-161 altogether suggest G2/M arrest occurred in A549 cells by MIR. DNA repair foci formation of DNA double-strand breaks (DSB) marker γ-H2AX and sensor 53BP1 was induced by MIR treatment, it implies the MIR induced G2/M cell cycle arrest resulted from DSB. This study illustrates a potential role for the use of MIR in lung cancer therapy by initiating DSB and blocking cell cycle progression. PMID:23335992

L-Cysteine supplementation increases adiponectin synthesis and secretion, and GLUT4 and glucose utilization by upregulating disulfide bond A-like protein expression mediated by MCP-1 inhibition in 3T3-L1 adipocytes exposed to high glucose.

PubMed

Achari, Arunkumar Elumalai; Jain, Sushil K

2016-03-01

Adiponectin is an anti-diabetic and anti-atherogenic adipokine; its plasma levels are decreased in obesity, insulin resistance, and type 2 diabetes. An adiponectin-interacting protein named disulfide bond A-like protein (DsbA-L) plays an important role in the assembly of adiponectin. This study examined the hypothesis that L-cysteine (LC) regulates glucose homeostasis through the DsbA-L upregulation and synthesis and secretion of adiponectin in diabetes. 3T3L1 adipocytes were treated with LC (250 and 500 µM, 2 h) and high glucose (HG, 25 mM, 20 h). Results showed that LC supplementation significantly (p < 0.05) upregulated the DsbA-L, adiponectin, and GLUT-4 protein expression and glucose utilization in HG-treated adipocytes. LC supplementation significantly (p < 0.05) promoted the secretion of total and HMW adiponectin secretion in HG-treated adipocytes. In addition, LC significantly (p < 0.05) decreased ROS production and MCP-1 secretion in HG-treated cells. We further investigated whether MCP-1 has any role of LC on DsbA-L expression and adiponectin levels in 3T3-L1 cells. Treatment with LC prevented the decrease in DsbA-L, adiponectin, and GLUT-4 expression in 3T3L1 adipocyte cells exposed to MCP-1. Thus, this study demonstrates that DsbA-L and adiponectin upregulation mediates the beneficial effects of LC on glucose utilization by inhibiting MCP-1 secretion in adipocytes and provides a novel mechanism by which LC supplementation can improve insulin sensitivity in diabetes.

Increased DNA double-strand break was associated with downregulation of repair and upregulation of apoptotic factors in rat hippocampus after alcohol exposure.

PubMed

Suman, Shubhankar; Kumar, Santosh; N'Gouemo, Prosper; Datta, Kamal

2016-08-01

Binge drinking is known to cause damage in critical areas of the brain, including the hippocampus, which is important for relational memory and is reported to be sensitive to alcohol toxicity. However, the roles of DNA double-strand break (DSB) and its repair pathways, homologous recombination (HR), and non-homologous end joining (NHEJ) in alcohol-induced hippocampal injury remain to be elucidated. The purpose of this first study was to assess alcohol-induced DNA DSB and the mechanism by which alcohol affects DSB repair pathways in rat hippocampus. Male Sprague-Dawley rats (8-10 weeks old) were put on a 4-day binge ethanol treatment regimen. Control animals were maintained under similar conditions but were given the vehicle without ethanol. All animals were humanely euthanized 24 h after the last dose of ethanol administration and the hippocampi were dissected for immunoblot and immunohistochemistry analysis. Ethanol exposure caused increased 4-hydroxynonenal (4-HNE) staining as well as elevated γH2AX and 53BP1 foci in hippocampal cells. Immunoblot analysis showed decreased Mre11, Rad51, Rad50, and Ku86 as well as increased Bax and p21 in samples from ethanol-treated rats. Additionally, we also observed increased activated caspase3 staining in hippocampal cells 24 h after ethanol withdrawal. Taken together, our data demonstrated that ethanol concurrently induced DNA DSB, downregulated DSB repair pathway proteins, and increased apoptotic factors in hippocampal cells. We believe these findings will provide the impetus for further research on DNA DSB and its repair pathways in relation to alcohol toxicity in brain. Copyright © 2016 Elsevier Inc. All rights reserved.

4β-Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells.

PubMed

Tang, Jen-Yang; Huang, Hurng-Wern; Wang, Hui-Ru; Chan, Ya-Ching; Haung, Jo-Wen; Shu, Chih-Wen; Wu, Yang-Chang; Chang, Hsueh-Wei

2018-03-01

Reactive oxygen species (ROS) induction had been previously reported in 4β-hydroxywithanolide (4βHWE)-induced selective killing of oral cancer cells, but the mechanism involving ROS and the DNA damage effect remain unclear. This study explores the role of ROS and oxidative DNA damage of 4βHWE in the selective killing of oral cancer cells. Changes in cell viability, morphology, ROS, DNA double strand break (DSB) signaling (γH2AX foci in immunofluorescence and DSB signaling in western blotting), and oxidative DNA damage (8-oxo-2'deoxyguanosine [8-oxodG]) were detected in 4βHWE-treated oral cancer (Ca9-22) and/or normal (HGF-1) cells. 4βHWE decreased cell viability, changed cell morphology and induced ROS generation in oral cancer cells rather than oral normal cells, which were recovered by a free radical scavenger N-acetylcysteine (NAC). For immunofluorescence, 4βHWE also accumulated more of the DSB marker, γH2AX foci, in oral cancer cells than in oral normal cells. For western blotting, DSB signaling proteins such as γH2AX and MRN complex (MRE11, RAD50, and NBS1) were overexpressed in 4βHWE-treated oral cancer cells in different concentrations and treatment time. In the formamidopyrimidine-DNA glycolyase (Fpg)-based comet assay and 8-oxodG-based flow cytometry, the 8-oxodG expressions were higher in 4βHWE-treated oral cancer cells than in oral normal cells. All the 4βHWE-induced DSB and oxidative DNA damage to oral cancer cells were recovered by NAC pretreatment. Taken together, the 4βHWE selectively induced DSB and oxidative DNA damage for the ROS-mediated selective killing of oral cancer cells. © 2017 Wiley Periodicals, Inc.

Correlations between radiation-induced double strand breaks, cell division delay, and cyclin-dependent signaling in x-irradiated NIH3T3 fibroblasts

NASA Astrophysics Data System (ADS)

Cariveau, Mickael J.

2005-07-01

Molecular responses to radiation-induced DNA double strand breaks (DSB) are mediated by the phosphorylation of the histone variant H2AX which forms identifiable gamma-H2AX foci at the site of the DSB. This event is thought to be linked with the down-regulation of signaling proteins contributing to the checkpoints regulating cell cycle progression and, vis-a-vis , the induction of cell division delay. However, it is unclear whether this division delay is directly related to the number of DSB (gamma-H2AX foci) sustained by an irradiated cell and, if so, whether this number drives cells into cell cycle delay or apoptosis. For this reason, studies were conducted in the immortalized NIH/3T3 fibroblast cell in order to establish correlations between the temporal appearance of the gamma-H2AX foci (a DSB) and the expression of the cell cycle regulatory proteins, cyclin E, A, B1, and their cyclin kinase inhibitor, p21. Cell cycle kinetics and flow cytometry were used to establish radiation-induced division delay over a dose range of 1--6 Gy where a mitotic delay of 2.65 min/cGy was established. Correlations between the expression of cyclin E, A, B1, p21, and the generation of DSB were established in NIH/3T3 cells exposed to 2 or 4 Gy x-irradiation. The data suggest that the G1/S and S phase delay (cyclin E and cyclin A protein levels) are dependent on the dose of radiation while the G2/M (cyclin B1 protein levels) delay is dependent on the quantity of DSB sustained by the irradiated cell.

Genome Editing: A New Approach to Human Therapeutics.

PubMed

Porteus, Matthew

2016-01-01

The ability to manipulate the genome with precise spatial and nucleotide resolution (genome editing) has been a powerful research tool. In the past decade, the tools and expertise for using genome editing in human somatic cells and pluripotent cells have increased to such an extent that the approach is now being developed widely as a strategy to treat human disease. The fundamental process depends on creating a site-specific DNA double-strand break (DSB) in the genome and then allowing the cell's endogenous DSB repair machinery to fix the break such that precise nucleotide changes are made to the DNA sequence. With the development and discovery of several different nuclease platforms and increasing knowledge of the parameters affecting different genome editing outcomes, genome editing frequencies now reach therapeutic relevance for a wide variety of diseases. Moreover, there is a series of complementary approaches to assessing the safety and toxicity of any genome editing process, irrespective of the underlying nuclease used. Finally, the development of genome editing has raised the issue of whether it should be used to engineer the human germline. Although such an approach could clearly prevent the birth of people with devastating and destructive genetic diseases, questions remain about whether human society is morally responsible enough to use this tool.

RAD51 Is a Selective DNA Repair Target to Radiosensitize Glioma Stem Cells.

PubMed

King, Harry O; Brend, Tim; Payne, Helen L; Wright, Alexander; Ward, Thomas A; Patel, Karan; Egnuni, Teklu; Stead, Lucy F; Patel, Anjana; Wurdak, Heiko; Short, Susan C

2017-01-10

Patients with glioblastoma die from local relapse despite surgery and high-dose radiotherapy. Resistance to radiotherapy is thought to be due to efficient DNA double-strand break (DSB) repair in stem-like cells able to survive DNA damage and repopulate the tumor. We used clinical samples and patient-derived glioblastoma stem cells (GSCs) to confirm that the DSB repair protein RAD51 is highly expressed in GSCs, which are reliant on RAD51-dependent DSB repair after radiation. RAD51 expression and RAD51 foci numbers fall when these cells move toward astrocytic differentiation. In GSCs, the small-molecule RAD51 inhibitors RI-1 and B02 prevent RAD51 focus formation, reduce DNA DSB repair, and cause significant radiosensitization. We further demonstrate that treatment with these agents combined with radiation promotes loss of stem cells defined by SOX2 expression. This indicates that RAD51-dependent repair represents an effective and specific target in GSCs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Chromosome integrity at a double-strand break requires exonuclease 1 and MRX

PubMed Central

Nakai, Wataru; Westmoreland, Jim; Yeh, Elaine; Bloom, Kerry; Resnick, Michael A.

2010-01-01

The continuity of duplex DNA is generally considered a prerequisite for chromosome continuity. However, as previously shown in yeast as well as human cells, the introduction of a double-strand break (DSB) does not generate a chromosome break (CRB) in yeast or human cells. The transition from DSB to CRB was found to be under limited control by the tethering function of the RAD50/MRE11/XRS2 (MRX) complex. Using a system for differential fluorescent marking of both sides of an endonuclease-induced DSB in single cells, we found that nearly all DSBs are converted to CRBs in cells lacking both exonuclease 1 (EXO1) activity and MRX complex. Thus, it appears that some feature of exonuclease processing or resection at a DSB is critical for maintaining broken chromosome ends in close proximity. In addition, we discovered a thermal sensitive (cold) component to CRB formation in an MRX mutant that has implications for chromosome end mobility and/or end-processing. PMID:21115410

Heavy ion induced DNA-DSB in yeast and mammalian cells

NASA Technical Reports Server (NTRS)

Loebrich, M.; Ikpeme, S.; Kiefer, J.

1994-01-01

Molecular changes at the DNA are assumed to be the main cause for radiation effects in a number of organisms. During the course of the last decades techniques have been developed for measuring DNA double-strand breaks (dsb), generally assumed to be the most critical DNA lesions. The outcome of all those different approaches portrays a collection of data useful for a theoretical description of radiation action mechanisms. However, in the case of heavy ion induced DNA dsb the picture is not quite clear yet and further projects and strategies have to be developed. The biological systems studied in our group are yeast and mammalian cells. While in the case of yeast cells technical and methodical reasons highlight these organisms mammalian cells reach greater importance when dsb repair studies are performed. In both types of organisms the technique of pulsed-field gel electrophoresis (PFGE) is applied, although with different modifications and evaluation procedures mainly due to the different genome sizes.

Different genome maintenance strategies in human and tobacco cells.

PubMed

Pelczar, Pawel; Kalck, Véronique; Kovalchuk, Igor

2003-08-22

In this work, genome maintenance strategies of organisms belonging to different kingdoms (animals versus plants) but of similar genome size were investigated using a novel, universal double-strand break (DSB) repair assay. Different plasmids linearised with KpnI, Acc65I or EcoRV yielding either 3' or 5' protruding or blunt DNA termini, respectively, were transfected into HeLa cells and Nicotiana plumbaginifolia protoplasts and assayed for the efficiency and fidelity of DSB repair. We show that the mechanism of break sealing is similar but that drastic differences are seen in the fidelity of repair: in HeLa cells, 50-55% DSBs were repaired precisely, compared to as little as 15-30% in tobacco cells. Moreover, the DSB repair in plants resulted in 30-40% longer deletions and significantly shorter insertions. Combined, these led to more than twofold larger net DNA loss in tobacco cells. Our observations point to possible differences in the strategies of DSB repair and genome maintenance in plants and animals.

DNA-DSB in CHO-K1 cells induced by heavy-ions: Break rejoining and residual damage (GSI)

NASA Technical Reports Server (NTRS)

Taucher-Scholz, G.; Heilmann, J.; Becher, G.; Kraft, G.

1994-01-01

DNA double strand breaks (DSB's) are the critical lesions involved in cellular effects of ionizing radiation. Therefore, the evaluation of DSB induction in mammalian cells after heavy ion irradiation is an essential task for the assessment of high-LET radiation risk in space. Of particular interest has been the question of how the biological efficiency for the cellular inactivation endpoint relates to the initial lesions (DSBs) at varying LETs. For cell killing, an increased Relative Biological Efficiency (RBE) has been determined for highLET radiation around 100-200 keV/mu m. At higher LET, the RBE's decrease again to values below one for the very heavy particles. At GSI, DSB-induction was measured in CHO-K1 cells following irradiation with accelerated particles covering a wide LET range. The electrophoretic elution of fragmented DNA out of agarose plugs in a constant electrical field was applied for the detection of DSB's. The fraction of DNA retained was determined considering the relative intensities of ethidium bromide fluorescence in the well and in the gel lane. Dose-effect curves were established, from which the RBE for DSB induction was calculated at a fraction of 0.7 of DNA retained In summary, these rejoining studies are in line with an enhanced severity of the DNA DSB's at higher LET's, resulting in a decreased repairability of the induced lesions. However, no information concerning the fidelity of strand breaks rejoining is provided in these studies. To assess correct rejoining of DNA fragments an experimental system involving individual DNA hybridization bands has been set up. In preliminary experiments Sal I generated DNA fragments of 0.9 Mbp were irradiated with xrays and incubated for repair However, restitution of the original signals was not observed, probably due to the high radiation dose necessary for breakage of a fragment of this size. A banding pattern with NotI hybridization signals in a higher MW range (3Mbp) has been obtained by varying the electrophoretic conditions and correct rejoining studies will be further developed in this system.

SU-F-BRD-16: Relative Biological Effectiveness of Double-Strand Break Induction for Modeling Cell Survival in Pristine Proton Beams of Different Dose-Averaged Linear Energy Transfers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peeler, C; Bronk, L; UT Graduate School of Biomedical Sciences at Houston, Houston, TX

2015-06-15

Purpose: High throughput in vitro experiments assessing cell survival following proton radiation indicate that both the alpha and the beta parameters of the linear quadratic model increase with increasing proton linear energy transfer (LET). We investigated the relative biological effectiveness (RBE) of double-strand break (DSB) induction as a means of explaining the experimental results. Methods: Experiments were performed with two lung cancer cell lines and a range of proton LET values (0.94 – 19.4 keV/µm) using an experimental apparatus designed to irradiate cells in a 96 well plate such that each column encounters protons of different dose-averaged LET (LETd). Traditionalmore » linear quadratic survival curve fitting was performed, and alpha, beta, and RBE values obtained. Survival curves were also fit with a model incorporating RBE of DSB induction as the sole fit parameter. Fitted values of the RBE of DSB induction were then compared to values obtained using Monte Carlo Damage Simulation (MCDS) software and energy spectra calculated with Geant4. Other parameters including alpha, beta, and number of DSBs were compared to those obtained from traditional fitting. Results: Survival curve fitting with RBE of DSB induction yielded alpha and beta parameters that increase with proton LETd, which follows from the standard method of fitting; however, relying on a single fit parameter provided more consistent trends. The fitted values of RBE of DSB induction increased beyond what is predicted from MCDS data above proton LETd of approximately 10 keV/µm. Conclusion: In order to accurately model in vitro proton irradiation experiments performed with high throughput methods, the RBE of DSB induction must increase more rapidly than predicted by MCDS above LETd of 10 keV/µm. This can be explained by considering the increased complexity of DSBs or the nature of intra-track pairwise DSB interactions in this range of LETd values. NIH Grant 2U19CA021239-35.« less

Comprehensive track-structure based evaluation of DNA damage by light ions from radiotherapy-relevant energies down to stopping

PubMed Central

Friedland, W.; Schmitt, E.; Kundrát, P.; Dingfelder, M.; Baiocco, G.; Barbieri, S.; Ottolenghi, A.

2017-01-01

Track structures and resulting DNA damage in human cells have been simulated for hydrogen, helium, carbon, nitrogen, oxygen and neon ions with 0.25–256 MeV/u energy. The needed ion interaction cross sections have been scaled from those of hydrogen; Barkas scaling formula has been refined, extending its applicability down to about 10 keV/u, and validated against established stopping power data. Linear energy transfer (LET) has been scored from energy deposits in a cell nucleus; for very low-energy ions, it has been defined locally within thin slabs. The simulations show that protons and helium ions induce more DNA damage than heavier ions do at the same LET. With increasing LET, less DNA strand breaks are formed per unit dose, but due to their clustering the yields of double-strand breaks (DSB) increase, up to saturation around 300 keV/μm. Also individual DSB tend to cluster; DSB clusters peak around 500 keV/μm, while DSB multiplicities per cluster steadily increase with LET. Remarkably similar to patterns known from cell survival studies, LET-dependencies with pronounced maxima around 100–200 keV/μm occur on nanometre scale for sites that contain one or more DSB, and on micrometre scale for megabasepair-sized DNA fragments. PMID:28345622

APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair

PubMed Central

Nowarski, Roni; Wilner, Ofer I.; Cheshin, Ori; Shahar, Or D.; Kenig, Edan; Baraz, Leah; Britan-Rosich, Elena; Nagler, Arnon; Harris, Reuben S.; Goldberg, Michal; Willner, Itamar

2012-01-01

APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy. PMID:22645179

APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair.

PubMed

Nowarski, Roni; Wilner, Ofer I; Cheshin, Ori; Shahar, Or D; Kenig, Edan; Baraz, Leah; Britan-Rosich, Elena; Nagler, Arnon; Harris, Reuben S; Goldberg, Michal; Willner, Itamar; Kotler, Moshe

2012-07-12

APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy.

RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

PubMed

Manfrini, Nicola; Trovesi, Camilla; Wery, Maxime; Martina, Marina; Cesena, Daniele; Descrimes, Marc; Morillon, Antonin; d'Adda di Fagagna, Fabrizio; Longhese, Maria Pia

2015-02-01

Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability. © 2014 The Authors.

Relative biological effectiveness for photons: implication of complex DNA double-strand breaks as critical lesions

NASA Astrophysics Data System (ADS)

Liang, Ying; Fu, Qibin; Wang, Xudong; Liu, Feng; Yang, Gen; Luo, Chunxiong; Ouyang, Qi; Wang, Yugang

2017-03-01

Current knowledge in radiobiology ascribes the adverse biological effects of ionizing radiation primarily to the induction of DNA double-strand breaks (DSBs), which is supposed to be potentially lethal and may be converted to lethal damage due to misrepair. Soft and ultrasoft x-rays have been found to bear elevated biological effectiveness for cell killing compared with conventional x-rays or 60Co γ-rays. This phenomenon is qualitatively interpreted as the increased level of DSB induction for low energy photons, however, a thorough quantitative reasoning is lacking. Here, we systematically compared the relative biological effectiveness (RBE) with relative DSB induction for photons from several hundreds of eV up to MeV. Although there is an approximate two-fold increase in the yields of DSB for low energy photons found in our calculation and a large number of experimental measurements, it is far from enough to account for the three- to four-fold increase in RBE. Further theoretical investigations show that DSB complexity (additional single-strand breaks and base damage within 10 base pairs) increases notably for low energy photons, which largely reconciles the discrepancy between RBE and DSB induction. Our theoretical results are in line with accumulating experimental evidence that complex DSBs are refractory to repair machinery and may contribute predominantly to the formation of lethal damage.

Exploiting Tumor-Activated Testes Proteins to Enhance Efficacy of First-Line Chemotherapeutics in NSCLC

DTIC Science & Technology

2015-10-01

TERMS Cancer Testis Antigen (CTA), Fanconia- Anemia (FA), DNA Damage, Genomic Instability, DNA Double Strand Break (DSB) 16. SECURITY CLASSIFICATION OF...Cancer Testis Antigen (CTA) o Fanconia- Anemia (FA) o DNA Damage o Genomic Instability o DNA Double Strand Break (DSB) 3. Accomplishments • What

Exploiting Tumor Activated Testes Proteins To Enhance Efficacy of First Line Chemotherapeutics in NSCLC

DTIC Science & Technology

2016-10-01

Antigen (CTA), Fanconia- Anemia (FA), DNA Damage, Genomic Instability, DNA Double Strand Break (DSB) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION...Fanconia- Anemia (FA) o DNA Damage o Genomic Instability o DNA Double Strand Break (DSB) 3. Accomplishments • What were the major goals and objectives of

Hot-electron bolometer terahertz mixers for the Herschel Space Observatory.

PubMed

Cherednichenko, Sergey; Drakinskiy, Vladimir; Berg, Therese; Khosropanah, Pourya; Kollberg, Erik

2008-03-01

We report on low noise terahertz mixers (1.4-1.9 THz) developed for the heterodyne spectrometer onboard the Herschel Space Observatory. The mixers employ double slot antenna integrated superconducting hot-electron bolometers (HEBs) made of thin NbN films. The mixer performance was characterized in terms of detection sensitivity across the entire rf band by using a Fourier transform spectrometer (from 0.5 to 2.5 THz, with 30 GHz resolution) and also by measuring the mixer noise temperature at a limited number of discrete frequencies. The lowest mixer noise temperature recorded was 750 K [double sideband (DSB)] at 1.6 THz and 950 K DSB at 1.9 THz local oscillator (LO) frequencies. Averaged across the intermediate frequency band of 2.4-4.8 GHz, the mixer noise temperature was 1100 K DSB at 1.6 THz and 1450 K DSB at 1.9 THz LO frequencies. The HEB heterodyne receiver stability has been analyzed and compared to the HEB stability in the direct detection mode. The optimal local oscillator power was determined and found to be in a 200-500 nW range.

UVA irradiation of BrU-substituted DNA in the presence of Hoechst 33258.

PubMed

Saha, Abhijit; Kizaki, Seiichiro; Han, Ji Hoon; Yu, Zutao; Sugiyama, Hiroshi

2018-01-01

Given that our knowledge of DNA repair is limited because of the complexity of the DNA system, a technique called UVA micro-irradiation has been developed that can be used to visualize the recruitment of DNA repair proteins at double-strand break (DSB) sites. Interestingly, Hoechst 33258 was used under micro-irradiation to sensitize 5-bromouracil ( Br U)-labelled DNA, causing efficient DSBs. However, the molecular basis of DSB formation under UVA micro-irradiation remains unknown. Herein, we investigated the mechanism of DSB formation under UVA micro-irradiation conditions. Our results suggest that the generation of a uracil-5-yl radical through electron transfer from Hoechst 33258 to Br U caused DNA cleavage preferentially at self-complementary 5'-AA Br U Br U-3' sequences to induce DSB. We also investigated the DNA cleavage in the context of the nucleosome to gain a better understanding of UVA micro-irradiation in a cell-like model. We found that DNA cleavage occurred in both core and linker DNA regions although its efficiency reduced in core DNA. Copyright © 2017 Elsevier Ltd. All rights reserved.

A histone H3K36 chromatin switch coordinates DNA double-strand break repair pathway choice.

PubMed

Pai, Chen-Chun; Deegan, Rachel S; Subramanian, Lakxmi; Gal, Csenge; Sarkar, Sovan; Blaikley, Elizabeth J; Walker, Carol; Hulme, Lydia; Bernhard, Eric; Codlin, Sandra; Bähler, Jürg; Allshire, Robin; Whitehall, Simon; Humphrey, Timothy C

2014-06-09

DNA double-strand break (DSB) repair is a highly regulated process performed predominantly by non-homologous end joining (NHEJ) or homologous recombination (HR) pathways. How these pathways are coordinated in the context of chromatin is unclear. Here we uncover a role for histone H3K36 modification in regulating DSB repair pathway choice in fission yeast. We find Set2-dependent H3K36 methylation reduces chromatin accessibility, reduces resection and promotes NHEJ, while antagonistic Gcn5-dependent H3K36 acetylation increases chromatin accessibility, increases resection and promotes HR. Accordingly, loss of Set2 increases H3K36Ac, chromatin accessibility and resection, while Gcn5 loss results in the opposite phenotypes following DSB induction. Further, H3K36 modification is cell cycle regulated with Set2-dependent H3K36 methylation peaking in G1 when NHEJ occurs, while Gcn5-dependent H3K36 acetylation peaks in S/G2 when HR prevails. These findings support an H3K36 chromatin switch in regulating DSB repair pathway choice.

Mitosis, double strand break repair, and telomeres: a view from the end: how telomeres and the DNA damage response cooperate during mitosis to maintain genome stability.

PubMed

Cesare, Anthony J

2014-11-01

Double strand break (DSB) repair is suppressed during mitosis because RNF8 and downstream DNA damage response (DDR) factors, including 53BP1, do not localize to mitotic chromatin. Discovery of the mitotic kinase-dependent mechanism that inhibits DSB repair during cell division was recently reported. It was shown that restoring mitotic DSB repair was detrimental, resulting in repair dependent genome instability and covalent telomere fusions. The telomere DDR that occurs naturally during cellular aging and in cancer is known to be refractory to G2/M checkpoint activation. Such DDR-positive telomeres, and those that occur as part of the telomere-dependent prolonged mitotic arrest checkpoint, normally pass through mitosis without covalent ligation, but result in cell growth arrest in G1 phase. The discovery that suppressing DSB repair during mitosis may function primarily to protect DDR-positive telomeres from fusing during cell division reinforces the unique cooperation between telomeres and the DDR to mediate tumor suppression. © 2014 The Author. Bioessays published by WILEY Periodicals, Inc.

MOF phosphorylation by ATM regulates 53BP1-mediated DSB repair pathway choice

PubMed Central

Gupta, Arun; Hunt, Clayton R.; Hegdec, Muralidhar L.; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh1, Mayank; Ramnarain, Deepti B.; Hittelman, Walter N.; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K.; Ludwig, Thomas; Pandita, Raj K.; Tyler, Jessica K.; Pandita, Tej K.

2014-01-01

Cell cycle phase is a critical determinant of the choice between DNA damage repair by non-homologous end joining (NHEJ) or homologous recombination (HR). Here we report that DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF co-localizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S- and G2-phase but not G1-phase cells. Expression of MOF-T392A also reverses the reduction in DSB associated 53BP1 seen in wild type S/G2-phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair and decreased cell survival following irradiation. These data support a model whereby ATM mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2-phase. PMID:24953651

PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

PubMed Central

Wang, Minli; Wu, Weizhong; Wu, Wenqi; Rosidi, Bustanur; Zhang, Lihua; Wang, Huichen; Iliakis, George

2006-01-01

Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer. PMID:17088286

Positive regulation of meiotic DNA double-strand break formation by activation of the DNA damage checkpoint kinase Mec1(ATR).

PubMed

Gray, Stephen; Allison, Rachal M; Garcia, Valerie; Goldman, Alastair S H; Neale, Matthew J

2013-07-31

During meiosis, formation and repair of programmed DNA double-strand breaks (DSBs) create genetic exchange between homologous chromosomes-a process that is critical for reductional meiotic chromosome segregation and the production of genetically diverse sexually reproducing populations. Meiotic DSB formation is a complex process, requiring numerous proteins, of which Spo11 is the evolutionarily conserved catalytic subunit. Precisely how Spo11 and its accessory proteins function or are regulated is unclear. Here, we use Saccharomyces cerevisiae to reveal that meiotic DSB formation is modulated by the Mec1(ATR) branch of the DNA damage signalling cascade, promoting DSB formation when Spo11-mediated catalysis is compromised. Activation of the positive feedback pathway correlates with the formation of single-stranded DNA (ssDNA) recombination intermediates and activation of the downstream kinase, Mek1. We show that the requirement for checkpoint activation can be rescued by prolonging meiotic prophase by deleting the NDT80 transcription factor, and that even transient prophase arrest caused by Ndt80 depletion is sufficient to restore meiotic spore viability in checkpoint mutants. Our observations are unexpected given recent reports that the complementary kinase pathway Tel1(ATM) acts to inhibit DSB formation. We propose that such antagonistic regulation of DSB formation by Mec1 and Tel1 creates a regulatory mechanism, where the absolute frequency of DSBs is maintained at a level optimal for genetic exchange and efficient chromosome segregation.

Correlation of Meiotic DSB Formation and Transcription Initiation Around Fission Yeast Recombination Hotspots.

PubMed

Yamada, Shintaro; Okamura, Mika; Oda, Arisa; Murakami, Hiroshi; Ohta, Kunihiro; Yamada, Takatomi

2017-06-01

Meiotic homologous recombination, a critical event for ensuring faithful chromosome segregation and creating genetic diversity, is initiated by programmed DNA double-strand breaks (DSBs) formed at recombination hotspots. Meiotic DSB formation is likely to be influenced by other DNA-templated processes including transcription, but how DSB formation and transcription interact with each other has not been understood well. In this study, we used fission yeast to investigate a possible interplay of these two events. A group of hotspots in fission yeast are associated with sequences similar to the cyclic AMP response element and activated by the ATF/CREB family transcription factor dimer Atf1-Pcr1. We first focused on one of those hotspots, ade6-3049 , and Atf1. Our results showed that multiple transcripts, shorter than the ade6 full-length messenger RNA, emanate from a region surrounding the ade6-3049 hotspot. Interestingly, we found that the previously known recombination-activation region of Atf1 is also a transactivation domain, whose deletion affected DSB formation and short transcript production at ade6-3049 These results point to a possibility that the two events may be related to each other at ade6-3049 In fact, comparison of published maps of meiotic transcripts and hotspots suggested that hotspots are very often located close to meiotically transcribed regions. These observations therefore propose that meiotic DSB formation in fission yeast may be connected to transcription of surrounding regions. Copyright © 2017 by the Genetics Society of America.

DNA strand-exchange patterns associated with double-strand break-induced and spontaneous mitotic crossovers in Saccharomyces cerevisiae

PubMed Central

2018-01-01

Mitotic recombination can result in loss of heterozygosity and chromosomal rearrangements that shape genome structure and initiate human disease. Engineered double-strand breaks (DSBs) are a potent initiator of recombination, but whether spontaneous events initiate with the breakage of one or both DNA strands remains unclear. In the current study, a crossover (CO)-specific assay was used to compare heteroduplex DNA (hetDNA) profiles, which reflect strand exchange intermediates, associated with DSB-induced versus spontaneous events in yeast. Most DSB-induced CO products had the two-sided hetDNA predicted by the canonical DSB repair model, with a switch in hetDNA position from one product to the other at the position of the break. Approximately 40% of COs, however, had hetDNA on only one side of the initiating break. This anomaly can be explained by a modified model in which there is frequent processing of an early invasion (D-loop) intermediate prior to extension of the invading end. Finally, hetDNA tracts exhibited complexities consistent with frequent expansion of the DSB into a gap, migration of strand-exchange junctions, and template switching during gap-filling reactions. hetDNA patterns in spontaneous COs isolated in either a wild-type background or in a background with elevated levels of reactive oxygen species (tsa1Δ mutant) were similar to those associated with the DSB-induced events, suggesting that DSBs are the major instigator of spontaneous mitotic recombination in yeast. PMID:29579095

mre11S—a yeast mutation that blocks double-strand-break processing and permits nonhomologous synapsis in meiosis

PubMed Central

Nairz, Knud; Klein, Franz

1997-01-01

During meiotic prophase the repair of self-inflicted DNA double-strand break (DSB) damage leads to meiotic recombination in yeast. We employed a genetic screen to specifically characterize cellular functions that become essential after this DSB formation. As a result a new allele of MRE11, termed mre11S (for Separation of functions) was isolated that allows initiation but not processing and repair of meiotic DSBs similar to the well-characterized rad50S allele. In contrast, the mre11-1 allele blocks initiation of meiotic DSBs as reported previously by others. The mre11S allele, which is mutated in the 5′ part of the gene, can partially complement mre11 alleles disrupted close to the 3′ end that cannot initiate DSBs when homozygous. This suggests homodimerization of the Mre11 protein and the presence of separate domains for DSB initiation and 5′ resection. The fact that two genes, RAD50 and MRE11, required for DSB processing are also essential for DSB initiation dictates a model in which a bifunctional initiation/repair complex is required to initiate meiotic recombination. A subset of mre11S nuclei was shown to perform extensive but partially nonhomologous synapsis. We propose that the unprocessed DSBs present in mre11S allow for synapsis, but that homologous synapsis is only ensured at a later stage of recombination. PMID:9303542

Dynamic dependence on ATR and ATM for double-strand break repair in human embryonic stem cells and neural descendants.

PubMed

Adams, Bret R; Golding, Sarah E; Rao, Raj R; Valerie, Kristoffer

2010-04-02

The DNA double-strand break (DSB) is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR) is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ) is dominant. We have characterized the DNA damage response (DDR) and quality of DNA double-strand break (DSB) repair in human embryonic stem cells (hESCs), and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF) was used as a surrogate for DSB repair. The resolution of gamma-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs) and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR), showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ]) in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.

p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage.

PubMed

Gong, Lu; Gong, Hongjian; Pan, Xiao; Chang, Changqing; Ou, Zhao; Ye, Shengfan; Yin, Le; Yang, Lina; Tao, Ting; Zhang, Zhenhai; Liu, Cong; Lane, David P; Peng, Jinrong; Chen, Jun

2015-03-01

The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.

SU-F-T-128: Dose-Volume Constraints for Particle Therapy Treatment Planning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, R; Smith, W; Hendrickson, K

2016-06-15

Purpose: Determine equivalent Organ at Risk (OAR) tolerance dose (TD) constraints for MV x-rays and particle therapy. Methods: Equivalent TD estimates for MV x-rays are determined from an isoeffect, regression-analysis of published and in-house constraints for various fractionation schedules (n fractions). The analysis yields an estimate of (α/β) for an OAR. To determine equivalent particle therapy constraints, the MV x-ray TD(n) values are divided by the RBE for DSB induction (RBE{sub DSB}) or cell survival (RBE{sub S}). Estimates of (RBE{sub DSB}) are computed using the Monte Carlo Damage Simulation, and estimates of RBES are computed using the Repair-Misrepair-Fixation (RMF) model.more » A research build of the RayStation™ treatment planning system implementing the above model is used to estimate (RBE{sub DSB}) for OARs of interest in 16 proton therapy patient plans (head and neck, thorax, prostate and brain). Results: The analysis gives an (α/β) estimate of about 20 Gy for the trachea and heart and 2–4 Gy for the esophagus, spine, and brachial plexus. Extrapolation of MV x-ray constraints (n = 1) to fast neutrons using RBE{sub DSB} = 2.7 are in excellent agreement with clinical experience (n = 10 to 20). When conventional (n > 30) x-ray treatments are used as the reference radiation, fast neutron RBE increased to a maximum of 6. For comparison to a constant RBE of 1.1, the RayStation™ analysis gave estimates of proton RBE{sub DSB} from 1.03 to 1.33 for OARs of interest. Conclusion: The presented system of models is a convenient formalism to synthesize from multiple sources of information a set of self-consistent plan constraints for MV x-ray and hadron therapy treatments. Estimates of RBE{sub DSB} from the RayStation™ analysis differ substantially from 1.1 and vary among patients and treatment sites. A treatment planning system that incorporates patient and anatomy-specific corrections in proton RBE would create opportunities to increase the therapeutic ratio. The research build of the RayStation used in the study was made available to the University of Washington free of charge. RaySearch Laboratories did not provide any monetary support for the reported studies.« less

75 FR 55577 - Office of the Secretary: Defense Science Board (DSB) Task Force on the Survivability of DoD...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-13

... DEPARTMENT OF DEFENSE Office of the Secretary: Defense Science Board (DSB) Task Force on the Survivability of DoD Systems and Assets to Electromagnetic Pulse (EMP) and Other Nuclear Weapons Effects AGENCY... Systems and Assets to Electromagnetic Pulse (EMP) and other Nuclear Weapons Effects (hereafter referred to...

Estimating the number of double-strand breaks formed during meiosis from partial observation.

PubMed

Toyoizumi, Hiroshi; Tsubouchi, Hideo

2012-12-01

Analyzing the basic mechanism of DNA double-strand breaks (DSB) formation during meiosis is important for understanding sexual reproduction and genetic diversity. The location and amount of meiotic DSBs can be examined by using a common molecular biological technique called Southern blotting, but only a subset of the total DSBs can be observed; only DSB fragments still carrying the region recognized by a Southern blot probe are detected. With the assumption that DSB formation follows a nonhomogeneous Poisson process, we propose two estimators of the total number of DSBs on a chromosome: (1) an estimator based on the Nelson-Aalen estimator, and (2) an estimator based on a record value process. Further, we compared their asymptotic accuracy.

Meiotic Recombination in the Giraffe (G. reticulata).

PubMed

Vozdova, Miluse; Fröhlich, Jan; Kubickova, Svatava; Sebestova, Hana; Rubes, Jiri

2017-01-01

Recently, the reticulated giraffe (G. reticulata) was identified as a distinct species, which emphasized the need for intensive research in this interesting animal. To shed light on the meiotic process as a source of biodiversity, we analysed the frequency and distribution of meiotic recombination in 2 reticulated giraffe males. We used immunofluorescence detection of synaptonemal complex protein (SYCP3), meiotic double strand breaks (DSB, marked as RAD51 foci) in leptonema, and crossovers (COs, as MLH1 foci) in pachynema. The mean number of autosomal MLH1 foci per cell (27), which resulted from a single, distally located MLH1 focus observed on most chromosome arms, is one of the lowest among mammalian species analysed so far. The CO/DSB conversion ratio was 0.32. The pseudoautosomal region was localised in the Xq and Yp termini by FISH and showed an MLH1 focus in 83% of the pachytene cells. Chromatin structures corresponding to the nucleolus organiser regions were observed in the pachytene spermatocytes. The results are discussed in the context of known data on meiosis in Cetartiodactyla, depicting that the variation in CO frequency among species of this taxonomic group is mostly associated with their diploid chromosome number. © 2017 S. Karger AG, Basel.

Regulation of Phosphodiesterase 3 in the Pulmonary Arteries During the Perinatal Period in Sheep

PubMed Central

Chen, Bernadette; Lakshminrusimha, Satyan; Czech, Lyubov; Groh, Beezly S.; Gugino, Sylvia F.; Russell, James A.; Farrow, Kathryn N.; Steinhorn, Robin H.

2009-01-01

The role of cAMP in the pulmonary vasculature during the transition from intrauterine to extrauterine life is poorly understood. We hypothesized that cAMP levels are regulated by alterations in phosphodiesterase 3 (PDE3), which hydrolyzes cAMP. PDE3 protein expression and hydrolytic activity were increased in resistance pulmonary arteries (PA) from spontaneously breathing one-day-old (1dSB) lambs relative to equivalent-gestation fetuses. This was accompanied by a decrease in steady-state cAMP. Ventilation with 21% O2 and 100% O2 for 24h disrupted the normal transition, whereas ventilation with 100% O2+inhaled NO (iNO) for 24h restored both PDE3 activity and cAMP to 1dSB levels. Consistent with these findings, relaxation to milrinone, a PDE3 inhibitor, was greater in PA isolated from 1dSB and 100% O2+iNO lambs, relative to fetal, 21% O2, and 100% O2 lambs. In conclusion, PDE3 expression and activity in PA dramatically increase after birth, with a concomitant decrease in steady-state cAMP. Ventilation with either 21% O2 or 100% O2 blunts this PDE3 increase, whereas iNO restores PDE3 activity to levels equivalent to 1dSB lambs. The vasodilatory effects of milrinone were most pronounced in vessels from lambs with the highest PDE3 activity, i.e. 1dSB and 100% O2+iNO lambs. Thus, milrinone may be most beneficial when used in conjunction with iNO. PMID:19707176

Development, optimization, and in vitro characterization of dasatinib-loaded PEG functionalized chitosan capped gold nanoparticles using Box-Behnken experimental design.

PubMed

Adena, Sandeep Kumar Reddy; Upadhyay, Mansi; Vardhan, Harsh; Mishra, Brahmeshwar

2018-03-01

The purpose of this research study was to develop, optimize, and characterize dasatinib loaded polyethylene glycol (PEG) stabilized chitosan capped gold nanoparticles (DSB-PEG-Ch-GNPs). Gold (III) chloride hydrate was reduced with chitosan and the resulting nanoparticles were coated with thiol-terminated PEG and loaded with dasatinib (DSB). Plackett-Burman design (PBD) followed by Box-Behnken experimental design (BBD) were employed to optimize the process parameters. Polynomial equations, contour, and 3D response surface plots were generated to relate the factors and responses. The optimized DSB-PEG-Ch-GNPs were characterized by FTIR, XRD, HR-SEM, EDX, TEM, SAED, AFM, DLS, and ZP. The results of the optimized DSB-PEG-Ch-GNPs showed particle size (PS) of 24.39 ± 1.82 nm, apparent drug content (ADC) of 72.06 ± 0.86%, and zeta potential (ZP) of -13.91 ± 1.21 mV. The responses observed and the predicted values of the optimized process were found to be close. The shape and surface morphology studies showed that the resulting DSB-PEG-Ch-GNPs were spherical and smooth. The stability and in vitro drug release studies confirmed that the optimized formulation was stable at different conditions of storage and exhibited a sustained drug release of the drug of up to 76% in 48 h and followed Korsmeyer-Peppas release kinetic model. A process for preparing gold nanoparticles using chitosan, anchoring PEG to the particle surface, and entrapping dasatinib in the chitosan-PEG surface corona was optimized.

Chlamydomonas chloroplasts can use short dispersed repeats and multiple pathways to repair a double-strand break in the genome.

PubMed

Odom, Obed W; Baek, Kwang-Hyun; Dani, Radhika N; Herrin, David L

2008-03-01

Certain group I introns insert into intronless DNA via an endonuclease that creates a double-strand break (DSB). There are two models for intron homing in phage: synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR). The Cr.psbA4 intron homes efficiently from a plasmid into the chloroplast psbA gene in Chlamydomonas, but little is known about the mechanism. Analysis of co-transformants selected using a spectinomycin-resistant 16S gene (16S(spec)) provided evidence for both pathways. We also examined the consequences of the donor DNA having only one-sided or no homology with the psbA gene. When there was no homology with the donor DNA, deletions of up to 5 kb involving direct repeats that flank the psbA gene were obtained. Remarkably, repeats as short as 15 bp were used for this repair, which is consistent with the single-strand annealing (SSA) pathway. When the donor had one-sided homology, the DSB in most co-transformants was repaired using two DNAs, the donor and the 16S(spec) plasmid, which, coincidentally, contained a region that is repeated upstream of psbA. DSB repair using two separate DNAs provides further evidence for the SDSA pathway. These data show that the chloroplast can repair a DSB using short dispersed repeats located proximally, distally, or even on separate molecules relative to the DSB. They also provide a rationale for the extensive repertoire of repeated sequences in this genome.

Glucose starvation impairs DNA repair in tumour cells selectively by blocking histone acetylation.

PubMed

Ampferl, Rena; Rodemann, Hans Peter; Mayer, Claus; Höfling, Tobias Tim Alexander; Dittmann, Klaus

2018-03-01

Tumour cells are characterized by aerobic glycolysis and thus have high glucose consumption. Because repairing radiation-induced DNA damage is an energy-demanding process, we hypothesized that glucose starvation combined with radiotherapy could be an effective strategy to selectively target tumour cells. We glucose-starved tumour cells (A549, FaDu) in vitro and analysed their radiation-induced cell responses compared to normal fibroblasts (HSF7). Irradiation depleted intracellular ATP levels preferentially in cancer cells. Consequently, glucose starvation impaired DNA double-strand break (DSB) repair and radiosensitized confluent tumour cells but not normal fibroblasts. In proliferating tumour cells glucose starvation resulted in a reduction of proliferation, but failed to radiosensitize cells. Glucose supply was indispensable during the late DSB repair in confluent tumour cells starting approximately 13 h after irradiation, and glucose starvation inhibited radiation-induced histone acetylation, which is essential for chromatin relaxation. Sirtinol - an inhibitor of histone deacetylases - reverted the effects of glucose depletion on histone acetylation and DNA DSB repair in tumour cells. Furthermore, a glucose concentration of 2.8 mmol/L was sufficient to impair DSB repair in tumour cells and reduced their clonogenic survival under a fractionated irradiation regimen. In resting tumour cells, glucose starvation combined with irradiation resulted in the impairment of late DSB repair and the reduction of clonogenic survival, which was associated with disrupted radiation-induced histone acetylation. However, in normal cells, DNA repair and radiosensitivity were not affected by glucose depletion. Copyright © 2017 Elsevier B.V. All rights reserved.

Repair of DNA double-strand breaks and cell killing by charged particles

NASA Astrophysics Data System (ADS)

Eguchi-Kasai, K.; Murakami, M.; Itsukaichi, H.; Fukutsu, K.; Yatagai, F.; Kanai, T.; Ohara, H.; Sato, K.

It has been suggested that it is not simple double-strand breaks (dsb) but the non-reparable breaks which correlate well with the high biological effectiveness of high LET radiations for cell killing. We have compared the effects of charged particles on cell death in 3 pairs of cell lines which are normal or defective in the repair of DNA dsbs. For the cell lines SL3-147, M10, and SX10 which are deficient in DNA dsb repair, RBE values were close to unity for cell killing induced by charged particles with linear energy transfer (LET) up to 200 keV/mum and were even smaller than unity for the LET region greater than 300 keV/mum. The inactivation cross section (ICS) increased with LET for all 3 pairs. The ICS of dsb repair deficient mutants was always larger than that of their parents for all the LET ranges, but with increasing LET the difference in ICS between the mutant and its parent became smaller. Since a small difference in ICS remained at LET of about 300 keV/mum, dsb repair may still take place at this high LET, even if its role is apparently small. These results suggest that the DNA repair system does not play a major role in protection against the attack of high LET radiations and that a main cause of cell death is non-reparable dsb which are produced at a higher yield compared with low LET radiations. No correlation was observed between DNA content or nuclear area and ICS.

Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination

PubMed Central

Zan, Hong; Tat, Connie; Qiu, Zhifang; Taylor, Julia R.; Guerrero, Justin A.; Shen, Tian; Casali, Paolo

2017-01-01

Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S–S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52+/+ counterparts, which display normal CSR, Rad52−/− B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S–S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S–S recombination, as emphasized by the significantly greater CSR reduction in Rad52−/− versus Rad52+/+ B cells on Ku86 knockdown. PMID:28176781

I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.

PubMed

Fenina, Myriam; Simon-Chazottes, Dominique; Vandormael-Pournin, Sandrine; Soueid, Jihane; Langa, Francina; Cohen-Tannoudji, Michel; Bernard, Bruno A; Panthier, Jean-Jacques

2012-01-01

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.

Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair

PubMed Central

Morales, Julio C.; Richard, Patricia; Rommel, Amy; Fattah, Farjana J.; Motea, Edward A.; Patidar, Praveen L.; Xiao, Ling; Leskov, Konstantin; Wu, Shwu-Yuan; Hittelman, Walter N.; Chiang, Cheng-Ming; Manley, James L.; Boothman, David A.

2014-01-01

Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair. PMID:24589584

Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination.

PubMed

Zan, Hong; Tat, Connie; Qiu, Zhifang; Taylor, Julia R; Guerrero, Justin A; Shen, Tian; Casali, Paolo

2017-02-08

Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S-S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52 +/+ counterparts, which display normal CSR, Rad52 -/- B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S-S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S-S recombination, as emphasized by the significantly greater CSR reduction in Rad52 -/- versus Rad52 +/+ B cells on Ku86 knockdown.

SIRT1 collaborates with ATM and HDAC1 to maintain genomic stability in neurons

PubMed Central

Dobbin, Matthew M; Madabhushi, Ram; Pan, Ling; Chen, Yue; Kim, Dohoon; Gao, Jun; Ahononu, Biafra; Pao, Ping-Chieh; Qiu, Yi; Zhao, Yingming; Tsai, Li-Huei

2016-01-01

Summary Defects in DNA repair have been linked to cognitive decline with age and neurodegenerative disease. Yet the mechanisms that protect neurons from genotoxic stress remain largely obscure. In this report, we characterize the roles of the NAD+-dependent deacetylase, SIRT1, in the neuronal response to DNA double-strand breaks (DSBs). We show that SIRT1 is rapidly recruited to DSBs in postmitotic neurons, where it exhibits a synergistic relationship with ATM. SIRT1 recruitment to breaks is ATM-dependent; however, SIRT1 also stimulates ATM auto-phosphorylation and activity and stabilizes ATM at DSB sites. Upon DSB induction, SIRT1 also binds the neuroprotective class I histone deacetylase, HDAC1. We show that SIRT1 deacetylates HDAC1 and stimulates its enzymatic activity, which is necessary for DSB repair through the nonhomologous end-joining (NHEJ) pathway. HDAC1 mutants that mimic a constitutively acetylated state render neurons more susceptible to DNA damage, whereas pharmacological SIRT1 activators that promote HDAC1 deacetylation also reduce DNA damage in two mouse models of neurodegeneration. We propose that SIRT1 is an apical transducer of the DSB response and that SIRT1 activation offers an important therapeutic avenue in neurodegeneration. PMID:23852118

Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair.

PubMed

Morales, Julio C; Richard, Patricia; Rommel, Amy; Fattah, Farjana J; Motea, Edward A; Patidar, Praveen L; Xiao, Ling; Leskov, Konstantin; Wu, Shwu-Yuan; Hittelman, Walter N; Chiang, Cheng-Ming; Manley, James L; Boothman, David A

2014-04-01

Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.

Task Force on Defense Strategies for Ensuring the Resilience of National Space Capabilities. Executive Summary

DTIC Science & Technology

2017-03-21

March 2017 Task Force on Defense Strategies for Ensuring the Resilience of National Space Capabilities OFFICE OF THE UNDER SECRETARY OF...the Department of Defense. The DSB Task Force on Defense Strategies for Ensuring the Resilience of National Space Capabilities completed its formal...Ensuring the Resilience of National Space Capabilities | i DSB Task Force on Defense Strategies for Ensuring the

Cyber Defense Management

DTIC Science & Technology

2016-09-01

manage cyber security is often a verymanual and labor intensive process. When a crisis hits, DoD responses range from highly automatedand instrumented...DSB Task Force Report on Cyber Defense Management September 2016 (U) This page intentionally blank REPORT OF THE DEFENSE SCIENCE BOARD STUDY ON Cyber ...DEFENSE FOR ACQUISITION, TECHNOLOGY & LOGISTICS SUBJECT: Final Report of the Defense Science Board (DSB) Task Force on Cyber Defense Management I am

Progress towards understanding the nature of chromatid breakage.

PubMed

Bryant, P E; Gray, L J; Peresse, N

2004-01-01

The wide range of sensitivities of stimulated T-cells from different individuals to radiation-induced chromatid breakage indicates the involvement of several low penetrance genes that appear to link elevated chromatid breakage to cancer susceptibility. The mechanisms of chromatid breakage are not yet fully understood. However, evidence is accumulating that suggests chromatid breaks are not simply expanded DNA double-strand breaks (DSB). Three models of chromatid breakage are considered. The classical breakage-first and the Revell "exchange" models do not accord with current evidence. Therefore a derivative of Revell's model has been proposed whereby both spontaneous and radiation-induced chromatid breaks result from DSB signaling and rearrangement processes from within large looped chromatin domains. Examples of such rearrangements can be observed by harlequin staining whereby an exchange of strands occurs immediately adjacent to the break site. However, these interchromatid rearrangements comprise less than 20% of the total breaks. The rest are thought to result from intrachromatid rearrangements, including a very small proportion involving complete excision of a looped domain. Work is in progress with the aim of revealing these rearrangements, which may involve the formation of inversions adjacent to the break sites. It is postulated that the disappearance of chromatid breaks with time results from the completion of such rearrangements, rather than from the rejoining of DSB. Elevated frequencies of chromatid breaks occur in irradiated cells with defects in both nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways, however there is little evidence of a correlation between reduced DSB rejoining and disappearance of chromatid breaks. Moreover, at least one treatment which abrogates the disappearance of chromatid breaks with time leaves DSB rejoining unaffected. The I-SceI DSB system holds considerable promise for the elucidation of these mechanisms, although the break frequency is relatively low in the cell lines so far derived. Techniques to study and improve such systems are under way in different cell lines. Clearly, much remains to be done to clarify the mechanisms involved in chromatid breakage, but the experimental models are becoming available with which we can begin to answer some of the key questions. Copyright 2003 S. Karger AG, Basel

Rapid MCNP simulation of DNA double strand break (DSB) relative biological effectiveness (RBE) for photons, neutrons, and light ions.

PubMed

Stewart, Robert D; Streitmatter, Seth W; Argento, David C; Kirkby, Charles; Goorley, John T; Moffitt, Greg; Jevremovic, Tatjana; Sandison, George A

2015-11-07

To account for particle interactions in the extracellular (physical) environment, information from the cell-level Monte Carlo damage simulation (MCDS) for DNA double strand break (DSB) induction has been integrated into the general purpose Monte Carlo N-particle (MCNP) radiation transport code system. The effort to integrate these models is motivated by the need for a computationally efficient model to accurately predict particle relative biological effectiveness (RBE) in cell cultures and in vivo. To illustrate the approach and highlight the impact of the larger scale physical environment (e.g. establishing charged particle equilibrium), we examined the RBE for DSB induction (RBEDSB) of x-rays, (137)Cs γ-rays, neutrons and light ions relative to γ-rays from (60)Co in monolayer cell cultures at various depths in water. Under normoxic conditions, we found that (137)Cs γ-rays are about 1.7% more effective at creating DSB than γ-rays from (60)Co (RBEDSB  =  1.017) whereas 60-250 kV x-rays are 1.1 to 1.25 times more efficient at creating DSB than (60)Co. Under anoxic conditions, kV x-rays may have an RBEDSB up to 1.51 times as large as (60)Co γ-rays. Fission neutrons passing through monolayer cell cultures have an RBEDSB that ranges from 2.6 to 3.0 in normoxic cells, but may be as large as 9.93 for anoxic cells. For proton pencil beams, Monte Carlo simulations suggest an RBEDSB of about 1.2 at the tip of the Bragg peak and up to 1.6 a few mm beyond the Bragg peak. Bragg peak RBEDSB increases with decreasing oxygen concentration, which may create opportunities to apply proton dose painting to help address tumor hypoxia. Modeling of the particle RBE for DSB induction across multiple physical and biological scales has the potential to aid in the interpretation of laboratory experiments and provide useful information to advance the safety and effectiveness of hadron therapy in the treatment of cancer.

Targeting DNA double strand break repair with hyperthermia and DNA-PKcs inhibition to enhance the effect of radiation treatment.

PubMed

van Oorschot, Bregje; Granata, Giovanna; Di Franco, Simone; Ten Cate, Rosemarie; Rodermond, Hans M; Todaro, Matilde; Medema, Jan Paul; Franken, Nicolaas A P

2016-10-04

Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 μM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

Increased sensitivity to ionizing radiation by targeting the homologous recombination pathway in glioma initiating cells.

PubMed

Lim, Yi Chieh; Roberts, Tara L; Day, Bryan W; Stringer, Brett W; Kozlov, Sergei; Fazry, Shazrul; Bruce, Zara C; Ensbey, Kathleen S; Walker, David G; Boyd, Andrew W; Lavin, Martin F

2014-12-01

Glioblastoma is deemed the most malignant form of brain tumour, particularly due to its resistance to conventional treatments. A small surviving group of aberrant stem cells termed glioma initiation cells (GICs) that escape surgical debulking are suggested to be the cause of this resistance. Relatively quiescent in nature, GICs are capable of driving tumour recurrence and undergo lineage differentiation. Most importantly, these GICs are resistant to radiotherapy, suggesting that radioresistance contribute to their survival. In a previous study, we demonstrated that GICs had a restricted double strand break (DSB) repair pathway involving predominantly homologous recombination (HR) associated with a lack of functional G1/S checkpoint arrest. This unusual behaviour led to less efficient non-homologous end joining (NHEJ) repair and overall slower DNA DSB repair kinetics. To determine whether specific targeting of the HR pathway with small molecule inhibitors could increase GIC radiosensitivity, we used the Ataxia-telangiectasia mutated inhibitor (ATMi) to ablate HR and the DNA-dependent protein kinase inhibitor (DNA-PKi) to inhibit NHEJ. Pre-treatment with ATMi prior to ionizing radiation (IR) exposure prevented HR-mediated DNA DSB repair as measured by Rad51 foci accumulation. Increased cell death in vitro and improved in vivo animal survival could be observed with combined ATMi and IR treatment. Conversely, DNA-PKi treatment had minimal impact on GICs ability to resolve DNA DSB after IR with only partial reduction in cell survival, confirming the major role of HR. These results provide a mechanistic insight into the predominant form of DNA DSB repair in GICs, which when targeted may be a potential translational approach to increase patient survival. Copyright © 2014. Published by Elsevier B.V.

Low-dose ionizing irradiation triggers a 53BP1 response to DNA double strand breaks in mouse spermatogonial stem cells.

PubMed

Le, Wei; Qi, Lixin; Li, Jiaxuan; Wu, DengIong; Xu, Jun; Zhang, Jinfu

2016-01-01

The present study aims to examine the effect of low-dose ionizing irradiation on DNA double strand breaks (DSB) in mouse spermatogonial stem cells (SSCs) and reveal the underlying pathways for the DNA repair for DSB in SSCs. Eighteen one-month-old mice were divided into 6 groups and sacrificed separately at 45 minutes, 2 hours, 24 hours, 48 hours, and 72 hours after 0.1Gy X-ray irradiation (mice without receiving ionizing irradiation served as control). After perfusion fixation, testes were removed, sectioned, and followed by staining of γH2AX, 53BP1, Caspase 3, and promyelocytic leukemia zinc-finger (PLZF) for analysis among the different groups. The staining was observed by immunofluorescence visualized by confocal laser scanning. After low-dose irradiation, only 53BP1, but not Caspase3 or γH2AX was upregulated in PLZF positive SSCs within 45 minutes. The expression level of 53BP1 gradually decreased 24 hours after irradiation. Moreover, low-dose irradiation had no effect on the cell number and apoptotic status of SSCs. However other spermatogenic cells highly expressed γH2AX shortly after irradiation which was dramatically reduced following the events of DNA repair. It appears that low-dose ionizing irradiation may cause the DNA DSB of mouse spermatogenic cells. 53BP1, but not γH2AX, is involved in the DNA repair for DSB in SSCs. Our data indicates that 53BP1 plays an important role in the pathophysiological repair of DNA DSB in SSCs. This may open a new avenue to understanding the mechanisms of DNA repair of SSCs and male infertility.

The Thioredoxin Domain of Neisseria Gonorrhoeae PilB can use Electrons from DsbD to Reduce Downstream Methionine Sulfoxide Reductases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brot,N.; Collet, J.; Johnson, L.

2006-01-01

The PilB protein from Neisseria gonorrhoeae is located in the periplasm and made up of three domains. The N-terminal, thioredoxin-like domain (NT domain) is fused to tandem methionine sulfoxide reductase A and B domains (MsrA/B). We show that the {alpha} domain of Escherichia coli DsbD is able to reduce the oxidized NT domain, which suggests that DsbD in Neisseria can transfer electrons from the cytoplasmic thioredoxin to the periplasm for the reduction of the MsrA/B domains. An analysis of the available complete genomes provides further evidence for this proposition in other bacteria where DsbD/CcdA, Trx, MsrA, and MsrB gene homologsmore » are all located in a gene cluster with a common transcriptional direction. An examination of wild-type PilB and a panel of Cys to Ser mutants of the full-length protein and the individually expressed domains have also shown that the NT domain more efficiently reduces the MsrA/B domains when in the polyprotein context. Within this framework there does not appear to be a preference for the NT domain to reduce the proximal MsrA domain over MsrB domain. Finally, we report the 1.6 {angstrom} crystal structure of the NT domain. This structure confirms the presence of a surface loop that makes it different from other membrane-tethered, Trx-like molecules including TlpA, CcmG and ResA. Subtle differences are observed in this loop when compared to the N. meningitidis NT domain structure. The data taken together supports the formation of specific NT domain interactions with the MsrA/B domains and its in vivo recycling partner, DsbD.« less

Double strand break induction and kinetics indicate preserved hypersensitivity in keratinocytes to subtherapeutic doses for 7weeks of radiotherapy.

PubMed

Qvarnström, Fredrik; Simonsson, Martin; Nyman, Jan; Hermansson, Ingegerd; Book, Majlis; Johansson, Karl-Axel; Turesson, Ingela

2017-01-01

Previously we reported that hyper-radiosensitivity (HRS) was evidenced by quantifying DNA double strand break (DSB) foci in epidermis biopsies collected after delivering radiotherapeutic one and five dose fractions. The aim of this study was to determine whether HRS was preserved throughout a 7-week radiotherapy treatment, and also to examine the rate of foci decline and foci persistence between dose fractions. 42 patients with prostate cancer received 7-week fractionated radiotherapy treatment (RT) with daily dose fractions of 0.05-1.10Gy to the skin. Before RT, and at several times throughout treatment, skin biopsies (n=452) were collected at 30min, and 2, 3, 24, and 72h after dose fractions. DSB-foci markers, γH2AX and 53BP1, were labelled in epidermal keratinocytes with immunofluorescence and immunohistochemical staining. Foci were counted both with digital image analysis and manually. HRS in keratinocytes was evidenced by the dose-response relationships of DSB foci, observed throughout the treatment course, independent of sampling time and quantification method. Foci observed at 24h after dose fractions indicated considerable DSB persistence. Accordingly, foci significantly accumulated after 5 consecutive dose fractions. For doses below 0.3Gy, persistent foci could be observed even at 72h after damage induction. A comparison of γH2AX and 53BP1 quantifications in double-stained biopsies showed similar HRS dose-response relationships. These results represented the first evidence of preserved HRS, assessed by γH2AX- and 53BP1-labelled DSB foci, throughout a 7-week treatment course with daily repeated subtherapeutic dose fractions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Rapid DNA double-strand breaks resulting from processing of Cr-DNA cross-links by both MutS dimers.

PubMed

Reynolds, Mindy F; Peterson-Roth, Elizabeth C; Bespalov, Ivan A; Johnston, Tatiana; Gurel, Volkan M; Menard, Haley L; Zhitkovich, Anatoly

2009-02-01

Mismatch repair (MMR) strongly enhances cyto- and genotoxicity of several chemotherapeutic agents and environmental carcinogens. DNA double-strand breaks (DSB) formed after two replication cycles play a major role in MMR-dependent cell death by DNA alkylating drugs. Here, we examined DNA damage detection and the mechanisms of the unusually rapid induction of DSB by MMR proteins in response to carcinogenic chromium(VI). We found that MSH2-MSH6 (MutSalpha) dimer effectively bound DNA probes containing ascorbate-Cr-DNA and cysteine-Cr-DNA cross-links. Binary Cr-DNA adducts, the most abundant form of Cr-DNA damage, were poor substrates for MSH2-MSH6, and their toxicity in cells was weak and MMR independent. Although not involved in the initial recognition of Cr-DNA damage, MSH2-MSH3 (MutSbeta) complex was essential for the induction of DSB, micronuclei, and apoptosis in human cells by chromate. In situ fractionation of Cr-treated cells revealed MSH6 and MSH3 chromatin foci that originated in late S phase and did not require replication of damaged DNA. Formation of MSH3 foci was MSH6 and MLH1 dependent, whereas MSH6 foci were unaffected by MSH3 status. DSB production was associated with progression of cells from S into G(2) phase and was completely blocked by the DNA synthesis inhibitor aphidicolin. Interestingly, chromosome 3 transfer into MSH3-null HCT116 cells activated an alternative, MSH3-like activity that restored dinucleotide repeat stability and sensitivity to chromate. Thus, sequential recruitment and unprecedented cooperation of MutSalpha and MutSbeta branches of MMR in processing of Cr-DNA cross-links is the main cause of DSB and chromosomal breakage at low and moderate Cr(VI) doses.

The pathomechanism of cytochrome c oxidase deficiency includes nuclear DNA damage.

PubMed

Douiev, Liza; Saada, Ann

2018-06-07

Mitochondrial cytochrome c oxidase (COX, respiratory chain complex IV), contributes to ATP production via oxidative phosphorylation (OXPHOS). Clinical presentation of COX deficiency is heterogeneous ranging from mild to severe neuromuscular diseases. Anemia is among the symptoms and we have previously reported Fanconi anemia like features in COX4-1 deficiency, suggesting genomic instability and our preliminary results detected nuclear double stranded DNA breaks (DSB). We now quantified the DSB by phospho histone H2AX Ser139 staining of COX4-1 and COX6B1 deficient fibroblasts (225% and 215% of normal, respectively) and confirmed their occurrence by neutral comet assay. We further explored the mechanism of DNA damage by studying normal fibroblasts treated with micromolar concentrations of cyanide (KCN). Present results demonstrate elevated nuclear DSB in cells treated with 50 μM KCN for 24 h (170% of normal) in high-glucose medium conditions where ROS and ATP remain normal, although Glutathione content was partially decreased. In glucose-free and serum-free medium, where growth is hampered, DSB were not elevated. Additionally we demonstrate the benefit of nicotinamide riboside (NR) which ameliorated DSB in COX4-1, COX6B1 and KCN treated cells (130%, 154% and 87% of normal cells, respectively). Conversely a negative effect of a poly[ADP-ribose] polymerase (PARP) inhibitor was found. Although additional investigation is needed, our findings raise the possibility that the pathomechanism of COX deficiency and possibly also in other OXPHOS defects, include nuclear DNA damage resulting from nicotinamide adenine dinucleotide (NAD + ) deficit combined with a replicative state, rather than oxidative stress and energy depletion. Copyright © 2018 Elsevier B.V. All rights reserved.

The antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of Mycobacterium tuberculosis.

PubMed

Rosado, Leonardo Astolfi; Wahni, Khadija; Degiacomi, Giulia; Pedre, Brandán; Young, David; de la Rubia, Alfonso G; Boldrin, Francesca; Martens, Edo; Marcos-Pascual, Laura; Sancho-Vaello, Enea; Albesa-Jové, David; Provvedi, Roberta; Martin, Charlotte; Makarov, Vadim; Versées, Wim; Verniest, Guido; Guerin, Marcelo E; Mateos, Luis M; Manganelli, Riccardo; Messens, Joris

2017-08-11

The Mycobacterium tuberculosis rv2466c gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in M. tuberculosis have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under H 2 O 2 stress. Further, we discovered that Rv2466c lacks oxidase activity; rather, it receives electrons through the mycothiol/mycothione reductase/NADPH pathway to activate TP053, preferentially via a dithiol-disulfide mechanism. We also found that Rv2466c uses a monothiol-disulfide exchange mechanism to reduce S -mycothiolated mixed disulfides and intramolecular disulfides. Genetic, phylogenetic, bioinformatics, structural, and biochemical analyses revealed that Rv2466c is a novel mycothiol-dependent reductase, which represents a mycoredoxin cluster of enzymes within the DsbA family different from the glutaredoxin cluster to which mycoredoxin-1 (Mrx1 or Rv3198A) belongs. To validate this DsbA-mycoredoxin cluster, we also characterized a homologous enzyme of Corynebacterium glutamicum (NCgl2339) and observed that it demycothiolates and reduces a mycothiol arsenate adduct with kinetic properties different from those of Mrx1. In conclusion, our work has uncovered a DsbA-like mycoredoxin that promotes mycobacterial resistance to oxidative stress and reacts with free mycothiol and mycothiolated targets. The characterization of the DsbA-like mycoredoxin cluster reported here now paves the way for correctly classifying similar enzymes from other organisms. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

REC-1 and HIM-5 distribute meiotic crossovers and function redundantly in meiotic double-strand break formation in Caenorhabditis elegans

PubMed Central

Chung, George; Rose, Ann M.; Petalcorin, Mark I.R.; Martin, Julie S.; Kessler, Zebulin; Sanchez-Pulido, Luis; Ponting, Chris P.; Yanowitz, Judith L.; Boulton, Simon J.

2015-01-01

The Caenorhabditis elegans gene rec-1 was the first genetic locus identified in metazoa to affect the distribution of meiotic crossovers along the chromosome. We report that rec-1 encodes a distant paralog of HIM-5, which was discovered by whole-genome sequencing and confirmed by multiple genome-edited alleles. REC-1 is phosphorylated by cyclin-dependent kinase (CDK) in vitro, and mutation of the CDK consensus sites in REC-1 compromises meiotic crossover distribution in vivo. Unexpectedly, rec-1; him-5 double mutants are synthetic-lethal due to a defect in meiotic double-strand break formation. Thus, we uncovered an unexpected robustness to meiotic DSB formation and crossover positioning that is executed by HIM-5 and REC-1 and regulated by phosphorylation. PMID:26385965

In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites

PubMed Central

Grey, Corinne; Clément, Julie A.J.; Buard, Jérôme; Leblanc, Benjamin; Gut, Ivo; Gut, Marta; Duret, Laurent

2017-01-01

In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed an extensive analysis of PRDM9 binding in mouse spermatocytes. Unexpectedly, we identified a noncanonical recruitment of PRDM9 to sites that lack recombination activity and the PRDM9 binding consensus motif. These sites include gene promoters, where PRDM9 is recruited in a DSB-dependent manner. Another subset reveals DSB-independent interactions between PRDM9 and genomic sites, such as the binding sites for the insulator protein CTCF. We propose that these DSB-independent sites result from interactions between hotspot-bound PRDM9 and genomic sequences located on the chromosome axis. PMID:28336543

Chemical Warfare: Many Unanswered Questions.

DTIC Science & Technology

1983-04-29

of Defense DSB Defense Science Board GAO U.S. General Accounting Office IDA Institute for Defense Analyses JCS Joint Chiefs of Staff SIPRI Stockholm...Defense Science Board in 1980, DOD specifically asked it to review intelligence data on chemical warfare, and DSB’s 1981 report accordingly presents its...Defense Science Board report suggests, however, that its com- - ments are based on a composite of intelligence information on Soviet chemical warfare

Defense Science Board Task Force Report on Cyber Defense Management

DTIC Science & Technology

2016-09-01

manage cyber security is often a verymanual and labor intensive process. When a crisis hits, DoD responses range from highly automatedand instrumented...DSB Task Force Report on Cyber Defense Management September 2016 (U) This page intentionally blank REPORT OF THE DEFENSE SCIENCE BOARD STUDY ON Cyber ...DEFENSE FOR ACQUISITION, TECHNOLOGY & LOGISTICS SUBJECT: Final Report of the Defense Science Board (DSB) Task Force on Cyber Defense Management I am

The discovery of a class of novel HIV-1 maturation inhibitors and their potential in the therapy of HIV.

PubMed

Yu, Donglei; Wild, Carl T; Martin, David E; Morris-Natschke, Susan L; Chen, Chin-Ho; Allaway, Graham P; Lee, Kuo-Hsiung

2005-06-01

Although HIV infection is now primarily treated with reverse transcriptase and protease inhibitors, HIV therapy must look toward new drugs with novel mechanism(s) of action to both improve efficacy and address the growing problem of drug resistance. Using natural products as a source of biologically active compounds, our drug discovery program has successfully optimised the natural product betulinic acid to the first-in-class maturation inhibitor 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB). DSB's unique viral target has been identified as a late step in Gag processing. Specifically, it inhibits the cleavage of the capsid precursor, CA-SP1, resulting in a block to the processing of mature capsid protein leading to a defect in viral core condensation. DSB represents a unique class of anti-HIV compounds that inhibit virus maturation and provide additional opportunities for anti-HIV therapy. In this review, the discovery of DSB and its mode of action are summarised. Anti-AIDS Agents part 64. For part 63 in the series, see YU D, LEE KH: Recent progress and prospects on plant-derived anti-HIV agents and analogs. In: Medicinal Chemistry of Bioactive Natural Products. XT Liang, WS Fang (Eds), Wiley, New York, USA (2005) (In Press).

Nucleotide excision repair-dependent DNA double-strand break formation and ATM signaling activation in mammalian quiescent cells.

PubMed

Wakasugi, Mitsuo; Sasaki, Takuma; Matsumoto, Megumi; Nagaoka, Miyuki; Inoue, Keiko; Inobe, Manabu; Horibata, Katsuyoshi; Tanaka, Kiyoji; Matsunaga, Tsukasa

2014-10-10

Histone H2A variant H2AX is phosphorylated at Ser(139) in response to DNA double-strand break (DSB) and single-stranded DNA (ssDNA) formation. UV light dominantly induces pyrimidine photodimers, which are removed from the mammalian genome by nucleotide excision repair (NER). We previously reported that in quiescent G0 phase cells, UV induces ATR-mediated H2AX phosphorylation plausibly caused by persistent ssDNA gap intermediates during NER. In this study, we have found that DSB is also generated following UV irradiation in an NER-dependent manner and contributes to an earlier fraction of UV-induced H2AX phosphorylation. The NER-dependent DSB formation activates ATM kinase and triggers the accumulation of its downstream factors, MRE11, NBS1, and MDC1, at UV-damaged sites. Importantly, ATM-deficient cells exhibited enhanced UV sensitivity under quiescent conditions compared with asynchronously growing conditions. Finally, we show that the NER-dependent H2AX phosphorylation is also observed in murine peripheral T lymphocytes, typical nonproliferating quiescent cells in vivo. These results suggest that in vivo quiescent cells may suffer from NER-mediated secondary DNA damage including ssDNA and DSB. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Efficiencies of induction of DNA double strand breaks in solution by photoabsorption at phosphorus and platinum.

PubMed

Maeda, Munetoshi; Kobayashi, Katsumi; Hieda, Kotaro

2004-01-01

This paper aims at determining and comparing the cross sections and quantum yields for DNA strand break induction by the Auger effect at the K-shell of phosphorus and at the LIII-shell of platinum. Using synchrotron radiation, free and Pt-bound pBR322 plasmid DNA were irradiated in solution with monochromatic X-rays, the energies of which were 2.153 and 2.147 keV, corresponding to "on" and "below" the phosphorus K-shell photoabsorption, and 11.566 and 11.542 keV for "above" and "below" the L(III)-shell photoabsorption of platinum, respectively. To suppress indirect effects by hydroxyl radicals, DMSO (1M) was used as a scavenger. The inner-shell photoabsorption of phosphorus and of platinum significantly increased the induction of DNA double strand breaks (DSB), whereas it had little effect on single strand break (SSB) induction. The quantum yields for the induction of DSB were calculated to be 0.017 and 1.13, in the case of phosphorus and platinum, respectively. CONCLSIONS: The value of the quantum yield for the DSB induction of platinum was about 66-fold larger than that for the phosphorus. These results clearly demonstrate that the quantum yield of DSB depends upon the magnitude of the Auger cascade.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

Genomic features shaping the landscape of meiotic double-strand-break hotspots in maize.

PubMed

He, Yan; Wang, Minghui; Dukowic-Schulze, Stefanie; Zhou, Adele; Tiang, Choon-Lin; Shilo, Shay; Sidhu, Gaganpreet K; Eichten, Steven; Bradbury, Peter; Springer, Nathan M; Buckler, Edward S; Levy, Avraham A; Sun, Qi; Pillardy, Jaroslaw; Kianian, Penny M A; Kianian, Shahryar F; Chen, Changbin; Pawlowski, Wojciech P

2017-11-14

Meiotic recombination is the most important source of genetic variation in higher eukaryotes. It is initiated by formation of double-strand breaks (DSBs) in chromosomal DNA in early meiotic prophase. The DSBs are subsequently repaired, resulting in crossovers (COs) and noncrossovers (NCOs). Recombination events are not distributed evenly along chromosomes but cluster at recombination hotspots. How specific sites become hotspots is poorly understood. Studies in yeast and mammals linked initiation of meiotic recombination to active chromatin features present upstream from genes, such as absence of nucleosomes and presence of trimethylation of lysine 4 in histone H3 (H3K4me3). Core recombination components are conserved among eukaryotes, but it is unclear whether this conservation results in universal characteristics of recombination landscapes shared by a wide range of species. To address this question, we mapped meiotic DSBs in maize, a higher eukaryote with a large genome that is rich in repetitive DNA. We found DSBs in maize to be frequent in all chromosome regions, including sites lacking COs, such as centromeres and pericentromeric regions. Furthermore, most DSBs are formed in repetitive DNA, predominantly Gypsy retrotransposons, and only one-quarter of DSB hotspots are near genes. Genic and nongenic hotspots differ in several characteristics, and only genic DSBs contribute to crossover formation. Maize hotspots overlap regions of low nucleosome occupancy but show only limited association with H3K4me3 sites. Overall, maize DSB hotspots exhibit distribution patterns and characteristics not reported previously in other species. Understanding recombination patterns in maize will shed light on mechanisms affecting dynamics of the plant genome.

Nontoxic concentration of DNA-PK inhibitor NU7441 radio-sensitizes lung tumor cells with little effect on double strand break repair.

PubMed

Sunada, Shigeaki; Kanai, Hideki; Lee, Younghyun; Yasuda, Takeshi; Hirakawa, Hirokazu; Liu, Cuihua; Fujimori, Akira; Uesaka, Mitsuru; Okayasu, Ryuichi

2016-09-01

High-linear energy transfer (LET) heavy ions have been increasingly employed as a useful alternative to conventional photon radiotherapy. As recent studies suggested that high LET radiation mainly affects the nonhomologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair, we further investigated this concept by evaluating the combined effect of an NHEJ inhibitor (NU7441) at a non-toxic concentration and carbon ions. NU7441-treated non-small cell lung cancer (NSCLC) A549 and H1299 cells were irradiated with X-rays and carbon ions (290 MeV/n, 50 keV/μm). Cell survival was measured by clonogenic assay. DNA DSB repair, cell cycle distribution, DNA fragmentation and cellular senescence induction were studied using a flow cytometer. Senescence-associated protein p21 was detected by western blotting. In the present study, 0.3 μM of NU7441, nontoxic to both normal and tumor cells, caused a significant radio-sensitization in tumor cells exposed to X-rays and carbon ions. This concentration did not seem to cause inhibition of DNA DSB repair but induced a significant G2/M arrest, which was particularly emphasized in p53-null H1299 cells treated with NU7441 and carbon ions. In addition, the combined treatment induced more DNA fragmentation and a higher degree of senescence in H1299 cells than in A549 cells, indicating that DNA-PK inhibitor contributes to various modes of cell death in a p53-dependent manner. In summary, NSCLC cells irradiated with carbon ions were radio-sensitized by a low concentration of DNA-PK inhibitor NU7441 through a strong G2/M cell cycle arrest. Our findings may contribute to further effective radiotherapy using heavy ions. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Moraxella osloensis gene expression in the slug host Deroceras reticulatum.

PubMed

An, Ruisheng; Sreevatsan, Srinand; Grewal, Parwinder S

2008-01-28

The bacterium Moraxella osloensis is a mutualistic symbiont of the slug-parasitic nematode Phasmarhabditis hermaphrodita. In nature, P. hermaphrodita vectors M. osloensis into the shell cavity of the slug host Deroceras reticulatum in which the bacteria multiply and kill the slug. As M. osloensis is the main killing agent, genes expressed by M. osloensis in the slug are likely to play important roles in virulence. Studies on pathogenic interactions between bacteria and lower order hosts are few, but such studies have the potential to shed light on the evolution of bacterial virulence. Therefore, we investigated such an interaction by determining gene expression of M. osloensis in its slug host D. reticulatum by selectively capturing transcribed sequences. Thirteen M. osloensis genes were identified to be up-regulated post infection in D. reticulatum. Compared to the in vitro expressed genes in the stationary phase, we found that genes of ubiquinone synthetase (ubiS) and acyl-coA synthetase (acs) were up-regulated in both D. reticulatum and stationary phase in vitro cultures, but the remaining 11 genes were exclusively expressed in D. reticulatum and are hence infection specific. Mutational analysis on genes of protein-disulfide isomerase (dsbC) and ubiS showed that the virulence of both mutants to slugs was markedly reduced and could be complemented. Further, compared to the growth rate of wild-type M. osloensis, the dsbC and ubiS mutants showed normal and reduced growth rate in vitro, respectively. We conclude that 11 out of the 13 up-regulated M. osloensis genes are infection specific. Distribution of these identified genes in various bacterial pathogens indicates that the virulence genes are conserved among different pathogen-host interactions. Mutagenesis, growth rate and virulence bioassays further confirmed that ubiS and dsbC genes play important roles in M. osloensis survival and virulence, respectively in D. reticulatum.

Moraxella osloensis Gene Expression in the Slug Host Deroceras reticulatum

PubMed Central

An, Ruisheng; Sreevatsan, Srinand; Grewal, Parwinder S

2008-01-01

Background The bacterium Moraxella osloensis is a mutualistic symbiont of the slug-parasitic nematode Phasmarhabditis hermaphrodita. In nature, P. hermaphrodita vectors M. osloensis into the shell cavity of the slug host Deroceras reticulatum in which the bacteria multiply and kill the slug. As M. osloensis is the main killing agent, genes expressed by M. osloensis in the slug are likely to play important roles in virulence. Studies on pathogenic interactions between bacteria and lower order hosts are few, but such studies have the potential to shed light on the evolution of bacterial virulence. Therefore, we investigated such an interaction by determining gene expression of M. osloensis in its slug host D. reticulatum by selectively capturing transcribed sequences. Results Thirteen M. osloensis genes were identified to be up-regulated post infection in D. reticulatum. Compared to the in vitro expressed genes in the stationary phase, we found that genes of ubiquinone synthetase (ubiS) and acyl-coA synthetase (acs) were up-regulated in both D. reticulatum and stationary phase in vitro cultures, but the remaining 11 genes were exclusively expressed in D. reticulatum and are hence infection specific. Mutational analysis on genes of protein-disulfide isomerase (dsbC) and ubiS showed that the virulence of both mutants to slugs was markedly reduced and could be complemented. Further, compared to the growth rate of wild-type M. osloensis, the dsbC and ubiS mutants showed normal and reduced growth rate in vitro, respectively. Conclusion We conclude that 11 out of the 13 up-regulated M. osloensis genes are infection specific. Distribution of these identified genes in various bacterial pathogens indicates that the virulence genes are conserved among different pathogen-host interactions. Mutagenesis, growth rate and virulence bioassays further confirmed that ubiS and dsbC genes play important roles in M. osloensis survival and virulence, respectively in D. reticulatum. PMID:18226222

Excited state absorption spectra of dissolved and aggregated distyrylbenzene: A TD-DFT state and vibronic analysis

NASA Astrophysics Data System (ADS)

Oliveira, Eliezer Fernando; Shi, Junqing; Lavarda, Francisco Carlos; Lüer, Larry; Milián-Medina, Begoña; Gierschner, Johannes

2017-07-01

A time-dependent density functional theory study is performed to reveal the excited state absorption (ESA) features of distyrylbenzene (DSB), a prototype π-conjugated organic oligomer. Starting with a didactic insight to ESA based on simple molecular orbital and configuration considerations, the performance of various density functional theory functionals is tested to reveal the full vibronic ESA features of DSB at short and long probe delay times.

Advanced Technology Direction and Control Communications Systems

DTIC Science & Technology

1979-07-16

Leupert amesD./Morrell ~I~ Frederick . euprJae DCPAaol-78-C-1 259] ,. IERFORMlk6 ORGANIZATION NAME AND ADDRESS %a. P ROGRAM ELEMiENT. PROJIECT. TASK AREA...without the change in communicatios LCciniLlucs b•;i,,g aipiarci-IL. A JTIDS net is organized on the principle of time division. Various elements are...sideband (DSB-AI) "* Amplitude modulation, vestigial sideband (VSB-AM) * Frequency sI Shift modulation (FSK) "* Phase shift modulation (PSK) Ti DSB

DNA-dependent protein kinase in nonhomologous end joining: a lock with multiple keys?

PubMed

Weterings, Eric; Chen, David J

2007-10-22

The DNA-dependent protein kinase (DNA-PK) is one of the central enzymes involved in DNA double-strand break (DSB) repair. It facilitates proper alignment of the two ends of the broken DNA molecule and coordinates access of other factors to the repair complex. We discuss the latest findings on DNA-PK phosphorylation and offer a working model for the regulation of DNA-PK during DSB repair.

Screening of Pesticides with the Potential of Inducing DSB and Successive Recombinational Repair

PubMed Central

2017-01-01

A study was realized to ascertain whether eight selected pesticides would induce double strand breaks (DSB) in lymphocyte cultures and whether this damage would induce greater levels of proteins Rad51 participating in homologous recombination or of p-Ku80 participating in nonhomologous end joining. Only five pesticides were found to induce DSB of which only glyphosate and paraoxon induced a significant increase of p-Ku80 protein, indicating that nonhomologous end joining recombinational DNA repair system would be activated. The type of gamma-H2AX foci observed was comparable to that induced by etoposide at similar concentrations. These results are of importance since these effects occurred at low concentrations in the micromolar range, in acute treatments to the cells. Effects over longer exposures in actual environmental settings are expected to produce cumulative damage if repeated events of recombination take place over time. PMID:29129974

SPOP mutation leads to genomic instability in prostate cancer

PubMed Central

Boysen, Gunther; Barbieri, Christopher E; Prandi, Davide; Blattner, Mirjam; Chae, Sung-Suk; Dahija, Arun; Nataraj, Srilakshmi; Huang, Dennis; Marotz, Clarisse; Xu, Limei; Huang, Julie; Lecca, Paola; Chhangawala, Sagar; Liu, Deli; Zhou, Pengbo; Sboner, Andrea; de Bono, Johann S

2015-01-01

Genomic instability is a fundamental feature of human cancer often resulting from impaired genome maintenance. In prostate cancer, structural genomic rearrangements are a common mechanism driving tumorigenesis. However, somatic alterations predisposing to chromosomal rearrangements in prostate cancer remain largely undefined. Here, we show that SPOP, the most commonly mutated gene in primary prostate cancer modulates DNA double strand break (DSB) repair, and that SPOP mutation is associated with genomic instability. In vivo, SPOP mutation results in a transcriptional response consistent with BRCA1 inactivation resulting in impaired homology-directed repair (HDR) of DSB. Furthermore, we found that SPOP mutation sensitizes to DNA damaging therapeutic agents such as PARP inhibitors. These results implicate SPOP as a novel participant in DSB repair, suggest that SPOP mutation drives prostate tumorigenesis in part through genomic instability, and indicate that mutant SPOP may increase response to DNA-damaging therapeutics. DOI: http://dx.doi.org/10.7554/eLife.09207.001 PMID:26374986

New and old ways to control meiotic recombination.

PubMed

Phadnis, Naina; Hyppa, Randy W; Smith, Gerald R

2011-10-01

The unique segregation of homologs, rather than sister chromatids, at the first meiotic division requires the formation of crossovers (COs) between homologs by meiotic recombination in most species. Crossovers do not form at random along chromosomes. Rather, their formation is carefully controlled, both at the stage of formation of DNA double-strand breaks (DSBs) that can initiate COs and during the repair of these DSBs. Here, we review control of DSB formation and two recently recognized controls of DSB repair: CO homeostasis and CO invariance. Crossover homeostasis maintains a constant number of COs per cell when the total number of DSBs in a cell is experimentally or stochastically reduced. Crossover invariance maintains a constant CO density (COs per kb of DNA) across much of the genome despite strong DSB hotspots in some intervals. These recently uncovered phenomena show that CO control is even more complex than previously suspected. Copyright © 2011 Elsevier Ltd. All rights reserved.

Wavelength dependence of biological damage induced by UV radiation on bacteria.

PubMed

Santos, Ana L; Oliveira, Vanessa; Baptista, Inês; Henriques, Isabel; Gomes, Newton C M; Almeida, Adelaide; Correia, António; Cunha, Ângela

2013-01-01

The biological effects of UV radiation of different wavelengths (UVA, UVB and UVC) were assessed in nine bacterial isolates displaying different UV sensitivities. Biological effects (survival and activity) and molecular markers of oxidative stress [DNA strand breakage (DSB), generation of reactive oxygen species (ROS), oxidative damage to proteins and lipids, and the activity of antioxidant enzymes catalase and superoxide dismutase] were quantified and statistically analyzed in order to identify the major determinants of cell inactivation under the different spectral regions. Survival and activity followed a clear wavelength dependence, being highest under UVA and lowest under UVC. The generation of ROS, as well as protein and lipid oxidation, followed the same pattern. DNA damage (DSB) showed the inverse trend. Multiple stepwise regression analysis revealed that survival under UVA, UVB and UVC wavelengths was best explained by DSB, oxidative damage to lipids, and intracellular ROS levels, respectively.

Homeostatic regulation of meiotic DSB formation by ATM/ATR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cooper, Tim J.; Wardell, Kayleigh; Garcia, Valerie

2014-11-15

Ataxia–telangiectasia mutated (ATM) and RAD3-related (ATR) are widely known as being central players in the mitotic DNA damage response (DDR), mounting responses to DNA double-strand breaks (DSBs) and single-stranded DNA (ssDNA) respectively. The DDR signalling cascade couples cell cycle control to damage-sensing and repair processes in order to prevent untimely cell cycle progression while damage still persists [1]. Both ATM/ATR are, however, also emerging as essential factors in the process of meiosis; a specialised cell cycle programme responsible for the formation of haploid gametes via two sequential nuclear divisions. Central to achieving accurate meiotic chromosome segregation is the introduction ofmore » numerous DSBs spread across the genome by the evolutionarily conserved enzyme, Spo11. This review seeks to explore and address how cells utilise ATM/ATR pathways to regulate Spo11-DSB formation, establish DSB homeostasis and ensure meiosis is completed unperturbed.« less

BioSentinel: Mission Development of a Radiation Biosensor to Gauge DNA Damage and Repair Beyond Low Earth Orbit on a 6U Nanosatellite

NASA Technical Reports Server (NTRS)

Sanchez, Hugo; Lewis, Brian; Hanel, Robert

2015-01-01

We are designing and developing a 6U (10 x 22 x 34 cm; 14 kg) nanosatellite as a secondary payload to fly aboard NASAs Space Launch System (SLS) Exploration Mission (EM) 1, scheduled for launch in late 2017. For the first time in over forty years, direct experimental data from biological studies beyond low Earth orbit (LEO) will be obtained during BioSentinels 12- to 18-month mission. BioSentinel will measure the damage and repair of DNA in a biological organism and allow us to compare that to information from onboard physical radiation sensors. In order to understand the relative contributions of the space environments two dominant biological perturbations, reduced gravity and ionizing radiation, results from deep space will be directly compared to data obtained in LEO (on ISS) and on Earth. These data points will be available for validation of existing biological radiation damage and repair models, and for extrapolation to humans, to assist in mitigating risks during future long-term exploration missions beyond LEO. The BioSentinel Payload occupies 4U of the spacecraft and will utilize the monocellular eukaryotic organism Saccharomyces cerevisiae (yeast) to report DNA double-strand-break (DSB) events that result from ambient space radiation. DSB repair exhibits striking conservation of repair proteins from yeast to humans. Yeast was selected because of 1) its similarity to cells in higher organisms, 2) the well-established history of strains engineered to measure DSB repair, 3) its spaceflight heritage, and 4) the wealth of available ground and flight reference data. The S. cerevisiae flight strain will include engineered genetic defects to prevent growth and division until a radiation-induced DSB activates the yeasts DNA repair mechanisms. The triggered culture growth and metabolic activity directly indicate a DSB and its successful repair. The yeast will be carried in the dry state within the 1-atm PL container in 18 separate fluidics cards with each card having 16 independent culture microwells, with integral microchannels and filters to supply nutrients and reagents, confine the yeast to the wells, and enable optical measurement. The measurement subsystem will monitor each subgroup of culture wells continuously for several weeks, optically tracking DSB-triggered cell growth and metabolism. BioSentinel will also include physical radiation sensors based on the TimePix sensor, as implemented by JSCs RadWorks group, which record individual radiation events including estimates of their linear-energy-transfer (LET) values. Radiation-dose and LET data will be compared directly to the rate of DSB-and-repair events measured by the S. cerevisiae biosentinels.

Differential antimutagenicity of WR-1065 added after irradiation in L5178Y cell lines

NASA Technical Reports Server (NTRS)

Evans, H. H.; Horng, M. F.; Ricanati, M.; McCoy, E. C.

1999-01-01

The purpose of this study was to determine the antimutagenicity of WR-1065 added after irradiation of cells of cell lines differing in their ability to rejoin radiation-induced DNA double-strand breaks (DSBs). The postirradiation antimutagenicity of WR-1065 at the thymidine kinase locus was demonstrated for L5178Y (LY)-S1 cells that are deficient in repair of DNA DSBs. Less postirradiation antimutagenicity of WR-1065 was observed in LY-R16 and LY-SR1 cells, which are relatively efficient in DSB repair. Postirradiation treatment with WR-1065 had only a small stimulatory effect on DSB rejoining. A 3-h incubation of irradiated LY cells with WR-1065 caused slight changes in the distribution of cells in the phases of the cell cycle that differed between LY-S1 and LY-SR1 cells. Both LY-S1 and LY-SR1 cells were protected against the cytotoxic and mutagenic effects of radiation when WR-1065 was present 30 min before and during the irradiation. We conclude that the differential postirradiation effects of WR-1065 in the LY-S1 and LY-SR1 cells are not caused by differences in cellular uptake of the radioprotector or in its radical scavenging activity. Possible mechanisms for the postirradiation antimutagenicity of WR-1065 are discussed.

Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koike, Manabu, E-mail: m_koike@nirs.go.jp; Yutoku, Yasutomo; Graduate School of Science, Chiba University, Chiba 263-8522

2011-08-19

Highlights: {yields} p21 accumulated rapidly at laser-irradiated sites via its C-terminal region. {yields} p21 colocalized with the DSB marker {gamma}-H2AX and the DSB sensor Ku80. {yields} Accumulation of p21 is dependent on PCNA, but not p53 and the NHEJ core factors. {yields} Accumulation activity of p21 was conserved among human and animal cells. {yields} p21 is a useful tool as a detection marker of DNA damaged sites. -- Abstract: The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 focimore » arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker {gamma}-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1-146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful tool as a detection marker of DNA damaged sites.« less

DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts.

PubMed

Ahmed, Emad A; Vélaz, Eukene; Rosemann, Michael; Gilbertz, Klaus-P; Scherthan, Harry

2017-03-01

Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G 1 -like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc scid ) and wild-type Sertoli cells. In nonirradiated mice and Prkdc scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc scid . Applying the same dose of IR to Prdkc -/- and Ku -/- mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc -/- cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku -/- MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ.

REC-1 and HIM-5 distribute meiotic crossovers and function redundantly in meiotic double-strand break formation in Caenorhabditis elegans.

PubMed

Chung, George; Rose, Ann M; Petalcorin, Mark I R; Martin, Julie S; Kessler, Zebulin; Sanchez-Pulido, Luis; Ponting, Chris P; Yanowitz, Judith L; Boulton, Simon J

2015-09-15

The Caenorhabditis elegans gene rec-1 was the first genetic locus identified in metazoa to affect the distribution of meiotic crossovers along the chromosome. We report that rec-1 encodes a distant paralog of HIM-5, which was discovered by whole-genome sequencing and confirmed by multiple genome-edited alleles. REC-1 is phosphorylated by cyclin-dependent kinase (CDK) in vitro, and mutation of the CDK consensus sites in REC-1 compromises meiotic crossover distribution in vivo. Unexpectedly, rec-1; him-5 double mutants are synthetic-lethal due to a defect in meiotic double-strand break formation. Thus, we uncovered an unexpected robustness to meiotic DSB formation and crossover positioning that is executed by HIM-5 and REC-1 and regulated by phosphorylation. © 2015 Chung et al.; Published by Cold Spring Harbor Laboratory Press.

Cluster pattern analysis of energy deposition sites for the brachytherapy sources 103Pd, 125I, 192Ir, 137Cs, and 60Co.

PubMed

Villegas, Fernanda; Tilly, Nina; Bäckström, Gloria; Ahnesjö, Anders

2014-09-21

Analysing the pattern of energy depositions may help elucidate differences in the severity of radiation-induced DNA strand breakage for different radiation qualities. It is often claimed that energy deposition (ED) sites from photon radiation form a uniform random pattern, but there is indication of differences in RBE values among different photon sources used in brachytherapy. The aim of this work is to analyse the spatial patterns of EDs from 103Pd, 125I, 192Ir, 137Cs sources commonly used in brachytherapy and a 60Co source as a reference radiation. The results suggest that there is both a non-uniform and a uniform random component to the frequency distribution of distances to the nearest neighbour ED. The closest neighbouring EDs show high spatial correlation for all investigated radiation qualities, whilst the uniform random component dominates for neighbours with longer distances for the three higher mean photon energy sources (192Ir, 137Cs, and 60Co). The two lower energy photon emitters (103Pd and 125I) present a very small uniform random component. The ratio of frequencies of clusters with respect to 60Co differs up to 15% for the lower energy sources and less than 2% for the higher energy sources when the maximum distance between each pair of EDs is 2 nm. At distances relevant to DNA damage, cluster patterns can be differentiated between the lower and higher energy sources. This may be part of the explanation to the reported difference in RBE values with initial DSB yields as an endpoint for these brachytherapy sources.

Cluster pattern analysis of energy deposition sites for the brachytherapy sources 103Pd, 125I, 192Ir, 137Cs, and 60Co

NASA Astrophysics Data System (ADS)

Villegas, Fernanda; Tilly, Nina; Bäckström, Gloria; Ahnesjö, Anders

2014-09-01

Analysing the pattern of energy depositions may help elucidate differences in the severity of radiation-induced DNA strand breakage for different radiation qualities. It is often claimed that energy deposition (ED) sites from photon radiation form a uniform random pattern, but there is indication of differences in RBE values among different photon sources used in brachytherapy. The aim of this work is to analyse the spatial patterns of EDs from 103Pd, 125I, 192Ir, 137Cs sources commonly used in brachytherapy and a 60Co source as a reference radiation. The results suggest that there is both a non-uniform and a uniform random component to the frequency distribution of distances to the nearest neighbour ED. The closest neighbouring EDs show high spatial correlation for all investigated radiation qualities, whilst the uniform random component dominates for neighbours with longer distances for the three higher mean photon energy sources (192Ir, 137Cs, and 60Co). The two lower energy photon emitters (103Pd and 125I) present a very small uniform random component. The ratio of frequencies of clusters with respect to 60Co differs up to 15% for the lower energy sources and less than 2% for the higher energy sources when the maximum distance between each pair of EDs is 2 nm. At distances relevant to DNA damage, cluster patterns can be differentiated between the lower and higher energy sources. This may be part of the explanation to the reported difference in RBE values with initial DSB yields as an endpoint for these brachytherapy sources.

Defense Science Board Task Force Report on Defense Strategies for Ensuring the Resilience of National Space Capabilities

DTIC Science & Technology

2017-03-01

Executive Summary is a product of the Defense Science Board (DSB). The DSB is a Federal Advisory Committee established to provide independent advice to the...Executive Summary Since 2000, when the Defense Science Board concluded space superiority was absolutely essential in achieving global awareness, information...control, critical enabling doctrine, and policy concepts. From April 2015 through September 2016, the Task Force received more than 40 briefings from

3D Visualization of Monte-Carlo Simulation's of HZE Track Structure and Initial Chemical Species

NASA Technical Reports Server (NTRS)

Plante, Ianik; Cucinotta, Francis A.

2009-01-01

Heavy ions biophysics is important for space radiation risk assessment [1] and hadron-therapy [2]. The characteristic of heavy ions tracks include a very high energy deposition region close to the track (<20 nm) denoted as the track core, and an outer penumbra region consisting of individual secondary electrons (6-rays). A still open question is the radiobiological effects of 6- rays relative to the track core. Of importance is the induction of double-strand breaks (DSB) [3] and oxidative damage to the biomolecules and the tissue matrix, considered the most important lesions for acute and long term effects of radiation. In this work, we have simulated a 56Fe26+ ion track of 1 GeV/amu with our Monte-Carlo code RITRACKS [4]. The simulation results have been used to calculate the energy depiction and initial chemical species in a "voxelized" space, which is then visualized in 3D. Several voxels with dose >1000 Gy are found in the penumbra, some located 0.1 mm from the track core. In computational models, the DSB induction probability is calculated with radial dose [6], which may not take into account the higher RBE of electron track ends for DSB induction. Therefore, these simulations should help improve models of DSB induction and our understanding of heavy ions biophysics.

Radiobiological Characterization of Tuberous Sclerosis: a Delay in the Nucleo-Shuttling of ATM May Be Responsible for Radiosensitivity.

PubMed

Ferlazzo, Mélanie L; Bach-Tobdji, Mohamed Kheir Eddine; Djerad, Amar; Sonzogni, Laurène; Devic, Clément; Granzotto, Adeline; Bodgi, Larry; Bachelet, Jean-Thomas; Djefal-Kerrar, Assia; Hennequin, Christophe; Foray, Nicolas

2018-06-01

The tuberous sclerosis complex (TSC) syndrome is associated with numerous cutaneous pathologies (notably on the face), epilepsy, intellectual disability and developmental retardation and, overall, high occurrence of benign tumors in several organs, like angiofibromas, giant cell astrocytomas, renal angiomyolipomas, and pulmonary lymphangioleiomyomatosis. TSC is caused by mutations of either of the hamartin or tuberin proteins that are mainly cytoplasmic. Some studies published in the 1980s reported that TSC is associated with radiosensitivity. However, its molecular basis in TSC cells is not documented enough. Here, we examined the functionality of the repair and signaling of radiation-induced DNA double-strand breaks (DSB) in fibroblasts derived from TSC patients. Quiescent TSC fibroblast cells elicited abnormally low rate of recognized DSB reflected by a low yield of nuclear foci formed by phosphorylated H2AX histones. Irradiated TSC cells also presented a delay in the nucleo-shuttling of the ATM kinase, potentially due to a specific binding of ATM to mutated TSC protein in cytoplasm. Lastly, TSC fibroblasts showed abnormally high MRE11 nuclease activity suggesting genomic instability. A combination of biphosphonates and statins complemented these impairments by facilitating the nucleoshuttling of ATM and increasing the yield of recognized DSB. Our results showed that TSC belongs to the group of syndromes associated with low but significant defect of DSB signaling and delay in the ATM nucleo-shuttling associated with radiosensitivity.

The rate of X-ray-induced DNA double-strand break repair in the embryonic mouse brain is unaffected by exposure to 50 Hz magnetic fields.

PubMed

Woodbine, Lisa; Haines, Jackie; Coster, Margaret; Barazzuol, Lara; Ainsbury, Elizabeth; Sienkiewicz, Zenon; Jeggo, Penny

2015-06-01

Following in utero exposure to low dose radiation (10-200 mGy), we recently observed a linear induction of DNA double-strand breaks (DSB) and activation of apoptosis in the embryonic neuronal stem/progenitor cell compartment. No significant induction of DSB or apoptosis was observed following exposure to magnetic fields (MF). In the present study, we exploited this in vivo system to examine whether exposure to MF before and after exposure to 100 mGy X-rays impacts upon DSB repair rates. 53BP1 foci were quantified following combined exposure to radiation and MF in the embryonic neuronal stem/progenitor cell compartment. Embryos were exposed in utero to 50 Hz MF at 300 μT for 3 h before and up to 9 h after exposure to 100 mGy X-rays. Controls included embryos exposed to MF or X-rays alone plus sham exposures. Exposure to MF before and after 100 mGy X-rays did not impact upon the rate of DSB repair in the embryonic neuronal stem cell compartment compared to repair rates following radiation exposure alone. We conclude that in this sensitive system MF do not exert any significant level of DNA damage and do not impede the repair of X-ray induced damage.

Central region component1, a novel synaptonemal complex component, is essential for meiotic recombination initiation in rice.

PubMed

Miao, Chunbo; Tang, Ding; Zhang, Honggen; Wang, Mo; Li, Yafei; Tang, Shuzhu; Yu, Hengxiu; Gu, Minghong; Cheng, Zhukuan

2013-08-01

In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated central region component1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID receptor-interacting protein13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with homologous pairing aberration in rice meiosis1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13.

53BP1 is a reader of the DNA damage-induced H2A Lys15 ubiquitin mark

PubMed Central

Fradet-Turcotte, Amélie; Canny, Marella D.; Escribano-Díaz, Cristina; Orthwein, Alexandre; Leung, Charles C.Y.; Huang, Hao; Landry, Marie-Claude; Kitevski-LeBlanc, Julianne; Noordermeer, Sylvie M.; Sicheri, Frank; Durocher, Daniel

2014-01-01

53BP1 (TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains enigmatic since its relocalization involves recognition of H4 Lys20 (H4K20) methylation by its Tudor domain. Here we elucidate how 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mono-nucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitylated on Lys15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a C-terminal extension, termed the ubiquitylation-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone “code” produced by DSB signaling. PMID:23760478

Mre11 and Exo1 contribute to the initiation and processivity of resection at meiotic double-strand breaks made independently of Spo11.

PubMed

Hodgson, Adam; Terentyev, Yaroslav; Johnson, Rebecca A; Bishop-Bailey, Anna; Angevin, Thibaut; Croucher, Adam; Goldman, Alastair S H

2011-02-07

During meiosis DNA double-strand breaks (DSBs) are induced and repaired by homologous recombination to create gene conversion and crossover products. Mostly these DSBs are made by Spo11, which covalently binds to the DSB ends. More rarely in Saccharomyces cerevisiae, other meiotic DSBs are formed by self-homing endonucleases such as VDE, which is site specific and does not covalently bind to the DSB ends. We have used experimentally located VDE-DSB sites to analyse an intermediate step in homologous recombination, resection of the single-strand ending 5' at the DSB site. Analysis of strains with different mutant alleles of MRE11 (mre11-58S and mre11-H125N) and deleted for EXO1 indicated that these two nucleases make significant contributions to repair of VDE-DSBs. Physical analysis of single-stranded repair intermediates indicates that efficient initiation and processivity of resection at VDE-DSBs require both Mre11 and Exo1, with loss of function for either protein causing severe delay in resection. We propose that these experiments model what happens at Spo11-DSBs after removal of the covalently bound protein, and that Mre11 and Exo1 are the major nucleases involved in creating resection tracts of widely varying lengths typical of meiotic recombination. Copyright © 2010 Elsevier B.V. All rights reserved.

CENTRAL REGION COMPONENT1, a Novel Synaptonemal Complex Component, Is Essential for Meiotic Recombination Initiation in Rice[C][W

PubMed Central

Miao, Chunbo; Tang, Ding; Zhang, Honggen; Wang, Mo; Li, Yafei; Tang, Shuzhu; Yu, Hengxiu; Gu, Minghong; Cheng, Zhukuan

2013-01-01

In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated CENTRAL REGION COMPONENT1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID RECEPTOR-INTERACTING PROTEIN13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13. PMID:23943860

Geant4-DNA simulation of DNA damage caused by direct and indirect radiation effects and comparison with biological data.

NASA Astrophysics Data System (ADS)

Villagrasa, Carmen; Meylan, Sylvain; Gonon, Geraldine; Gruel, Gaëtan; Giesen, Ulrich; Bueno, Marta; Rabus, Hans

2017-09-01

In this work we present results obtained in the frame of the BioQuaRT project. The objective of the study was the correlation between the number of radiation-induced double strand breaks (DSB) of the DNA molecule and the probability of detecting nuclear foci after targeted microbeam irradiation of cells with protons and alpha particles of different LET. The former were obtained by simulation with new methods integrated into Geant4-DNA that permit calculating the number of DSB in a DNA target model induced by direct and indirect radiation effects. A particular focus was laid in this work on evaluating the influence of different criteria applied to the simulated results for predicting the formation of a direct SSB. Indeed, these criteria have an important impact on the predicted number of DSB per particle track and its dependence with LET. Among the criteria tested in this work, the case that a direct radiation interaction leads to a strand break if the cumulative energy deposited in the backbone part of one nucleotide exceeds a threshold of 17.5 eV leads to the best agreement with the relative LET dependence of number of radiation induced foci. Further calculations and experimental data are nevertheless needed in order to fix the simulation parameters and to help interpreting the biological experimental data observed by immunofluorescence in terms of the DSB complexity.

Single- and Double-Strand Breaks of Dry DNA Exposed to Protons at Bragg-Peak Energies.

PubMed

Souici, Mounir; Khalil, Talat T; Muller, Dominique; Raffy, Quentin; Barillon, Rémi; Belafrites, Abdelfettah; Champion, Christophe; Fromm, Michel

2017-01-26

Ultrathin layers (<20 nm) of pBR322 plasmid DNA were deposited onto 2.5 μm thick polyester films and exposed to proton Bragg-peak energies (90-3000 keV) at various fluences. A quantitative analysis of radio-induced DNA damage is reported here in terms of single- and double-strand breaks (SSB and DSB, respectively). The corresponding yields as well as G-values and the cross sections exhibit fairly good agreement with the rare available data, stemming from close experimental conditions, namely, based on α particle irradiation. SSB/DSB rates appear to be linear when plotted against linear energy transfer (LET) in the whole energy range studied. All the data present a maximum in the 150-200 keV energy range; as for LET, it peaks at 90 keV. We also show that fragmentation starts to be significant for proton fluences greater than 1 × 10 11 cm -2 at the Bragg-peak energies. Finally, we determine the average proton track radial extension, r max , corresponding to an occupation probability of 100% DSB in the Bragg-peak region. The r max values determined are in excellent agreement with the radial extensions of proton tracks determined by simulation approaches in water. When plotted as a function of LET, both SSB and DSB cross sections bend back at high LETs.

DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions.

PubMed

Panchakshari, Rohit A; Zhang, Xuefei; Kumar, Vipul; Du, Zhou; Wei, Pei-Chi; Kao, Jennifer; Dong, Junchao; Alt, Frederick W

2018-01-23

Ig heavy chain (IgH) class switch recombination (CSR) in B lymphocytes switches IgH constant regions to change antibody functions. CSR is initiated by DNA double-strand breaks (DSBs) within a donor IgH switch (S) region and a downstream acceptor S region. CSR is completed by fusing donor and acceptor S region DSB ends by classical nonhomologous end-joining (C-NHEJ) and, in its absence, by alternative end-joining that is more biased to use longer junctional microhomologies (MHs). Deficiency for DSB response (DSBR) factors, including ataxia telangiectasia-mutated (ATM) and 53BP1, variably impair CSR end-joining, with 53BP1 deficiency having the greatest impact. However, studies of potential impact of DSBR factor deficiencies on MH-mediated CSR end-joining have been technically limited. We now use a robust DSB joining assay to elucidate impacts of deficiencies for DSBR factors on CSR and chromosomal translocation junctions in primary mouse B cells and CH12F3 B-lymphoma cells. Compared with wild-type, CSR and c-myc to S region translocation junctions in the absence of 53BP1, and, to a lesser extent, other DSBR factors, have increased MH utilization; indeed, 53BP1-deficient MH profiles resemble those associated with C-NHEJ deficiency. However, translocation junctions between c-myc DSB and general DSBs genome-wide are not MH-biased in ATM-deficient versus wild-type CH12F3 cells and are less biased in 53BP1- and C-NHEJ-deficient cells than CSR junctions or c-myc to S region translocation junctions. We discuss potential roles of DSBR factors in suppressing increased MH-mediated DSB end-joining and features of S regions that may render their DSBs prone to MH-biased end-joining in the absence of DSBR factors.

Activation of Telomerase by Ionizing Radiation: Differential Response to the Inhibition of DNA Double-Strand Break Repair by Abrogation of Poly(ADP-ribosyl)ation, by LY294002, or by Wortmannin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Neuhof, Dirk; Zwicker, Felix; Kuepper, Jan-Heiner

2007-11-01

Purpose: Telomerase activity represents a radiation-inducible function, which may be targeted by a double-strand break (DSB)-activated signal transduction pathway. Therefore, the effects of DNA-PK inhibitors (Wortmannin and LY294002) on telomerase upregulation after irradiation were studied. In addition, the role of trans-dominant inhibition of poly(ADP-ribosyl)ation, which strongly reduces DSB rejoining, was assessed in comparison with 3-aminobenzamide. Methods and Materials: COM3 rodent cells carry a construct for the dexamethasone-inducible overexpression of the DNA-binding domain of PARP1 and exhibit greatly impaired DSB rejoining after irradiation. Telomerase activity was measured using polymerase chain reaction ELISA 1 h after irradiation with doses up to 10more » Gy. Phosphorylation status of PKB/Akt and of PKC{alpha}/{beta}{sub II} was assessed by western blotting. Results: No telomerase upregulation was detectable for irradiated cells with undisturbed DSB rejoining. In contrast, incubation with LY294002 or dexamethasone yielded pronounced radiation induction of telomerase activity that could be suppressed by Wortmannin. 3-Aminobenzamide not only was unable to induce telomerase activity but also suppressed telomerase upregulation upon incubation with LY294002 or dexamethasone. Phospho-PKB was detectable independent of irradiation or dexamethasone pretreatment, but was undetectable upon incubations with LY294002 or Wortmannin, whereas phospho-PKC rested detectable. Conclusions: Telomerase activation postirradiation was triggered by different treatments that interfere with DNA DSB processing. This telomerase upregulation, however, was not reflected by the phosporylation status of the putative mediators of TERT activation, PKB and PKC. Although an involvement of PKB in TERT activation is not supported by the present findings, a respective role of PKC isoforms other than {alpha}/{beta}{sub II} cannot be ruled out.« less

Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks.

PubMed

Bañuelos, C A; Banáth, J P; MacPhail, S H; Zhao, J; Eaves, C A; O'Connor, M D; Lansdorp, P M; Olive, P L

2008-09-01

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed <10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However, the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses <20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells, mES cells lacking H2AX, a histone protein involved in the DNA damage response, were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs), an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.

Temporal analysis of meiotic DNA double-strand break formation and repair in Drosophila females.

PubMed

Mehrotra, S; McKim, K S

2006-11-24

Using an antibody against the phosphorylated form of His2Av (gamma-His2Av), we have described the time course for the series of events leading from the formation of a double-strand break (DSB) to a crossover in Drosophila female meiotic prophase. MEI-P22 is required for DSB formation and localizes to chromosomes prior to gamma-His2Av foci. Drosophila females, however, are among the group of organisms where synaptonemal complex (SC) formation is not dependent on DSBs. In the absence of two SC proteins, C(3)G and C(2)M, the number of DSBs in oocytes is significantly reduced. This is consistent with the appearance of SC protein staining prior to gamma-His2Av foci. However, SC formation is incomplete or absent in the neighboring nurse cells, and gamma-His2Av foci appear with the same kinetics as in oocytes and do not depend on SC proteins. Thus, competence for DSB formation in nurse cells occurs with a specific timing that is independent of the SC, whereas in the oocytes, some SC proteins may have a regulatory role to counteract the effects of a negative regulator of DSB formation. The SC is not sufficient for DSB formation, however, since DSBs were absent from the heterochromatin even though SC formation occurs in these regions. All gamma-His2Av foci disappear before the end of prophase, presumably as repair is completed and crossovers are formed. However, oocytes in early prophase exhibit a slower response to X-ray-induced DSBs compared to those in the late pachytene stage. Assuming all DSBs appear as gamma-His2Av foci, there is at least a 3:1 ratio of noncrossover to crossover products. From a comparison of the frequency of gamma-His2Av foci and crossovers, it appears that Drosophila females have only a weak mechanism to ensure a crossover in the presence of a low number of DSBs.

⁹⁹mTc pyrene derivative complex causes double-strand breaks in dsDNA mainly through cluster-mediated indirect effect in aqueous solution.

PubMed

Chung, Wei-Ju; Cui, Yujia; Huang, Feng-Yun J; Tu, Tzu-Hui; Yang, Tzu-Sen; Lo, Jem-Mau; Chiang, Chi-Shiun; Hsu, Ian C

2014-01-01

Radiation therapy for cancer patients works by ionizing damage to nuclear DNA, primarily by creating double-strand breaks (DSB). A major shortcoming of traditional radiation therapy is the set of side effect associated with its long-range interaction with nearby tissues. Low-energy Auger electrons have the advantage of an extremely short effective range, minimizing damage to healthy tissue. Consequently, the isotope ⁹⁹mTc, an Auger electron source, is currently being studied for its beneficial potential in cancer treatment. We examined the dose effect of a pyrene derivative ⁹⁹mTc complex on plasmid DNA by using gel electrophoresis in both aqueous and methanol solutions. In aqueous solutions, the average yield per decay for double-strand breaks is 0.011±0.005 at low dose range, decreasing to 0.0005±0.0003 in the presence of 1 M dimethyl sulfoxide (DMSO). The apparent yield per decay for single-strand breaks (SSB) is 0.04±0.02, decreasing to approximately a fifth with 1 M DMSO. In methanol, the average yield per decay of DSB is 0.54±0.06 and drops to undetectable levels in 2 M DMSO. The SSB yield per decay is 7.2±0.2, changing to 0.4±0.2 in the presence of 2 M DMSO. The 95% decrease in the yield of DSB in DMSO indicates that the main mechanism for DSB formation is through indirect effect, possibly by cooperative binding or clustering of intercalators. In the presence of non-radioactive ligands at a near saturation concentration, where radioactive Tc compounds do not form large clusters, the yield of SSB stays the same while the yield of DSB decreases to the value in DMSO. DSBs generated by ⁹⁹mTc conjugated to intercalators are primarily caused by indirect effects through clustering.

KARP-1 works as a heterodimer with Ku70, but the function of KARP-1 cannot perfectly replace that of Ku80 in DSB repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koike, Manabu, E-mail: m_koike@nirs.go.jp; Yutoku, Yasutomo; Graduate School of Science, Chiba University, Yayoicho, Inage-ku, Chiba 263-8522

2011-10-01

Ku, the heterodimer of Ku70 and Ku80, plays an essential role in the DNA double-strand break (DSB) repair pathway, i.e., non-homologous end-joining (NHEJ). Two isoforms of Ku80 encoded by the same genes, namely, Ku80 and KARP-1 are expressed and function in primate cells, but not in rodent cells. Ku80 works as a heterodimer with Ku70. However, it is not yet clear whether KARP-1 forms a heterodimer with Ku70 and works as a heterodimer. Although KARP-1 appears to work in NHEJ, its physiological role remains unclear. In this study, we established and characterized EGFP-KARP-1-expressing xrs-6 cell lines, EGFP-KARP-1/xrs-6. We found thatmore » nuclear localization signal (NLS) of KARP-1 is localized in the C-terminal region. Our data showed that KARP-1 localizes within the nucleus in NLS-dependent and NLS-independent manner and forms a heterodimer with Ku70, and stabilizes Ku70. On the other hand, EGFP-KARP-1 could not perfectly complement the radiosensitivity and DSB repair activity of Ku80-deficient xrs-6 cells. Furthermore, KARP-1 could not accumulate at DSBs faster than Ku80, although EGFP-KARP-1 accumulates at DSBs. Our data demonstrate that the function of KARP-1 could not perfectly replace that of Ku80 in DSB repair, although KARP-1 has some biochemical properties, which resemble those of Ku80, and works as a heterodimer with Ku70. On the other hand, the number of EGFP-KARP-1-expressing xrs-6 cells showing pan-nuclear {gamma}-H2AX staining significantly increases following X-irradiation, suggesting that KARP-1 may have a novel role in DSB response.« less

p73 coordinates with Δ133p53 to promote DNA double-strand break repair.

PubMed

Gong, Hongjian; Zhang, Yuxi; Jiang, Kunpeng; Ye, Shengfan; Chen, Shuming; Zhang, Qinghe; Peng, Jinrong; Chen, Jun

2018-03-06

Tumour repressor p53 isoform Δ133p53 is a target gene of p53 and an antagonist of p53-mediated apoptotic activity. We recently demonstrated that Δ133p53 promotes DNA double-strand break (DSB) repair by upregulating transcription of the repair genes RAD51, LIG4 and RAD52 in a p53-independent manner. However, Δ133p53 lacks the transactivation domain of full-length p53, and the mechanism by which it exerts transcriptional activity independently of full-length p53 remains unclear. In this report, we describe the accumulation of high levels of both Δ133p53 and p73 (a p53 family member) at 24 h post γ-irradiation (hpi). Δ133p53 can form a complex with p73 upon γ-irradiation. The co-expression of Δ133p53 and p73, but not either protein alone, can significantly promote DNA DSB repair mechanisms, including homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA). p73 and Δ133p53 act synergistically to promote the expression of RAD51, LIG4 and RAD52 by joining together to bind to region containing a Δ133p53-responsive element (RE) and a p73-RE in the promoters of all three repair genes. In addition to its accumulation at 24 hpi, p73 protein expression also peaks at 4 hpi. The depletion of p73 not only reduces early-stage apoptotic frequency (4-6 hpi), but also significantly increases later-stage DNA DSB accumulation (48 hpi), leading to cell cycle arrest in the G2 phase and, ultimately, cell senescence. In summary, the apoptotic regulator p73 also coordinates with Δ133p53 to promote DNA DSB repair, and the loss of function of p73 in DNA DSB repair may underlie spontaneous and carcinogen-induced tumorigenesis in p73 knockout mice.

Effects of heavy ions on inactivation and DNA double strand breaks in Deinococcus radiodurans R1.

PubMed

Zimmermann, H; Schafer, M; Schmitz, C; Bucker, H

1994-10-01

Inactivation and double strand break (dsb) induction after heavy ion irradiation were studied in stationary phase cells of the highly radiation resistant bacterium Deinococcus radiodurans R1. There is evidence that the radiation sensitivity of this bacterium is nearly independent on energy in the range of up to 15 MeV/u for lighter ions (Ar). The responses to dsb induction for charged particles show direct relationship between increasing radiation dose and residual intact DNA.

Analytical model of chemical phase and formation of DSB in chromosomes by ionizing radiation.

PubMed

Barilla, Jiří; Lokajíček, Miloš; Pisaková, Hana; Simr, Pavel

2013-03-01

Mathematical analytical model of the processes running in individual radical clusters during the chemical phase (under the presence of radiomodifiers) proposed by us earlier has been further developed and improved. It has been applied to the data presented by Blok and Loman characterizing the oxygen effect in SSB and DSB formation (in water solution and at low-LET radiation) also in the region of very small oxygen concentrations, which cannot be studied with the help of experiments done with living cells. In this new analysis the values of all reaction rates and diffusion parameters known from literature have been made use of. The great increase of SSB and DSB at zero oxygen concentration may follow from the fact that at small oxygen concentrations the oxygen absorbs other radicals while at higher concentrations the formation of oxygen radicals prevails. It explains the double oxygen effect found already earlier by Ewing. The model may be easily extended to include also the effects of other radiomodifiers present in medium during irradiation.

System and method for tuning adjusting the central frequency of a laser while maintaining frequency stabilization to an external reference

NASA Technical Reports Server (NTRS)

Livas, Jeffrey (Inventor); Thorpe, James I. (Inventor); Numata, Kenji (Inventor)

2011-01-01

A method and system for stabilizing a laser to a frequency reference with an adjustable offset. The method locks a sideband signal generated by passing an incoming laser beam through the phase modulator to a frequency reference, and adjusts a carrier frequency relative to the locked sideband signal by changing a phase modulation frequency input to the phase modulator. The sideband signal can be a single sideband (SSB), dual sideband (DSB), or an electronic sideband (ESB) signal. Two separate electro-optic modulators can produce the DSB signal. The two electro-optic modulators can be a broadband modulator and a resonant modulator. With a DSB signal, the method can introduce two sinusoidal phase modulations at the phase modulator. With ESB signals, the method can further drive the optical phase modulator with an electrical signal with nominal frequency OMEGA(sub 1) that is phase modulated at a frequency OMEGA(sub 2)

Microhomology-mediated end joining induces hypermutagenesis at breakpoint junctions

PubMed Central

Li, Fuyang; Villarreal, Diana; Shim, Jae Hoon; Myung, Kyungjae; Shim, Eun Yong; Lee, Sang Eun

2017-01-01

Microhomology (MH) flanking a DNA double-strand break (DSB) drives chromosomal rearrangements but its role in mutagenesis has not yet been analyzed. Here we determined the mutation frequency of a URA3 reporter gene placed at multiple locations distal to a DSB, which is flanked by different sizes (15-, 18-, or 203-bp) of direct repeat sequences for efficient repair in budding yeast. Induction of a DSB accumulates mutations in the reporter gene situated up to 14-kb distal to the 15-bp MH, but more modestly to those carrying 18- and 203-bp or no homology. Increased mutagenesis in MH-mediated end joining (MMEJ) appears coupled to its slower repair kinetics and the extensive resection occurring at flanking DNA. Chromosomal translocations via MMEJ also elevate mutagenesis of the flanking DNA sequences 7.1 kb distal to the breakpoint junction as compared to those without MH. The results suggest that MMEJ could destabilize genomes by triggering structural alterations and increasing mutation burden. PMID:28419093

Localization and roles of Ski8p protein in Sordaria meiosis and delineation of three mechanistically distinct steps of meiotic homolog juxtaposition.

PubMed

Tessé, Sophie; Storlazzi, Aurora; Kleckner, Nancy; Gargano, Silvana; Zickler, Denise

2003-10-28

Ski8p is implicated in degradation of non-poly(A) and double-stranded RNA, and in meiotic DNA recombination. We have identified the Sordaria macrospora SKI8 gene. Ski8p is cytoplasmically localized in all vegetative and sexual cycle cells, and is nuclear localized, specifically in early-mid-meiotic prophase, in temporal correlation with Spo11p, the meiotic double-strand break (DSB) transesterase. Localizations of Ski8p and Spo11p are mutually interdependent. ski8 mutants exhibit defects in vegetative growth, entry into the sexual program, and sporulation. Diverse meiotic defects, also seen in spo11 mutants, are diagnostic of DSB absence, and they are restored by exogenous DSBs. These results suggest that Ski8p promotes meiotic DSB formation by acting directly within meiotic prophase chromosomes. Mutant phenotypes also divide meiotic homolog juxtaposition into three successive, mechanistically distinct steps; recognition, presynaptic alignment, and synapsis, which are distinguished by their differential dependence on DSBs.

Localization and roles of Ski8p protein in Sordaria meiosis and delineation of three mechanistically distinct steps of meiotic homolog juxtaposition

PubMed Central

Tessé, Sophie; Storlazzi, Aurora; Kleckner, Nancy; Gargano, Silvana; Zickler, Denise

2003-01-01

Ski8p is implicated in degradation of non-poly(A) and double-stranded RNA, and in meiotic DNA recombination. We have identified the Sordaria macrospora SKI8 gene. Ski8p is cytoplasmically localized in all vegetative and sexual cycle cells, and is nuclear localized, specifically in early-mid-meiotic prophase, in temporal correlation with Spo11p, the meiotic double-strand break (DSB) transesterase. Localizations of Ski8p and Spo11p are mutually interdependent. ski8 mutants exhibit defects in vegetative growth, entry into the sexual program, and sporulation. Diverse meiotic defects, also seen in spo11 mutants, are diagnostic of DSB absence, and they are restored by exogenous DSBs. These results suggest that Ski8p promotes meiotic DSB formation by acting directly within meiotic prophase chromosomes. Mutant phenotypes also divide meiotic homolog juxtaposition into three successive, mechanistically distinct steps; recognition, presynaptic alignment, and synapsis, which are distinguished by their differential dependence on DSBs. PMID:14563920

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

DOE PAGES

Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.; ...

2016-02-22

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

Mechanisms of double-strand-break repair during gene targeting in mammalian cells.

PubMed Central

Ng, P; Baker, M D

1999-01-01

In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism. PMID:10049929

Reactive oxygen species regulate DNA copy number in isolated yeast mitochondria by triggering recombination-mediated replication.

PubMed

Hori, Akiko; Yoshida, Minoru; Shibata, Takehiko; Ling, Feng

2009-02-01

Mitochondrial DNA (mtDNA) encodes proteins that are essential for cellular ATP production. Reactive oxygen species (ROS) are respiratory byproducts that damage mtDNA and other cellular components. In Saccharomyces cerevisiae, the oxidized base excision-repair enzyme Ntg1 introduces a double-stranded break (DSB) at the mtDNA replication origin ori5; this DSB initiates the rolling-circle mtDNA replication mediated by the homologous DNA pairing protein Mhr1. Thus, ROS may play a role in the regulation of mtDNA copy number. Here, we show that the treatment of isolated mitochondria with low concentrations of hydrogen peroxide increased mtDNA copy number in an Ntg1- and Mhr1-dependent manner. This treatment elevated the DSB levels at ori5 of hypersuppressive [rho(-)] mtDNA only if Ntg1 was active. In vitro Ntg1-treatment of hypersuppressive [rho(-)] mtDNA extracted from hydrogen peroxide-treated mitochondria revealed increased oxidative modifications at ori5 loci. We also observed that purified Ntg1 created breaks in single-stranded DNA harboring oxidized bases, and that ori5 loci have single-stranded character. Furthermore, chronic low levels of hydrogen peroxide increased in vivo mtDNA copy number. We therefore propose that ROS act as a regulator of mtDNA copy number, acting through the Mhr1-dependent initiation of rolling-circle replication promoted by Ntg1-induced DSB in the single-stranded regions at ori5.

Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9.

PubMed

Chung, Mu-En; Yeh, I-Hsin; Sung, Li-Yu; Wu, Meng-Ying; Chao, Yun-Peng; Ng, I-Son; Hu, Yu-Chen

2017-01-01

Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA-guided system which enables site-specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9-triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ-Red proteins and linear dsDNA, CRISPR/Cas9-induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9-assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172-183. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The purine scaffold Hsp90 inhibitor PU-H71 sensitizes cancer cells to heavy ion radiation by inhibiting DNA repair by homologous recombination and non-homologous end joining.

PubMed

Lee, Younghyun; Li, Huizi Keiko; Masaoka, Aya; Sunada, Shigeaki; Hirakawa, Hirokazu; Fujimori, Akira; Nickoloff, Jac A; Okayasu, Ryuichi

2016-10-01

PU-H71 is a purine-scaffold Hsp90 inhibitor developed to overcome limitations of conventional Hsp90 inhibitors. This study was designed to investigate the combined effect of PU-H71 and heavy ion irradiation on human tumor and normal cells. The effects of PU-H71 were determined by monitoring cell survival by colony formation, and DNA double-strand break (DSB) repair by γ-H2AX foci and immuno-blotting DSB repair proteins. The mode of cell death was evaluated by sub-G1 DNA content (as an indicator for apoptosis), and mitotic catastrophe. PU-H71 enhanced heavy ion irradiation-induced cell death in three human cancer cell lines, but the drug did not radiosensitize normal human fibroblasts. In irradiated tumor cells, PU-H71 increased the persistence of γ-H2AX foci, and it reduced RAD51 foci and phosphorylated DNA-PKcs, key DSB repair proteins involved in homologous recombination (HR) and non-homologous end joining (NHEJ). In some tumor cell lines, PU-H71 altered the sub-G1 cell fraction and mitotic catastrophe following carbon ion irradiation. Our results demonstrate that PU-H71 sensitizes human cancer cells to heavy ion irradiation by inhibiting both HR and NHEJ DSB repair pathways. PU-H71 holds promise as a radiosensitizer for enhancing the efficacy of heavy ion radiotherapy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Repair of DNA damage induced by accelerated heavy ions--a mini review.

PubMed

Okayasu, Ryuichi

2012-03-01

Increasing use of heavy ions for cancer therapy and concerns from exposure to heavy charged particles in space necessitate the study of the basic biological mechanisms associated with exposure to heavy ions. As the most critical damage induced by ionizing radiation is DNA double strand break (DSB), this review focuses on DSBs induced by heavy ions and their repair processes. Compared with X- or gamma-rays, high-linear energy transfer (LET) heavy ion radiation induces more complex DNA damage, categorized into DSBs and non-DSB oxidative clustered DNA lesions (OCDL). This complexity makes the DNA repair process more difficult, partially due to retarded enzymatic activities, leading to increased chromosome aberrations and cell death. In general, the repair process following heavy ion exposure is LET-dependent, but with nonhomologous end joining defective cells, this trend is less emphasized. The variation in cell survival levels throughout the cell cycle is less prominent in cells exposed to high-LET heavy ions when compared with low LET, but this mechanism has not been well understood until recently. Involvement of several DSB repair proteins is suggested to underlie this interesting phenomenon. Recent improvements in radiation-induced foci studies combined with high-LET heavy ion exposure could provide a useful opportunity for more in depth study of DSB repair processes. Accelerated heavy ions have become valuable tools to investigate the molecular mechanisms underlying repair of DNA DSBs, the most crucial form of DNA damage induced by radiation and various chemotherapeutic agents. Copyright © 2011 UICC.

What Does It Take to Search Organized? The Cognitive Correlates of Search Organization During Cancellation After Stroke.

PubMed

Ten Brink, Antonia F; Visser-Meily, Johanna M A; Nijboer, Tanja C W

2018-05-01

Stroke could lead to deficits in organization of visual search. Cancellation tests are frequently used in standard neuropsychological assessment and appear suitable to measure search organization. The current aim was to evaluate which cognitive functions are associated with cancellation organization measures after stroke. Stroke patients admitted to inpatient rehabilitation were included in this retrospective study. We performed exploratory factor analyses to explore cognitive domains. A digital shape cancellation test (SC) was administered, and measures of search organization (intersections rate and best r) were computed. The following cognitive functions were measured by neuropsychological testing: neglect (SC, line bisection; LB, Catherine Bergego Scale; CBS, and Balloons Test), visuospatial perception and construction (Rey Complex Figure Test, RCFT), psychomotor speed (Trail Making Test; TMT-A), executive functioning/working memory (TMT-B), spatial planning (Tower Test), rule learning (Brixton Test), short-term auditory memory (Digit Span Forward; DSF), and verbal working memory (Digit Span Backward; DSB). In total, 439 stroke patients were included in our analyses. Four clusters were separated: "Executive functioning" (TMT-A, TMT-B, Brixton Test, and Tower Test), "Verbal memory" (DSF and DSB), "Search organization" (intersections rate and best r), and "Neglect" (CBS, RCFT copy, Balloons Test, SC, and LB). Search organization during cancellation, as measured with intersections rate and best r, seems a distinct cognitive construct compared to existing cognitive domains that are tested during neuropsychological assessment. Administering cancellation tests and analyzing measures of search organization could provide useful additional insights into the visuospatial processes of stroke patients. (JINS, 2018, 24, 424-436).

Crustal structure of the southern Dead Sea basin derived from project DESIRE wide-angle seismic data

NASA Astrophysics Data System (ADS)

Mechie, J.; Abu-Ayyash, K.; Ben-Avraham, Z.; El-Kelani, R.; Qabbani, I.; Weber, M.

2009-07-01

As part of the DEad Sea Integrated REsearch project (DESIRE) a 235 km long seismic wide-angle reflection/refraction (WRR) profile was completed in spring 2006 across the Dead Sea Transform (DST) in the region of the southern Dead Sea basin (DSB). The DST with a total of about 107 km multi-stage left-lateral shear since about 18 Ma ago, accommodates the movement between the Arabian and African plates. It connects the spreading centre in the Red Sea with the Taurus collision zone in Turkey over a length of about 1100 km. With a sedimentary infill of about 10 km in places, the southern DSB is the largest pull-apart basin along the DST and one of the largest pull-apart basins on Earth. The WRR measurements comprised 11 shots recorded by 200 three-component and 400 one-component instruments spaced 300 m to 1.2 km apart along the whole length of the E-W trending profile. Models of the P-wave velocity structure derived from the WRR data show that the sedimentary infill associated with the formation of the southern DSB is about 8.5 km thick beneath the profile. With around an additional 2 km of older sediments, the depth to the seismic basement beneath the southern DSB is about 11 km below sea level beneath the profile. Seismic refraction data from an earlier experiment suggest that the seismic basement continues to deepen to a maximum depth of about 14 km, about 10 km south of the DESIRE profile. In contrast, the interfaces below about 20 km depth, including the top of the lower crust and the Moho, probably show less than 3 km variation in depth beneath the profile as it crosses the southern DSB. Thus the Dead Sea pull-apart basin may be essentially an upper crustal feature with upper crustal extension associated with the left-lateral motion along the DST. The boundary between the upper and lower crust at about 20 km depth might act as a decoupling zone. Below this boundary the two plates move past each other in what is essentially a shearing motion. Thermo-mechanical modelling of the DSB supports such a scenario. As the DESIRE seismic profile crosses the DST about 100 km north of where the DESERT seismic profile crosses the DST, it has been possible to construct a crustal cross-section of the region before the 107 km left-lateral shear on the DST occurred.

Verbal short-term memory development and spoken language outcomes in deaf children with cochlear implants.

PubMed

Harris, Michael S; Kronenberger, William G; Gao, Sujuan; Hoen, Helena M; Miyamoto, Richard T; Pisoni, David B

2013-01-01

Cochlear implants (CIs) help many deaf children achieve near-normal speech and language (S/L) milestones. Nevertheless, high levels of unexplained variability in S/L outcomes are limiting factors in improving the effectiveness of CIs in deaf children. The objective of this study was to longitudinally assess the role of verbal short-term memory (STM) and working memory (WM) capacity as a progress-limiting source of variability in S/L outcomes after CI in children. Longitudinal study of 66 children with CIs for prelingual severe-to-profound hearing loss. Outcome measures included performance on digit span forward (DSF), digit span backward (DSB), and four conventional S/L measures that examined spoken-word recognition (Phonetically Balanced Kindergarten word test), receptive vocabulary (Peabody Picture Vocabulary Test ), sentence-recognition skills (Hearing in Noise Test), and receptive and expressive language functioning (Clinical Evaluation of Language Fundamentals Fourth Edition Core Language Score; CELF). Growth curves for DSF and DSB in the CI sample over time were comparable in slope, but consistently lagged in magnitude relative to norms for normal-hearing peers of the same age. For DSF and DSB, 50.5% and 44.0%, respectively, of the CI sample scored more than 1 SD below the normative mean for raw scores across all ages. The first (baseline) DSF score significantly predicted all endpoint scores for the four S/L measures, and DSF slope (growth) over time predicted CELF scores. DSF baseline and slope accounted for an additional 13 to 31% of variance in S/L scores after controlling for conventional predictor variables such as: chronological age at time of testing, age at time of implantation, communication mode (auditory-oral communication versus total communication), and maternal education. Only DSB baseline scores predicted endpoint language scores on Peabody Picture Vocabulary Test and CELF. DSB slopes were not significantly related to any endpoint S/L measures. DSB baseline scores and slopes taken together accounted for an additional 4 to 19% of variance in S/L endpoint measures after controlling for the conventional predictor variables. Verbal STM/WM scores, process measures of information capacity, develop at an average rate in the years after cochlear implantation, but were found to consistently lag in absolute magnitude behind those reported for normal-hearing peers. Baseline verbal STM/WM predicted long-term endpoint S/L outcomes, but verbal STM slopes predicted only endpoint language outcomes. Verbal STM/WM processing skills reflect important underlying core elementary neurocognitive functions and represent potential intervention targets for improving endpoint S/L outcomes in pediatric CI users.

Phenotypic Analysis of ATM Protein Kinase in DNA Double-Strand Break Formation and Repair.

PubMed

Mian, Elisabeth; Wiesmüller, Lisa

2017-01-01

Ataxia telangiectasia mutated (ATM) encodes a serine/threonine protein kinase, which is involved in various regulatory processes in mammalian cells. Its best-known role is apical activation of the DNA damage response following generation of DNA double-strand breaks (DSBs). When DSBs appear, sensor and mediator proteins are recruited, activating transducers such as ATM, which in turn relay a widespread signal to a multitude of downstream effectors. ATM mutation causes Ataxia telangiectasia (AT), whereby the disease phenotype shows differing characteristics depending on the underlying ATM mutation. However, all phenotypes share progressive neurodegeneration and marked predisposition to malignancies at the organismal level and sensitivity to ionizing radiation and chromosome aberrations at the cellular level. Expression and localization of the ATM protein can be determined via western blotting and immunofluorescence microscopy; however, detection of subtle alterations such as resulting from amino acid exchanges rather than truncating mutations requires functional testing. Previous studies on the role of ATM in DSB repair, which connects with radiosensitivity and chromosomal stability, gave at first sight contradictory results. To systematically explore the effects of clinically relevant ATM mutations on DSB repair, we engaged a series of lymphoblastoid cell lines (LCLs) derived from AT patients and controls. To examine DSB repair both in a quantitative and qualitative manners, we used an EGFP-based assay comprising different substrates for distinct DSB repair mechanisms. In this way, we demonstrated that particular signaling defects caused by individual ATM mutations led to specific DSB repair phenotypes. To explore the impact of ATM on carcinogenic chromosomal aberrations, we monitored chromosomal breakage at a breakpoint cluster region hotspot within the MLL gene that has been associated with therapy-related leukemia. PCR-based MLL-breakage analysis of HeLa cells treated with and without pharmacological kinase inhibitors revealed ATM-dependent chromatin remodeling at the MLL break site giving access to DNA repair proteins but also nucleases triggering MLL rearrangements. This chapter summarizes these methods for functional characterization of ATM in patient LCLs and human cell lines.

The DESIRE Airborne gravity project in the Dead Sea Basin and 3D numerical gravity modeling

NASA Astrophysics Data System (ADS)

Choi, S.; Goetze, H.; Meyer, U.; Group, D.

2008-12-01

This geo-scientific research focuses on the geological setting of the Dead Sea Transform (DST) and the Dead Sea Basin (DSB) and its resulting pull-apart basins. Since the late 1970s, crustal scale geophysical experiments have been carried out in this region. However, the nature of the crust underlying the eastern and western shoulders of the DSB and underneath the DST itself is still a hotly debated topic among researchers. To address one of the central questions of plate tectonics - How do large transform systems work and what are their typical features? - An international geoscientific Dead Sea Integrated Research project (DESIRE) is being conducted by colleagues from Germany, Israel, Palestine, and Jordan. In order to provide a high resolution gravity database that support 3D numerical modeling and hence a more comprehensive understanding of the nature and segmentation of the DST, an airborne gravity survey as a part of the DESIRE project has been carried out from February to March 2007. The airborne gravity survey covered the DST from Elat/Aqaba in the South to the northern rim of the Dead Sea. The low speed and terrain-following helicopter gravity flights were performed to acquire the highest possible data quality. In total, 32 north-south profiles and 16 west-east profiles crossing the DST have been measured. Most of the profiles concentrated in areas that lacked terrestrial gravity data coverage, e. g. over the shoulders of the DSB. The airborne gravity data are merged with existing conventional (terrestrial) data sets to provide a seamless gravity map of the area of interest. Using that combined gravity dataset and DESIRE wide angle refractions seismic interpretation we modified density structures in the DSB. As results we estimated that (1) the Moho depth varies from 26 km in the Israel side to 34 km in the Jordan side. (2) The maximum thickness of the Dead Sea sediment Basin is about 15 km. (3) The salt rock with an average thickness of about 5 km is present not only in the southern part of the DSB but also in northern part underlying the entire Dead Sea.

Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells

NASA Technical Reports Server (NTRS)

Dar, M. E.; Winters, T. A.; Jorgensen, T. J.

1997-01-01

Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human disease. Homozygotes suffer from a number of neurological disorders, as well as very high cancer incidence. Heterozygotes may also have a higher than normal risk of cancer, particularly for the breast. The gene responsible for the disease (ATM) has been cloned, but its role in mechanisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defect; however, overall levels of DSB rejoining appear normal. We used the shuttle vector, pZ189, containing an oxidatively-induced DSB, to compare the integrity of DSB rejoining in one normal and two A-T fibroblast cells lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutations found in all three cell lines were deletions (44-63%). The DNA sequence analysis indicated that 17 of the 17 plasmids with deletion mutations in normal cells occurred between short direct-repeat sequences (removing one of the repeats plus the intervening sequences), implicating illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve direct-repeats sequences, implicating a defect in the illegitimate recombination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a mechanistic understanding of this lethal disease.

Impact of Charged Particle Exposure on Homologous DNA Double-Strand Break Repair in Human Blood-Derived Cells

PubMed Central

Rall, Melanie; Kraft, Daniela; Volcic, Meta; Cucu, Aljona; Nasonova, Elena; Taucher-Scholz, Gisela; Bönig, Halvard; Wiesmüller, Lisa; Fournier, Claudia

2015-01-01

Ionizing radiation generates DNA double-strand breaks (DSB) which, unless faithfully repaired, can generate chromosomal rearrangements in hematopoietic stem and/or progenitor cells (HSPC), potentially priming the cells towards a leukemic phenotype. Using an enhanced green fluorescent protein (EGFP)-based reporter system, we recently identified differences in the removal of enzyme-mediated DSB in human HSPC versus mature peripheral blood lymphocytes (PBL), particularly regarding homologous DSB repair (HR). Assessment of chromosomal breaks via premature chromosome condensation or γH2AX foci indicated similar efficiency and kinetics of radiation-induced DSB formation and rejoining in PBL and HSPC. Prolonged persistence of chromosomal breaks was observed for higher LET charged particles which are known to induce more complex DNA damage compared to X-rays. Consistent with HR deficiency in HSPC observed in our previous study, we noticed here pronounced focal accumulation of 53BP1 after X-ray and carbon ion exposure (intermediate LET) in HSPC versus PBL. For higher LET, 53BP1 foci kinetics was similarly delayed in PBL and HSPC suggesting similar failure to repair complex DNA damage. Data obtained with plasmid reporter systems revealed a dose- and LET-dependent HR increase after X-ray, carbon ion and higher LET exposure, particularly in HR-proficient immortalized and primary lymphocytes, confirming preferential use of conservative HR in PBL for intermediate LET damage repair. HR measured adjacent to the leukemia-associated MLL breakpoint cluster sequence in reporter lines revealed dose dependency of potentially leukemogenic rearrangements underscoring the risk of leukemia-induction by radiation treatment. PMID:26618143

Bacterial expression and purification of a heterodimeric adrenomedullin receptor extracellular domain complex using DsbC-assisted disulfide shuffling.

PubMed

Hill, Heather E; Pioszak, Augen A

2013-03-01

Adrenomedullin (AM) is a peptide hormone that is a potent vasodilator and is essential for vascular development. The AM receptor is a heterodimeric cell surface receptor composed of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, in association with either of two receptor activity modifying protein (RAMP) coreceptors, RAMP2 or -3. The extracellular domains (ECDs) of CLR and the RAMPs form the primary AM binding site. Here, we present novel methodology for expression and purification of a heterodimeric AM receptor ECD complex as an MBP-CLR ECD fusion protein in association with the RAMP2 ECD. Co-expression of the RAMP2 ECD with the disulfide bond isomerase DsbC in the oxidizing cytoplasm of E. coli trxB gor enabled proper disulfide formation in vivo. The isolated RAMP2 ECD was purified to homogeneity. Co-expression of a soluble MBP-CLR ECD fusion protein with DsbC in E. coli trxB gor yielded a heterogeneous mixture of species with misfolded ECD. Incubation of affinity-purified MBP-CLR ECD in vitro with purified RAMP2 ECD, DsbC, and glutathione redox buffer promoted proper folding of the CLR ECD and formation of a stable MBP-CLR ECD:RAMP2 ECD complex that was purified by size-exclusion chromatography and which exhibited specific AM binding. Approximately 40mg of highly purified complex was obtained starting with 6L bacterial cultures for each protein. The methodology reported here will facilitate structure/function studies of the AM receptor. Copyright © 2012 Elsevier Inc. All rights reserved.

ERCC1-XPF Endonuclease Facilitates DNA Double-Strand Break Repair▿ †

PubMed Central

Ahmad, Anwaar; Robinson, Andria Rasile; Duensing, Anette; van Drunen, Ellen; Beverloo, H. Berna; Weisberg, David B.; Hasty, Paul; Hoeijmakers, Jan H. J.; Niedernhofer, Laura J.

2008-01-01

ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and γH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1−/− Ku86−/− fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3′ overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent. PMID:18541667

Germ line insertion of mtDNA at the breakpoint junction of a reciprocal constitutional translocation.

PubMed

Willett-Brozick, J E; Savul, S A; Richey, L E; Baysal, B E

2001-08-01

Constitutional chromosomal translocations are relatively common causes of human morbidity, yet the DNA double-strand break (DSB) repair mechanisms that generate them are incompletely understood. We cloned, sequenced and analyzed the breakpoint junctions of a familial constitutional reciprocal translocation t(9;11)(p24;q23). Within the 10-kb region flanking the breakpoints, chromosome 11 had 25% repeat elements, whereas chromosome 9 had 98% repeats, 95% of which were L1-type LINE elements. The breakpoints occurred within an L1-type repeat element at 9p24 and at the 3'-end of an Alu sequence at 11q23. At the breakpoint junction of derivative chromosome 9, we discovered an unusually large 41-bp insertion, which showed 100% identity to 12S mitochondrial DNA (mtDNA) between nucleotides 896 and 936 of the mtDNA sequence. Analysis of the human genome failed to show the preexistence of the inserted sequence at normal chromosomes 9 and 11 breakpoint junctions or elsewhere in the genome, strongly suggesting that the insertion was derived from human mtDNA and captured into the junction during the DSB repair process. To our knowledge, these findings represent the first observation of spontaneous germ line insertion of modern human mtDNA sequences and suggest that DSB repair may play a role in inter-organellar gene transfer in vivo. Our findings also provide evidence for a previously unrecognized insertional mechanism in human, by which non-mobile extra-chromosomal fragments can be inserted into the genome at DSB repair junctions.

Nucleosomes suppress the formation of double-strand DNA breaks during attempted base excision repair of clustered oxidative damages.

PubMed

Cannan, Wendy J; Tsang, Betty P; Wallace, Susan S; Pederson, David S

2014-07-18

Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Nucleosomes Suppress the Formation of Double-strand DNA Breaks during Attempted Base Excision Repair of Clustered Oxidative Damages*

PubMed Central

Cannan, Wendy J.; Tsang, Betty P.; Wallace, Susan S.; Pederson, David S.

2014-01-01

Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling. PMID:24891506

Deoxysphingoid bases as plasma markers in diabetes mellitus.

PubMed

Bertea, Mariana; Rütti, Markus F; Othman, Alaa; Marti-Jaun, Jaqueline; Hersberger, Martin; von Eckardstein, Arnold; Hornemann, Thorsten

2010-08-16

Sphingoid bases are formed from the precursors L-serine and palmitoyl-CoA-a reaction which is catalyzed by the serine-palmitoyltransferase (SPT). SPT metabolizes, besides palmitoyl-CoA also other acyl-CoAs but shows also variability towards the use of other amino acid substrates. The enzyme is also able to metabolize alanine, which results in the formation of an atypical deoxy-sphingoid base (DSB). This promiscuous activity is greatly increased in the case of the sensory neuropathy HSAN1, and pathologically elevated DSB levels have been identified as the cause of this disease. Clinically, HSAN1 shows a pronounced similarity to the diabetic sensory neuropathy (DSN), which is the most common chronic complication of diabetes mellitus. Since serine and alanine metabolism is functionally linked to carbohydrate metabolism by their precursors 3-phosphoglycerate and pyruvate, we were interested to see whether the levels of certain sphingoid base metabolites are altered in patients with diabetes. In a case-control study we compared plasma sphingoid base levels between healthy and diabetic individuals. DSB levels were higher in the diabetic group whereas C16 and C18 sphingoid bases were not significantly different. Plasma serine, but not alanine levels were lower in the diabetic group. A subsequent lipoprotein fractionation showed that the DSBs are primarily present in the LDL and VLDL fraction. Our results suggest that DSBs are a novel category of plasma biomarkers in diabetes which reflect functional impairments of carbohydrate metabolism. Furthermore, elevated DSB levels as we see them in diabetic patients might also contribute to the progression of the diabetic sensory neuropathy, the most frequent complication of diabetes.

The oxygen enhancement ratio for single- and double-strand breaks induced by tritium incorporated in DNA of cultured human T1 cells. Impact of the transmutation effect.

PubMed

Tisljar-Lentulis, G; Henneberg, P; Feinendegen, L E; Commerford, S L

1983-04-01

The effect of oxygen, expressed as the oxygen enhancement ratio (OER), on the number of single-strand breaks (SSB) and double-strand breaks (DSB) induced in DNA by the radioactive decay of tritium was measured in human T1 cells whose DNA had been labeled with tritium at carbon atom number 6 of thymidine. Decays were accumulated in vivo under aerobic conditions at 0-1 degrees C and at -196 degrees C and in a nitrogen atmosphere at 0-1 degrees C. The number of SSB and DSB produced was analyzed by sucrose gradient centrifugation. For each tritium decay there were 0.25 DSB in cells exposed to air at 0-1 degrees C and 0.07 in cells kept under nitrogen, indicating an OER of 3.6, a value expected for such low-LET radiation. However, for each tritium decay there were 1.25 SSB in cells exposed to air at 0-1 degrees C and 0.76 in cells kept under nitrogen indicating an OER of only 1.7. The corresponding values for 60Co gamma radiation, expressed as SSB per 100 eV absorbed energy, were 4.5 and 1.0, giving an OER of 4.5. The low OER value found for SSB induced by tritium decay can be explained if 31% of the total SSB produced in air result from transmutation by a mechanism which does not produce DSB and is unaffected by oxygen.

Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events.

PubMed Central

Kramer, K M; Brock, J A; Bloom, K; Moore, J K; Haber, J E

1994-01-01

In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells. PMID:8289808

Alternative end-joining pathway(s): bricolage at DNA breaks.

PubMed

Frit, Philippe; Barboule, Nadia; Yuan, Ying; Gomez, Dennis; Calsou, Patrick

2014-05-01

To cope with DNA double strand break (DSB) genotoxicity, cells have evolved two main repair pathways: homologous recombination which uses homologous DNA sequences as repair templates, and non-homologous Ku-dependent end-joining involving direct sealing of DSB ends by DNA ligase IV (Lig4). During the last two decades a third player most commonly named alternative end-joining (A-EJ) has emerged, which is defined as any Ku- or Lig4-independent end-joining process. A-EJ increasingly appears as a highly error-prone bricolage on DSBs and despite expanding exploration, it still escapes full characterization. In the present review, we discuss the mechanism and regulation of A-EJ as well as its biological relevance under physiological and pathological situations, with a particular emphasis on chromosomal instability and cancer. Whether or not it is a genuine DSB repair pathway, A-EJ is emerging as an important cellular process and understanding A-EJ will certainly be a major challenge for the coming years. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes

PubMed Central

Chakraborty, Anirban; Tapryal, Nisha; Venkova, Tatiana; Horikoshi, Nobuo; Pandita, Raj K.; Sarker, Altaf H.; Sarkar, Partha S.; Pandita, Tej K.; Hazra, Tapas K.

2016-01-01

DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome. PMID:27703167

Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off.

PubMed

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2012-08-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.

Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair.

PubMed

Zapotoczny, Grzegorz; Sekelsky, Jeff

2017-04-03

DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells. Copyright © 2017 Zapotoczny and Sekelsky.

Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair

PubMed Central

Zapotoczny, Grzegorz; Sekelsky, Jeff

2017-01-01

DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila. To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells. PMID:28179392

DNA Repair: The Search for Homology.

PubMed

Haber, James E

2018-05-01

The repair of chromosomal double-strand breaks (DSBs) by homologous recombination is essential to maintain genome integrity. The key step in DSB repair is the RecA/Rad51-mediated process to match sequences at the broken end to homologous donor sequences that can be used as a template to repair the lesion. Here, in reviewing research about DSB repair, I consider the many factors that appear to play important roles in the successful search for homology by several homologous recombination mechanisms. See also the video abstract here: https://youtu.be/vm7-X5uIzS8. © 2018 WILEY Periodicals, Inc.

A low noise 410-495 heterodyne two tuner mixer, using submicron Nb/Al2O3/Nb tunneljunctions

NASA Technical Reports Server (NTRS)

Delange, G.; Honingh, C. E.; Dierichs, M. M. T. M.; Panhuyzen, R. A.; Schaeffer, H. H. A.; Klapwijk, T. M.; Vandestadt, H.; Degraauw, M. W. M.

1992-01-01

A 410-495 GHz heterodyne receiver, with an array of two Nb/Al2O3/Nb tunneljunctions as mixing element is described. The noise temperature of this receiver is below 230 K (DSB) over the whole frequency range, and has lowest values of 160 K in the 435-460 GHz range. The calculated DSB mixergain over the whole frequency range varies from -11.9 plus or minus 0.6 dB to -12.6 plus or minus 0.6 dB and the mixer noise is 90 plus or minus 30 K.

Distyrylbenzene-aldehydes: identification of proteins in water.

PubMed

Kumpf, Jan; Freudenberg, Jan; Bunz, Uwe H F

2015-05-07

Three different, water soluble, aldehyde-appended distyrylbenzene (DSB) derivatives were prepared. Their interaction with different albumin variants (human, porcine, bovine, lactalbumin, ovalbumin) was investigated (pH 11). All three fluorophores exhibit graded, protein-dependent fluorescence turn-on at slightly differing wavelengths. Linear discriminant analysis (LDA) differentiated all of the investigated albumins and was used to discern commercially available protein shakes. The three DSB derivatives barely react with the constituting amino acids but cysteine. In the proteins significant fluorescence signals are generated, probably due to a combination of imine/N,S-aminal formation and hydrophobic interactions between the DSBs and the proteins.

Double dipole antenna SIS receivers at 100 and 400 GHz

NASA Technical Reports Server (NTRS)

Skalare, A.; Vandestadt, H.; Degraauw, T.; Panhuyzen, R. A.; Dierichs, M. M. T. M.

1992-01-01

Antenna patterns were measured between 95 and 120 GHz for a double dipole antenna / ellipsoidal lens combination. The structure produces a non-astigmatic beam with low side lobe levels over that whole band. A heterodyne SIS receiver based on this concept gave a best noise temperature of 145 K DSB at 98 GHz. Measurements were also made with a 400 GHz heterodyne SIS receiver, using a double dipole antenna in conjunction with a hyperhemispherical lens. The best noise temperature was 220 K DSB at 402 GHz. On-chip stubs were used to tune out the SIS junction capacitance.

Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks.

PubMed

Rybak, Paulina; Hoang, Agnieszka; Bujnowicz, Lukasz; Bernas, Tytus; Berniak, Krzysztof; Zarębski, Mirosław; Darzynkiewicz, Zbigniew; Dobrucki, Jerzy

2016-08-02

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities.

Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration of a transgene

PubMed Central

Furukawa, Tomoyuki; Angelis, Karel J.; Britt, Anne B.

2015-01-01

The DNA double-strand break (DSB) is a critical type of damage, and can be induced by both endogenous sources (e.g., errors of oxidative metabolism, transposable elements, programmed meiotic breaks, or perturbation of the DNA replication fork) and exogenous sources (e.g., ionizing radiation or radiomimetic chemicals). Although higher plants, like mammals, are thought to preferentially repair DSBs via nonhomologous end joining (NHEJ), much remains unclear about plant DSB repair pathways. Our reverse genetic approach suggests that DNA polymerase λ is involved in DSB repair in Arabidopsis. The Arabidopsis T-DNA insertion mutant (atpolλ-1) displayed sensitivity to both gamma-irradiation and treatment with radiomimetic reagents, but not to other DNA damaging treatments. The atpolλ-1 mutant showed a moderate sensitivity to DSBs, while Arabidopsis Ku70 and DNA ligase 4 mutants (atku70-3 and atlig4-2), both of which play critical roles in NHEJ, exhibited a hypersensitivity to these treatments. The atpolλ-1/atlig4-2 double mutant exhibited a higher sensitivity to DSBs than each single mutant, but the atku70/atpolλ-1 showed similar sensitivity to the atku70-3 mutant. We showed that transcription of the DNA ligase 1, DNA ligase 6, and Wee1 genes was quickly induced by BLM in several NHEJ deficient mutants in contrast to wild-type. Finally, the T-DNA transformation efficiency dropped in NHEJ deficient mutants and the lowest transformation efficiency was scored in the atpolλ-1/atlig4-2 double mutant. These results imply that AtPolλ is involved in both DSB repair and DNA damage response pathway. PMID:26074930

PARP1 inhibitor olaparib (Lynparza) exerts synthetic lethal effect against ligase 4-deficient melanomas

PubMed Central

Czyż, Małgorzata; Toma, Monika; Gajos-Michniewicz, Anna; Majchrzak, Kinga; Hoser, Grazyna; Szemraj, Janusz; Nieborowska-Skorska, Margaret; Cheng, Phil; Gritsyuk, Daniel; Levesque, Mitchell; Dummer, Reinhard; Sliwinski, Tomasz; Skorski, Tomasz

2016-01-01

Cancer including melanoma may be “addicted” to double strand break (DSB) repair and targeting this process could sensitize them to the lethal effect of DNA damage. PARP1 exerts an important impact on DSB repair as it binds to both single- and double- strand breaks. PARP1 inhibitors might be highly effective drugs triggering synthetic lethality in patients whose tumors have germline or somatic defects in DNA repair genes. We hypothesized that PARP1-dependent synthetic lethality could be induced in melanoma cells displaying downregulation of DSB repair genes. We observed that PARP1 inhibitor olaparib sensitized melanomas with reduced expression of DNA ligase 4 (LIG4) to an alkylatimg agent dacarbazine (DTIC) treatment in vitro, while normal melanocytes remained intact. PARP1 inhibition caused accumulation of DSBs, which was associated with apoptosis in LIG4 deficient melanoma cells. Our hypothesis that olaparib is synthetic lethal with LIG4 deficiency in melanoma cells was supported by selective anti-tumor effects of olaparib used either alone or in combination with dacarbazine (DTIC) in LIG4 deficient, but not LIG4 proficient cells. In addition, olaparib combined with DTIC inhibited the growth of LIG4 deficient human melanoma xenografts. This work for the first time demonstrates the effectiveness of a combination of PARP1 inhibitor olaparib and alkylating agent DTIC for treating LIG4 deficient melanomas. In addition, analysis of the TCGA and transcriptome microarray databases revealed numerous individual melanoma samples potentially displaying specific defects in DSB repair pathways, which may predispose them to synthetic lethality triggered by PARP1 inhibitor combined with a cytotoxic drug. PMID:27705909

Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70.

PubMed

Boboila, Cristian; Jankovic, Mila; Yan, Catherine T; Wang, Jing H; Wesemann, Duane R; Zhang, Tingting; Fazeli, Alex; Feldman, Lauren; Nussenzweig, Andre; Nussenzweig, Michel; Alt, Frederick W

2010-02-16

Class switch recombination (CSR) in B lymphocytes is initiated by introduction of multiple DNA double-strand breaks (DSBs) into switch (S) regions that flank immunoglobulin heavy chain (IgH) constant region exons. CSR is completed by joining a DSB in the donor S mu to a DSB in a downstream acceptor S region (e.g., S gamma1) by end-joining. In normal cells, many CSR junctions are mediated by classical nonhomologous end-joining (C-NHEJ), which employs the Ku70/80 complex for DSB recognition and XRCC4/DNA ligase 4 for ligation. Alternative end-joining (A-EJ) mediates CSR, at reduced levels, in the absence of C-NHEJ, even in combined absence of Ku70 and ligase 4, demonstrating an A-EJ pathway totally distinct from C-NHEJ. Multiple DSBs are introduced into S mu during CSR, with some being rejoined or joined to each other to generate internal switch deletions (ISDs). In addition, S-region DSBs can be joined to other chromosomes to generate translocations, the level of which is increased by absence of a single C-NHEJ component (e.g., XRCC4). We asked whether ISD and S-region translocations occur in the complete absence of C-NHEJ (e.g., in Ku70/ligase 4 double-deficient B cells). We found, unexpectedly, that B-cell activation for CSR generates substantial ISD in both S mu and S gamma1 and that ISD in both is greatly increased by the absence of C-NHEJ. IgH chromosomal translocations to the c-myc oncogene also are augmented in the combined absence of Ku70 and ligase 4. We discuss the implications of these findings for A-EJ in normal and abnormal DSB repair.

Detection of DNA Double Strand Breaks by γH2AX Does Not Result in 53bp1 Recruitment in Mouse Retinal Tissues

PubMed Central

Müller, Brigitte; Ellinwood, N. M.; Lorenz, Birgit; Stieger, Knut

2018-01-01

Gene editing is an attractive potential treatment of inherited retinopathies. However, it often relies on endogenous DNA repair. Retinal DNA repair is incompletely characterized in humans and animal models. We investigated recruitment of the double stranded break (DSB) repair complex of γH2AX and 53bp1 in both developing and mature mouse neuroretinas. We evaluated the immunofluorescent retinal expression of these proteins during development (P07-P30) in normal and retinal degeneration models, as well as in potassium bromate induced DSB repair in normal adult (3 months) retinal explants. The two murine retinopathy models used had different mutations in Pde6b: the severe rd1 and the milder rd10 models. Compared to normal adult retina, we found increased numbers of γH2AX positive foci in all retinal neurons of the developing retina in both model and control retinas, as well as in wild type untreated retinal explant cultures. In contrast, the 53bp1 staining of the retina differed both in amount and character between cell types at all ages and in all model systems. There was strong pan nuclear staining in ganglion, amacrine, and horizontal cells, and cone photoreceptors, which was attenuated. Rod photoreceptors did not stain unequivocally. In all samples, 53bp1 stained foci only rarely occurred. Co-localization of 53bp1 and γH2AX staining was a very rare event (< 1% of γH2AX foci in the ONL and < 3% in the INL), suggesting the potential for alternate DSB sensing and repair proteins in the murine retina. At a minimum, murine retinal DSB repair does not appear to follow canonical pathways, and our findings suggests further investigation is warranted. PMID:29765300

Induction and repair of DNA double strand breaks: the increasing spectrum of non-homologous end joining pathways.

PubMed

Mladenov, Emil; Iliakis, George

2011-06-03

A defining characteristic of damage induced in the DNA by ionizing radiation (IR) is its clustered character that leads to the formation of complex lesions challenging the cellular repair mechanisms. The most widely investigated such complex lesion is the DNA double strand break (DSB). DSBs undermine chromatin stability and challenge the repair machinery because an intact template strand is lacking to assist restoration of integrity and sequence in the DNA molecule. Therefore, cells have evolved a sophisticated machinery to detect DSBs and coordinate a response on the basis of inputs from various sources. A central function of cellular responses to DSBs is the coordination of DSB repair. Two conceptually different mechanisms can in principle remove DSBs from the genome of cells of higher eukaryotes. Homologous recombination repair (HRR) uses as template a homologous DNA molecule and is therefore error-free; it functions preferentially in the S and G2 phases. Non-homologous end joining (NHEJ), on the other hand, simply restores DNA integrity by joining the two ends, is error prone as sequence is only fortuitously preserved and active throughout the cell cycle. The basis of DSB repair pathway choice remains unknown, but cells of higher eukaryotes appear programmed to utilize preferentially NHEJ. Recent work suggests that when the canonical DNA-PK dependent pathway of NHEJ (D-NHEJ), becomes compromised an alternative NHEJ pathway and not HRR substitutes in a quasi-backup function (B-NHEJ). Here, we outline aspects of DSB induction by IR and review the mechanisms of their processing in cells of higher eukaryotes. We place particular emphasis on backup pathways of NHEJ and summarize their increasing significance in various cellular processes, as well as their potential contribution to carcinogenesis. 2011 Elsevier B.V. All rights reserved.

Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage

PubMed Central

Yang, Yang; Durando, Michael; Smith-Roe, Stephanie L.; Sproul, Chris; Greenwalt, Alicia M.; Kaufmann, William; Oh, Sehyun; Hendrickson, Eric A.; Vaziri, Cyrus

2013-01-01

The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H2O2-induced DNA damage. UVC and H2O2 treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G0, G1 and S-phase. Rad18 was important for repressing H2O2-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G1, indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H2O2-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H2O2-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G1. In contrast with G1-synchronized cultures, S-phase cells were H2O2-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G1 (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase. PMID:23295675

DOE Office of Scientific and Technical Information (OSTI.GOV)

Granzotto, Adeline; Benadjaoud, Mohamed Amine; Vogin, Guillaume

Purpose: Whereas post–radiation therapy overreactions (OR) represent a clinical and societal issue, there is still no consensual radiobiological endpoint to predict clinical radiosensitivity. Since 2003, skin biopsy specimens have been collected from patients treated by radiation therapy against different tumor localizations and showing a wide range of OR. Here, we aimed to establish quantitative links between radiobiological factors and OR severity grades that would be relevant to radioresistant and genetic hyperradiosensitive cases. Methods and Materials: Immunofluorescence experiments were performed on a collection of skin fibroblasts from 12 radioresistant, 5 hyperradiosensitive, and 100 OR patients irradiated at 2 Gy. The numbers ofmore » micronuclei, γH2AX, and pATM foci that reflect different steps of DNA double-strand breaks (DSB) recognition and repair were assessed from 10 minutes to 24 hours after irradiation and plotted against the severity grades established by the Common Terminology Criteria for Adverse Events and the Radiation Therapy Oncology Group. Results: OR patients did not necessarily show a gross DSB repair defect but a systematic delay in the nucleoshuttling of the ATM protein required for complete DSB recognition. Among the radiobiological factors, the maximal number of pATM foci provided the best discrimination among OR patients and a significant correlation with each OR severity grade, independently of tumor localization and of the early or late nature of reactions. Conclusions: Our results are consistent with a general classification of human radiosensitivity based on 3 groups: radioresistance (group I); moderate radiosensitivity caused by delay of nucleoshuttling of ATM, which includes OR patients (group II); and hyperradiosensitivity caused by a gross DSB repair defect, which includes fatal cases (group III).« less

Deoxysphingoid bases as plasma markers in Diabetes mellitus

PubMed Central

2010-01-01

Background Sphingoid bases are formed from the precursors L-serine and palmitoyl-CoA-a reaction which is catalyzed by the serine-palmitoyltransferase (SPT). SPT metabolizes, besides palmitoyl-CoA also other acyl-CoAs but shows also variability towards the use of other amino acid substrates. The enzyme is also able to metabolize alanine, which results in the formation of an atypical deoxy-sphingoid base (DSB). This promiscuous activity is greatly increased in the case of the sensory neuropathy HSAN1, and pathologically elevated DSB levels have been identified as the cause of this disease. Clinically, HSAN1 shows a pronounced similarity to the diabetic sensory neuropathy (DSN), which is the most common chronic complication of diabetes mellitus. Since serine and alanine metabolism is functionally linked to carbohydrate metabolism by their precursors 3-phosphoglycerate and pyruvate, we were interested to see whether the levels of certain sphingoid base metabolites are altered in patients with diabetes. Results In a case-control study we compared plasma sphingoid base levels between healthy and diabetic individuals. DSB levels were higher in the diabetic group whereas C16 and C18 sphingoid bases were not significantly different. Plasma serine, but not alanine levels were lower in the diabetic group. A subsequent lipoprotein fractionation showed that the DSBs are primarily present in the LDL and VLDL fraction. Conclusion Our results suggest that DSBs are a novel category of plasma biomarkers in diabetes which reflect functional impairments of carbohydrate metabolism. Furthermore, elevated DSB levels as we see them in diabetic patients might also contribute to the progression of the diabetic sensory neuropathy, the most frequent complication of diabetes. PMID:20712864

Sexual Behavior and Concerns in a Sample of Elderly, Former Indentured Swiss Child Laborers

PubMed Central

Burri, Andrea; Maercker, Andreas

2015-01-01

Abstract Introduction Past research suggests a link between post‐traumatic stress disorder (PTSD) and an increased risk for sexual problems. However, there is still no clear picture whether these higher rates are related to trauma exposure or to PTSD itself. Aim The aim of the present study was to complement existing knowledge on the relative impact of trauma and PTSD on sexuality in later life, considering different aspects of trauma exposure on both men and women. Methods The study was conducted on a unique population sample of former Swiss indentured child laborers (55 men, M age 78, age range 60–95 years) who have repeatedly experienced a variety of severe childhood traumas. Main Outcome Measures Sexual outcomes were measured using two scales from the Trauma Symptom Inventory—Dysfunctional Sexual Behavior (DSB) and Sexual Concerns (SC). PTSD symptoms and trauma were assessed with the Short Screening Scale for PTSD and the Composite International Diagnostic Interview, respectively. Results Twenty‐two individuals showed PTSD symptoms, and 53 reported having experienced childhood trauma. Significant differences between men and women were reported for DSB and SC. Men reported a significantly higher prevalence of both SC and DSB compared with women. Conclusions This is the very first study investigating DSB and SC in a sample of older adults exposed to similar traumatic experiences and settings. However, some study limitations need to be considered such as the small sample size. Additional studies are needed to further explore the relative role of traumatization and PTSD on sexual behavior and well‐being, especially to improve sexual therapy for patients who experience trauma. Rechsteiner K, Burri A, and Maercker A. Sexual behavior and concerns in a sample of elderly, former indentured Swiss child laborers. Sex Med 2015;3:305–314. PMID:26797066

SV40 Utilizes ATM Kinase Activity to Prevent Non-homologous End Joining of Broken Viral DNA Replication Products

PubMed Central

Sowd, Gregory A.; Mody, Dviti; Eggold, Joshua; Cortez, David; Friedman, Katherine L.; Fanning, Ellen

2014-01-01

Simian virus 40 (SV40) and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PKcs kinase activity, facilitates some aspects of double strand break (DSB) repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR) and do not colocalize with non-homologous end joining (NHEJ) factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PKcs and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5′ to 3′ end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication. PMID:25474690

Epigenetic therapy with inhibitors of histone methylation suppresses DNA damage signaling and increases glioma cell radiosensitivity.

PubMed

Gursoy-Yuzugullu, Ozge; Carman, Chelsea; Serafim, Rodolfo Bortolozo; Myronakis, Marios; Valente, Valeria; Price, Brendan D

2017-04-11

Radiation therapy is widely used to treat human malignancies, but many tumor types, including gliomas, exhibit significant radioresistance. Radiation therapy creates DNA double-strand breaks (DSBs), and DSB repair is linked to rapid changes in epigenetic modifications, including increased histone methylation. This increased histone methylation recruits DNA repair proteins which can then alter the local chromatin structure and promote repair. Consequently, combining inhibitors of specific histone methyltransferases with radiation therapy may increase tumor radiosensitivity, particularly in tumors with significant therapeutic resistance. Here, we demonstrate that inhibitors of the H4K20 methyltransferase SETD8 (UNC-0379) and the H3K9 methyltransferase G9a (BIX-01294) are effective radiosensitizers of human glioma cells. UNC-0379 blocked H4K20 methylation and reduced recruitment of the 53BP1 protein to DSBs, although this loss of 53BP1 caused only limited changes in radiosensitivity. In contrast, loss of H3K9 methylation through G9a inhibition with BIX-01294 increased radiosensitivity of a panel of glioma cells (SER2Gy range: 1.5 - 2.9). Further, loss of H3K9 methylation reduced DSB signaling dependent on H3K9, including reduced activation of the Tip60 acetyltransferase, loss of ATM signaling and reduced phosphorylation of the KAP-1 repressor. In addition, BIX-0194 inhibited DSB repair through both the homologous recombination and nonhomologous end-joining pathways. Inhibition of G9a and loss of H3K9 methylation is therefore an effective approach for increasing radiosensitivity of glioma cells. These results suggest that combining inhibitors of histone methyltransferases which are critical for DSB repair with radiation therapy may provide a new therapeutic route for sensitizing gliomas and other tumors to radiation therapy.

Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway.

PubMed

Nakada, Daisuke; Hirano, Yukinori; Sugimoto, Katsunori

2004-11-01

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.

Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast.

PubMed

Li, Ping; Jin, Hui; Yu, Hong-Guo

2014-10-01

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. © 2014 Li et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Development of a Bone-Conducted Ultrasonic Hearing Aid for the Profoundly Deaf: Evaluation of Sound Quality Using a Semantic Differential Method

NASA Astrophysics Data System (ADS)

Nakagawa, Seiji; Fujiyuki, Chika; Kagomiya, Takayuki

2013-07-01

Bone-conducted ultrasound (BCU) is perceived even by the profoundly sensorineural deaf. A novel hearing aid using the perception of amplitude-modulated BCU (BCU hearing aid: BCUHA) has been developed. However, there is room for improvement particularly in terms of sound quality. BCU speech is accompanied by a strong high-pitched tone and contain some distortion. In this study, the sound quality of BCU speech with several types of amplitude modulation [double-sideband with transmitted carrier (DSB-TC), double-sideband with suppressed carrier (DSB-SC), and transposed modulations] and air-conducted (AC) speech was quantitatively evaluated using semantic differential and factor analysis. The results showed that all the types of BCU speech had higher metallic and lower esthetic factor scores than AC speech. On the other hand, transposed speech was closer than the other types of BCU speech to AC speech generally; the transposed speech showed a higher powerfulness factor score than the other types of BCU speech and a higher esthetic factor score than DSB-SC speech. These results provide useful information for further development of the BCUHA.

SAW1 is required for SDSA double-strand break repair in S. cerevisiae.

PubMed

Diamante, Graciel; Phan, Claire; Celis, Angie S; Krueger, Jonas; Kelson, Eric P; Fischhaber, Paula L

2014-03-14

SAW1, coding for Saw1, is required for single-strand annealing (SSA) DNA double-strand break (DSB) repair in Saccharomycescerevisiae. Saw1 physically associates with Rad1 and Rad52 and recruits the Rad1-Rad10 endonuclease. Herein we show by fluorescence microscopy that SAW1 is similarly required for recruitment of Rad10 to sites of Synthesis-Dependent Strand Annealing (SDSA) and associates with sites of SDSA repair in a manner temporally overlapped with Rad10. The magnitude of induction of colocalized Saw1-CFP/Rad10-YFP/DSB-RFP foci in SDSA is more dramatic in S and G2 phase cells than in M phase, consistent with the known mechanism of SDSA. We observed a substantial fraction of foci in which Rad10 was localized to the repair site without Saw1, but few DSB sites that contained Saw1 without Rad10. Together these data are consistent with a model in which Saw1 recruits Rad1-Rad10 to SDSA sites, possibly even binding as a protein-protein complex, but departs the repair site in advance of Rad1-Rad10. Copyright © 2014 Elsevier Inc. All rights reserved.

Roles for the Rad27 Flap Endonuclease in Mitochondrial Mutagenesis and Double-Strand Break Repair in Saccharomyces cerevisiae.

PubMed

Nagarajan, Prabha; Prevost, Christopher T; Stein, Alexis; Kasimer, Rachel; Kalifa, Lidza; Sia, Elaine A

2017-06-01

The structure-specific nuclease, Rad27p/FEN1, plays a crucial role in DNA repair and replication mechanisms in the nucleus. Genetic assays using the rad27-∆ mutant have shown altered rates of DNA recombination, microsatellite instability, and point mutation in mitochondria. In this study, we examined the role of Rad27p in mitochondrial mutagenesis and double-strand break (DSB) repair in Saccharomyces cerevisiae Our findings show that Rad27p is essential for efficient mitochondrial DSB repair by a pathway that generates deletions at a region flanked by direct repeat sequences. Mutant analysis suggests that both exonuclease and endonuclease activities of Rad27p are required for its role in mitochondrial DSB repair. In addition, we found that the nuclease activities of Rad27p are required for the prevention of mitochondrial DNA (mtDNA) point mutations, and in the generation of spontaneous mtDNA rearrangements. Overall, our findings underscore the importance of Rad27p in the maintenance of mtDNA, and demonstrate that it participates in multiple DNA repair pathways in mitochondria, unlinked to nuclear phenotypes. Copyright © 2017 by the Genetics Society of America.

The Disulfide Bonding System Suppresses CsgD-Independent Cellulose Production in Escherichia coli

PubMed Central

Hufnagel, David A.; DePas, William H.

2014-01-01

The bacterial extracellular matrix encases cells and protects them from host-related and environmental insults. The Escherichia coli master biofilm regulator CsgD is required for the production of the matrix components curli and cellulose. CsgD activates the diguanylate cyclase AdrA, which in turn stimulates cellulose production through cyclic di-GMP (c-di-GMP). Here, we identified and characterized a CsgD- and AdrA-independent cellulose production pathway that was maximally active when cultures were grown under reducing conditions or when the disulfide bonding system (DSB) was compromised. The CsgD-independent cellulose activation pathway was dependent on a second diguanylate cyclase, called YfiN. c-di-GMP production by YfiN was repressed by the periplasmic protein YfiR, and deletion of yfiR promoted CsgD-independent cellulose production. Conversely, when YfiR was overexpressed, cellulose production was decreased. Finally, we found that YfiR was oxidized by DsbA and that intraprotein YfiR disulfide bonds stabilized YfiR in the periplasm. Altogether, we showed that reducing conditions and mutations in the DSB system caused hyperactivation of YfiN and subsequent CsgD-independent cellulose production. PMID:25112475

The disulfide bonding system suppresses CsgD-independent cellulose production in Escherichia coli.

PubMed

Hufnagel, David A; DePas, William H; Chapman, Matthew R

2014-11-01

The bacterial extracellular matrix encases cells and protects them from host-related and environmental insults. The Escherichia coli master biofilm regulator CsgD is required for the production of the matrix components curli and cellulose. CsgD activates the diguanylate cyclase AdrA, which in turn stimulates cellulose production through cyclic di-GMP (c-di-GMP). Here, we identified and characterized a CsgD- and AdrA-independent cellulose production pathway that was maximally active when cultures were grown under reducing conditions or when the disulfide bonding system (DSB) was compromised. The CsgD-independent cellulose activation pathway was dependent on a second diguanylate cyclase, called YfiN. c-di-GMP production by YfiN was repressed by the periplasmic protein YfiR, and deletion of yfiR promoted CsgD-independent cellulose production. Conversely, when YfiR was overexpressed, cellulose production was decreased. Finally, we found that YfiR was oxidized by DsbA and that intraprotein YfiR disulfide bonds stabilized YfiR in the periplasm. Altogether, we showed that reducing conditions and mutations in the DSB system caused hyperactivation of YfiN and subsequent CsgD-independent cellulose production. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Replication-mediated disassociation of replication protein A–XPA complex upon DNA damage: implications for RPA handing off

PubMed Central

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2013-01-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA–XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA–XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA–XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed. PMID:22578086

Disulfide Bond Formation and ToxR Activity in Vibrio cholerae

PubMed Central

Fengler, Vera H. I.; Boritsch, Eva C.; Tutz, Sarah; Seper, Andrea; Ebner, Hanna; Roier, Sandro; Schild, Stefan; Reidl, Joachim

2012-01-01

Virulence factor production in Vibrio cholerae is complex, with ToxRS being an important part of the regulatory cascade. Additionally, ToxR is the transcriptional regulator for the genes encoding the major outer membrane porins OmpU and OmpT. ToxR is a transmembrane protein and contains two cysteine residues in the periplasmic domain. This study addresses the influence of the thiol-disulfide oxidoreductase system DsbAB, ToxR cysteine residues and ToxR/ToxS interaction on ToxR activity. The results show that porin production correlates with ToxR intrachain disulfide bond formation, which depends on DsbAB. In contrast, formation of ToxR intrachain or interchain disulfide bonds is dispensable for virulence factor production and in vivo colonization. This study further reveals that in the absence of ToxS, ToxR interchain disulfide bond formation is facilitated, whereat cysteinyl dependent homo- and oligomerization of ToxR is suppressed if ToxS is coexpressed. In summary, new insights into gene regulation by ToxR are presented, demonstrating a mechanism by which ToxR activity is linked to a DsbAB dependent intrachain disulfide bond formation. PMID:23144706

MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

PubMed

Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

2014-07-10

Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Analysis of Terminal Deletions using a Generalized Time-Dependent Model of Radiation-Induced Formation of Chromosomal Aberrations

NASA Technical Reports Server (NTRS)

Ponomarev, Artem L.; George, K.; Cucinotta, Francis A.

2011-01-01

We have developed a model that can simulate different types of radiation induced chromosomal aberrations (CA's) and can provide predictions on the frequency and size of chromosomes with terminal deletions. Chromosomes with terminal deletions lack telomeres and this can elicit sister chromatid unions and the prolonged breakage/fusion/bridge (B/F/B) cycles that have been observed in mammalian tumors. The loss of a single telomere has been shown to cause extensive genomic instability through the B/F/B cycle process. Our model uses a stochastic process of DNA broken end joining, in which a realistic spectrum of CA's is created from improperly joined DNA free ends formed by DNA double strand breaks (DSBs). The distribution of the DNA free ends is given by a mechanistic model that takes into account the chromatin structure and track structure for high-LET radiation. The model allows for DSB clustering from high-LET radiation and simulates the formation of CA's in stages that correspond to the actual time after radiation exposure. The time scale for CA formation is derived from experimental data on DSB repair kinetics. At any given time a nucleus may have intact chromosomes, CA's, and/or unrepaired fragments, some of which are defined as terminal deletions, if they are capped by one telomere. The model produces a spectrum of terminal deletions with their corresponding probabilities and size distributions for different heavy ions exposures for the first division after exposure. This data provides valuable information because there is limited experimental data available in the literature on the on the actual size of terminal deletions. We compare our model output to the available experimental data and make a reasonable extrapolation on the number of chromosomes lacking telomeres in human lymphocytes exposed to heavy ions. This model generates data which may lead to predictions on the rate of genomic instability in cells after exposure to high charge and energy nuclei affecting astronauts during space missions.

Lentivirus-meditated frataxin gene delivery reverses genome instability in Friedreich ataxia patient and mouse model fibroblasts.

PubMed

Khonsari, H; Schneider, M; Al-Mahdawi, S; Chianea, Y G; Themis, M; Parris, C; Pook, M A; Themis, M

2016-12-01

Friedreich ataxia (FRDA) is a progressive neurodegenerative disease caused by deficiency of frataxin protein, with the primary sites of pathology being the large sensory neurons of the dorsal root ganglia and the cerebellum. FRDA is also often accompanied by severe cardiomyopathy and diabetes mellitus. Frataxin is important in mitochondrial iron-sulfur cluster (ISC) biogenesis and low-frataxin expression is due to a GAA repeat expansion in intron 1 of the FXN gene. FRDA cells are genomically unstable, with increased levels of reactive oxygen species and sensitivity to oxidative stress. Here we report the identification of elevated levels of DNA double strand breaks (DSBs) in FRDA patient and YG8sR FRDA mouse model fibroblasts compared to normal fibroblasts. Using lentivirus FXN gene delivery to FRDA patient and YG8sR cells, we obtained long-term overexpression of FXN mRNA and frataxin protein levels with reduced DSB levels towards normal. Furthermore, γ-irradiation of FRDA patient and YG8sR cells revealed impaired DSB repair that was recovered on FXN gene transfer. This suggests that frataxin may be involved in DSB repair, either directly by an unknown mechanism, or indirectly via ISC biogenesis for DNA repair enzymes, which may be essential for the prevention of neurodegeneration.

Meiotic Recombination Initiated by a Double-Strand Break in Rad50Δ Yeast Cells Otherwise Unable to Initiate Meiotic Recombination

PubMed Central

Malkova, A.; Ross, L.; Dawson, D.; Hoekstra, M. F.; Haber, J. E.

1996-01-01

Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SPO13 promoter, creating a DSB at a single site on one of yeast's 16 chromosomes. In Rad(+) strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13::HO induced gene conversions both in Rad(+) and in rad50Δ cells that cannot initiate normal meiotic DSBs. We find that the RAD50 gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad50Δ cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome does not signal an arrest of progression through meiosis. The recombination defect in rad50Δ diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination. PMID:8725223

Local and sex-specific biases in crossover vs. noncrossover outcomes at meiotic recombination hot spots in mice

PubMed Central

de Boer, Esther; Jasin, Maria; Keeney, Scott

2015-01-01

Meiotic recombination initiated by programmed double-strand breaks (DSBs) yields two types of interhomolog recombination products, crossovers and noncrossovers, but what determines whether a DSB will yield a crossover or noncrossover is not understood. In this study, we analyzed the influence of sex and chromosomal location on mammalian recombination outcomes by constructing fine-scale recombination maps in both males and females at two mouse hot spots located in different regions of the same chromosome. These include the most comprehensive maps of recombination hot spots in oocytes to date. One hot spot, located centrally on chromosome 1, behaved similarly in male and female meiosis: Crossovers and noncrossovers formed at comparable levels and ratios in both sexes. In contrast, at a distal hot spot, crossovers were recovered only in males even though noncrossovers were obtained at similar frequencies in both sexes. These findings reveal an example of extreme sex-specific bias in recombination outcome. We further found that estimates of relative DSB levels are surprisingly poor predictors of relative crossover frequencies between hot spots in males. Our results demonstrate that the outcome of mammalian meiotic recombination can be biased, that this bias can vary depending on location and cellular context, and that DSB frequency is not the only determinant of crossover frequency. PMID:26251527

Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination.

PubMed

Sugawara, N; Pâques, F; Colaiácovo, M; Haber, J E

1997-08-19

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles. When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3' tail.

Chromatin- and temperature-dependent modulation of radiation-induced double-strand breaks.

PubMed

Elmroth, K; Nygren, J; Stenerlöw, B; Hultborn, R

2003-10-01

To investigate the influence of chromatin organization and scavenging capacity in relation to irradiation temperature on the induction of double-strand breaks (DSB) in structures derived from human diploid fibroblasts. Agarose plugs with different chromatin structures (intact cells+/-wortmannin, permeabilized cells with condensed chromatin, nucleoids and DNA) were prepared and irradiated with X-rays at 2 or 37 degrees C and lysed using two different lysis protocols (new ice-cold lysis or standard lysis at 37 degrees C). Induction of DSB was determined by constant-field gel electrophoresis. The dose-modifying factor (DMF(temp)) for irradiation at 37 compared with 2 degrees C was 0.92 in intact cells (i.e. more DSB induced at 2 degrees C), but gradually increased to 1.5 in permeabilized cells, 2.2 in nucleoids and 2.6 in naked DNA, suggesting a role of chromatin organization for temperature modulation of DNA damage. In addition, DMF(temp) was influenced by the presence of 0.1 M DMSO or 30 mM glutathione, but not by post-irradiation temperature. The protective effect of low temperature was correlated to the indirect effects of ionizing radiation and was not dependent on post-irradiation temperature. Reasons for a dose modifying factor <1 in intact cells are discussed.

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

PubMed Central

Bolderson, Emma; Tomimatsu, Nozomi; Richard, Derek J.; Boucher, Didier; Kumar, Rakesh; Pandita, Tej K.; Burma, Sandeep; Khanna, Kum Kum

2010-01-01

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability. PMID:20019063

ATM and KAT5 safeguard replicating chromatin against formaldehyde damage

PubMed Central

Ortega-Atienza, Sara; Wong, Victor C.; DeLoughery, Zachary; Luczak, Michal W.; Zhitkovich, Anatoly

2016-01-01

Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA. PMID:26420831

Endogenous Hot Spots of De Novo Telomere Addition in the Yeast Genome Contain Proximal Enhancers That Bind Cdc13

PubMed Central

Obodo, Udochukwu C.; Epum, Esther A.; Platts, Margaret H.; Seloff, Jacob; Dahlson, Nicole A.; Velkovsky, Stoycho M.; Paul, Shira R.

2016-01-01

DNA double-strand breaks (DSBs) pose a threat to genome stability and are repaired through multiple mechanisms. Rarely, telomerase, the enzyme that maintains telomeres, acts upon a DSB in a mutagenic process termed telomere healing. The probability of telomere addition is increased at specific genomic sequences termed sites of repair-associated telomere addition (SiRTAs). By monitoring repair of an induced DSB, we show that SiRTAs on chromosomes V and IX share a bipartite structure in which a core sequence (Core) is directly targeted by telomerase, while a proximal sequence (Stim) enhances the probability of de novo telomere formation. The Stim and Core sequences are sufficient to confer a high frequency of telomere addition to an ectopic site. Cdc13, a single-stranded DNA binding protein that recruits telomerase to endogenous telomeres, is known to stimulate de novo telomere addition when artificially recruited to an induced DSB. Here we show that the ability of the Stim sequence to enhance de novo telomere addition correlates with its ability to bind Cdc13, indicating that natural sites at which telomere addition occurs at high frequency require binding by Cdc13 to a sequence 20 to 100 bp internal from the site at which telomerase acts to initiate de novo telomere addition. PMID:27044869

Controlled transition dipole alignment of energy donor and energy acceptor molecules in doped organic crystals, and the effect on intermolecular Förster energy transfer.

PubMed

Wang, Huan; Yue, Bailing; Xie, Zengqi; Gao, Bingrong; Xu, Yuanxiang; Liu, Linlin; Sun, Hongbo; Ma, Yuguang

2013-03-14

The orientation factor κ(2) ranging from 0 to 4, which depends on the relative orientation of the transition dipoles of the energy donor (D) and the energy acceptor (A) in space, is one of the pivotal factors deciding the efficiency and directionality of resonance energy transfer (RET) in a D-A molecular system. In this work, tetracene (Tc) and pentacene (Pc) are successfully doped in a trans-1,4-distyrylbenzene (DSB) crystalline lattice to form definite D-A mutually perpendicular transition dipole orientations. The cross D-A dipole arrangement results in an extremely small orientation factor, which is about two orders smaller than that in the disordered films. The energy transfer properties from the host (DSB) to the guest (Tc/Pc) were investigated in detail by steady-state as well as time-resolved fluorescence spectroscopy. Our experimental research results show that the small value of κ(2) allows less or partial energy transfer from the host (DSB) to the guest (Tc) in a wide range of guest concentration, with the Förster distance of around 1.5 nm. By controlling the doping concentrations in the Tc and Pc doubly doped DSB crystals, we demonstrate, as an example, for the first time the application of the restricted energy transfer by D-A cross transition dipole arrangement for preparation of a large-size, white-emissive organic crystal with the CIE coordinates of (0.36, 0.37) approaching an ideal white light. In contrast, Tc is also doped in an anthracene crystalline lattice to form head-to-tail D-A transition dipole alignment, which is proved to be highly effective to promote the intermolecular energy transfer. In this doped system, the orientation factor is relatively large and the Förster distance is around 7 nm.

Cisplatin Radiosensitization of DNA Irradiated with 2-20 eV Electrons: Role of Transient Anions.

PubMed

Bao, Qianhong; Chen, Yunfeng; Zheng, Yi; Sanche, Léon

2014-06-20

Platinum chemotherapeutic agents, such as cisplatin ( cis -diamminedichloroplatinum(II)), can act as radiosensitizers when bound covalently to nuclear DNA in cancer cells. This radiosensitization is largely due to an increase in DNA damage induced by low-energy secondary electrons, produced in large quantities by high-energy radiation. We report the yields of single- and double-strand breaks (SSB and DSB) and interduplex cross-links (CL) induced by electrons of 1.6-19.6 eV (i.e., the yield functions) incident on 5 monolayer (ML) films of cisplatin-DNA complexes. These yield functions are compared with those previously recorded with 5 ML films of unmodified plasmid DNA. Binding of five cisplatin molecules to plasmid DNA (3197 base pairs) enhances SSB, DSB, and CL by factors varying, from 1.2 to 2.8, 1.4 to 3.5, and 1.2 to 2.7, respectively, depending on electron energy. All yield functions exhibit structures around 5 and 10 eV that can be attributed to enhancement of bond scission, via the initial formation of core-excited resonances associated with π → π * transitions of the bases. This increase in damage is interpreted as arising from a modification of the parameters of the corresponding transient anions already present in nonmodified DNA, particularly those influencing molecular dissociation. Two additional resonances, specific to cisplatin-modified DNA, are formed at 13.6 and 17.6 eV in the yield function of SSB. Furthermore, cisplatin binding causes the induction of DSB by electrons of 1.6-3.6 eV, i.e., in an energy region where a DSB cannot be produced by a single electron in pure DNA. Breaking two bonds with a subexcitation-energy electron is tentatively explained by a charge delocalization mechanism, where a single electron occupies simultaneously two σ * bonds linking the Pt atom to guanine bases on opposite strands.

Cisplatin Radiosensitization of DNA Irradiated with 2–20 eV Electrons: Role of Transient Anions

PubMed Central

Bao, Qianhong; Chen, Yunfeng; Zheng, Yi; Sanche, Léon

2015-01-01

Platinum chemotherapeutic agents, such as cisplatin (cis-diamminedichloroplatinum(II)), can act as radiosensitizers when bound covalently to nuclear DNA in cancer cells. This radiosensitization is largely due to an increase in DNA damage induced by low-energy secondary electrons, produced in large quantities by high-energy radiation. We report the yields of single- and double-strand breaks (SSB and DSB) and interduplex cross-links (CL) induced by electrons of 1.6–19.6 eV (i.e., the yield functions) incident on 5 monolayer (ML) films of cisplatin–DNA complexes. These yield functions are compared with those previously recorded with 5 ML films of unmodified plasmid DNA. Binding of five cisplatin molecules to plasmid DNA (3197 base pairs) enhances SSB, DSB, and CL by factors varying, from 1.2 to 2.8, 1.4 to 3.5, and 1.2 to 2.7, respectively, depending on electron energy. All yield functions exhibit structures around 5 and 10 eV that can be attributed to enhancement of bond scission, via the initial formation of core-excited resonances associated with π → π* transitions of the bases. This increase in damage is interpreted as arising from a modification of the parameters of the corresponding transient anions already present in nonmodified DNA, particularly those influencing molecular dissociation. Two additional resonances, specific to cisplatin-modified DNA, are formed at 13.6 and 17.6 eV in the yield function of SSB. Furthermore, cisplatin binding causes the induction of DSB by electrons of 1.6–3.6 eV, i.e., in an energy region where a DSB cannot be produced by a single electron in pure DNA. Breaking two bonds with a subexcitation-energy electron is tentatively explained by a charge delocalization mechanism, where a single electron occupies simultaneously two σ* bonds linking the Pt atom to guanine bases on opposite strands. PMID:26793285

Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

2008-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hammel, Michal; Yu, Yaping; Radhakrishnan, Sarvan K.

DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). But, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcsmore » (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Our collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.« less

An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex

DOE PAGES

Hammel, Michal; Yu, Yaping; Radhakrishnan, Sarvan K.; ...

2016-11-14

DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). But, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcsmore » (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Our collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.« less

Calculation of DNA strand breaks due to direct and indirect effects of Auger electrons from incorporated 123I and 125I radionuclides using the Geant4 computer code.

PubMed

Raisali, Gholamreza; Mirzakhanian, Lalageh; Masoudi, Seyed Farhad; Semsarha, Farid

2013-01-01

In this work the number of DNA single-strand breaks (SSB) and double-strand breaks (DSB) due to direct and indirect effects of Auger electrons from incorporated (123)I and (125)I have been calculated by using the Geant4-DNA toolkit. We have performed and compared the calculations for several cases: (125)I versus (123)I, source positions and direct versus indirect breaks to study the capability of the Geant4-DNA in calculations of DNA damage yields. Two different simple geometries of a 41 base pair of B-DNA have been simulated. The location of (123)I has been considered to be in (123)IdUrd and three different locations for (125)I. The results showed that the simpler geometry is sufficient for direct break calculations while indirect damage yield is more sensitive to the helical shape of DNA. For (123)I Auger electrons, the average number of DSB due to the direct hits is almost twice the DSB due to the indirect hits. Furthermore, a comparison between the average number of SSB or DSB caused by Auger electrons of (125)I and (123)I in (125)IdUrd and (123)IdUrd shows that (125)I is 1.5 times more effective than (123)I per decay. The results are in reasonable agreement with previous experimental and theoretical results which shows the applicability of the Geant-DNA toolkit in nanodosimetry calculations which benefits from the open-source accessibility with the advantage that the DNA models used in this work enable us to save the computational time. Also, the results showed that the simpler geometry is suitable for direct break calculations, while for the indirect damage yield, the more precise model is preferred.

Excess single-stranded DNA inhibits meiotic double-strand break repair.

PubMed

Johnson, Rebecca; Borde, Valérie; Neale, Matthew J; Bishop-Bailey, Anna; North, Matthew; Harris, Sheila; Nicolas, Alain; Goldman, Alastair S H

2007-11-01

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.

Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair

PubMed Central

Bishop-Bailey, Anna; North, Matthew; Harris, Sheila; Nicolas, Alain; Goldman, Alastair S. H

2007-01-01

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1.We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Δ cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Δ cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins. PMID:18081428

Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukuchi, Mikoto; Wanotayan, Rujira; Liu, Sicheng

2015-06-12

XRCC4 and DNA Ligase IV (LIG4) cooperate to join two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). However, it is not fully understood how these proteins are localized to the nucleus. Here we created XRCC4{sup K271R} mutant, as Lys271 lies within the putative nuclear localization signal (NLS), and XRCC4{sup K210R} mutant, as Lys210 was reported to undergo SUMOylation, implicated in the nuclear localization of XRCC4. Wild-type and mutated XRCC4 with EGFP tag were introduced into HeLa cell, in which endogenous XRCC4 had been knocked down using siRNA directed to 3′-untranslated region,more » and tested for the nuclear localization function by fluorescence microscopy. XRCC4{sup K271R} was defective in the nuclear localization of itself and LIG4, whereas XRCC4{sup K210R} was competent for the nuclear localization with LIG4. To examine DSB repair function, wild-type and mutated XRCC4 were introduced into XRCC4-deficient M10. M10-XRCC4{sup K271R}, but not M10-XRCC4{sup K210R}, showed significantly reduced surviving fraction after 2 Gy γ-ray irradiation as compared to M10-XRCC4{sup WT}. The number of γ-H2AX foci remaining 2 h after 2 Gy γ-ray irradiation was significantly greater in M10-XRCC4{sup K271R} than in M10-XRCC4{sup WT}, whereas it was only marginally increased in M10-XRCC4{sup K210R} as compared to M10-XRCC4{sup WT}. The present results collectively indicated that Lys271, but not Lys210, of XRCC4 is required for the nuclear localization of XRCC4 and LIG4 and that the nuclear localizing ability is essential for DSB repair function of XRCC4. - Highlights: • XRCC4{sup K271R} is defective in the nuclear localization of itself and LIG4. • XRCC4{sup K210R} is competent for the nuclear localization of itself and LIG4. • XRCC4{sup K271R} is deficient in DSB repair function. • XRCC4{sup K210R} is mostly normal in DSB repair function.« less

Radar signal transmission and switching over optical networks

NASA Astrophysics Data System (ADS)

Esmail, Maged A.; Ragheb, Amr; Seleem, Hussein; Fathallah, Habib; Alshebeili, Saleh

2018-03-01

In this paper, we experimentally demonstrate a radar signal distribution over optical networks. The use of fiber enables us to distribute radar signals to distant sites with a low power loss. Moreover, fiber networks can reduce the radar system cost, by sharing precise and expensive radar signal generation and processing equipment. In order to overcome the bandwidth challenges in electrical switches, a semiconductor optical amplifier (SOA) is used as an all-optical device for wavelength conversion to the desired port (or channel) of a wavelength division multiplexing (WDM) network. Moreover, the effect of chromatic dispersion in double sideband (DSB) signals is combated by generating optical single sideband (OSSB) signals. The optimal values of the SOA device parameters required to generate an OSSB with a high sideband suppression ratio (SSR) are determined. We considered various parameters such as injection current, pump power, and probe power. In addition, the effect of signal wavelength conversion and transmission over fiber are studied in terms of signal dynamic range.

Dependence on radiation quality of DNA fragmentation spectra

NASA Astrophysics Data System (ADS)

Campa, Alessandro; Ottolenghi, Andrea; Alloni, Daniele; Ballarini, Francesca; Belli, Mauro; Esposito, Giuseppe; Facoetti, Angelica; Friedland, Werner; Liotta, Marco; Paretzke, Herwig

Energy deposition by radiation initially gives rise to cellular critical lesions such as DNA doublestrand breaks (DSB), that later lead to the formation of relevant biological endpoints. Studies on fragment size distributions induced by radiations of various qualities can be of great help in linking the characteristics of radiation to cellular endpoints, providing information for understanding the main mechanisms of cell damage. Here we are concerned with the damage induced by heavy charged particles; this issue is very important in the field of radioprotection of astronauts participating in long term space missions, besides being relevant also in other fields, like hadrontherapy. Galactic Cosmic Rays contain a large component of high-LET particles (HZE), e.g. helium and carbon ions, as well as highcharge particles such as iron ions. These particles are characterized by complex track structures with energy depositions not only along the path of the primary particle, but also at relatively large distance form the path, due to the presence of high energy secondary electrons. In this work we have simulated the irradiation of human fibroblasts with γ-rays, protons, helium, carbon and iron ions at a fixed dose with the biophysical Monte Carlo code PARTRAC,and calculated the induction of DSB. The PARTRAC code includes accurate representation of the chromatin geometry and of the physical and physico-chemical processes associated with the energy deposition by radiation. The results of a first validation of the code have been reported in A. Campa et al. (2005) and D. Alloni et al. (2007a, 2007b). DNA fragment spectra were calculated based on the DSB induction patterns and compared in particular for particles of the same specific energy and for particles of the same LET. Special emphasis has been directed to the calculation of very small fragments (< 1 kbp) that are not detectable by the most common experimental techniques and that can significantly influence the RBE (Relative Biological Effectiveness) of high LET radiation. This work was partially supported by EU ("RISC-RAD" project, Contract no. FI6R-CT 2003- 508842, and "NOTE" project, Contract no. FI6R-036465) and ASI (Italian Space Agency, "Mo-Ma/COUNT" project). References A. Campa, F. Ballarini, M. Belli, R. Cherubini, V. Dini, G. Esposito, W.Friedland, S. Gerardi, S. Molinelli, A. Ottolenghi, H. G. Paretzke, G. Simone and M. A. Tabocchini. DNA DSB induced in human cells by charged particles and gamma rays: experimental results and theoretical approaches. Int. J. Radiat.Biol. 81, 841-854 (2005). D. Alloni, F. Ballarini, M. Belli, A. Campa, G. Esposito, W. Friedland, M.Liotta, A. Ottolenghi and H. G. Paretzke. Modeling of DNA fragmentation induced in human fibroblasts by 56 Fe ions. Adv. Space Res. 40, 1401-1407 (2007a). D. Alloni, F. Antonelli, F. Ballarini, M. Belli, A. Campa, V. Dini, G.Esposito, W. Friedland, M. Liotta, A. Ottolenghi, H. G. Paretzke, G. Simone, E. Sorrentino and M. A. Tabocchini. Small DNA fragments induced in human fibroblasts by 56 Fe ions: experimental data and MC simulations. Proc. "Ion Beams in biology and medicine", Heidelberg, 26-29 September 2007, edited by J. Debus, K. Henrichs, G. Kraft, p. 164 (2207b).

Optical vector network analyzer based on double-sideband modulation.

PubMed

Jun, Wen; Wang, Ling; Yang, Chengwu; Li, Ming; Zhu, Ning Hua; Guo, Jinjin; Xiong, Liangming; Li, Wei

2017-11-01

We report an optical vector network analyzer (OVNA) based on double-sideband (DSB) modulation using a dual-parallel Mach-Zehnder modulator. The device under test (DUT) is measured twice with different modulation schemes. By post-processing the measurement results, the response of the DUT can be obtained accurately. Since DSB modulation is used in our approach, the measurement range is doubled compared with conventional single-sideband (SSB) modulation-based OVNA. Moreover, the measurement accuracy is improved by eliminating the even-order sidebands. The key advantage of the proposed scheme is that the measurement of a DUT with bandpass response can also be simply realized, which is a big challenge for the SSB-based OVNA. The proposed method is theoretically and experimentally demonstrated.

DNA mismatch repair and oligonucleotide end-protection promote base-pair substitution distal from a CRISPR/Cas9-induced DNA break

PubMed Central

Harmsen, Tim; Klaasen, Sjoerd; van de Vrugt, Henri; te Riele, Hein

2018-01-01

Abstract Single-stranded oligodeoxyribonucleotide (ssODN)-mediated repair of CRISPR/Cas9-induced DNA double-strand breaks (DSB) can effectively be used to introduce small genomic alterations in a defined locus. Here, we reveal DNA mismatch repair (MMR) activity is crucial for efficient nucleotide substitution distal from the Cas9-induced DNA break when the substitution is instructed by the 3′ half of the ssODN. Furthermore, protecting the ssODN 3′ end with phosphorothioate linkages enhances MMR-dependent gene editing events. Our findings can be exploited to optimize efficiencies of nucleotide substitutions distal from the DSB and imply that oligonucleotide-mediated gene editing is effectuated by templated break repair. PMID:29447381

A DsbA-Deficient Periplasm Enables Functional Display of a Protein with Redox-Sensitive Folding on M13 Phage.

PubMed

Chen, Minyong; Samuelson, James C

2016-06-14

The requirements for target protein folding in M13 phage display are largely underappreciated. Here we chose Fbs1, a carbohydrate binding protein, as a model to address this issue. Importantly, folding of Fbs1 is impaired in an oxidative environment. Fbs1 can be displayed on M13 phage using the SRP or Sec pathway. However, the displayed Fbs1 protein is properly folded only when Fbs1 is translocated via the SRP pathway and displayed using Escherichia coli cells with a DsbA-negative periplasm. This study indicates M13 phage display may be improved using a system specifically designed according to the folding requirements of each target protein.

Air Force Boom Event Analyzer Recorder (BEAR): System Description

DTIC Science & Technology

1989-08-01

8000008 ATTRY RAN START (OO87FDFO) 98 INDMBI IQU 87FDFOE KID OF BAT. RAN 53 (0087A030) 99 KIDDATA EQ 87A030H END OF VALID DATA (0087FEOO) 100 VABSTI 1QU...192 PORTC DS.B 1 PORT A COiD 008040 193 PODBD D.3 1 PORT B DATA 008041 194 PORN DS.B I PORT B COmmID 008042 195 W17IN DS.3 1 KETBOARD INPUT VALUE...RVO.L *EIDAT,DO GET KID 000482 223C 397 VDVI.L SOA5A51,Dl LOAD MAS 000488 30c1 398 1A*73 IO11.1 Dl,[AO)+ WRITE MASK 59 00048A DICO 399 CMI. DO,AO CHCK

Vertical Structure of the Urban Boundary Layer over Marseille Under Sea-Breeze Conditions

NASA Astrophysics Data System (ADS)

Lemonsu, Aude; Bastin, Sophie; Masson, Valéry; Drobinski, Philippe

2006-03-01

During the UBL-ESCOMPTE program (June July 2001), intensive observations were performed in Marseille (France). In particular, a Doppler lidar, located in the north of the city, provided radial velocity measurements on a 6-km radius area in the lowest 3 km of the troposphere. Thus, it is well adapted to document the vertical structure of the atmosphere above complex terrain, notably in Marseille, which is bordered by the Mediterranean sea and framed by numerous massifs. The present study focuses on the last day of the intensive observation period 2 (26 June 2001), which is characterized by a weak synoptic pressure gradient favouring the development of thermal circulations. Under such conditions, a complex stratification of the atmosphere is observed. Three-dimensional numerical simulations, with the Méso-NH atmospheric model including the town energy balance (TEB) urban parameterization, are conducted over south-eastern France. A complete evaluation of the model outputs was already performed at both regional and city scales. Here, the 250-m resolution outputs describing the vertical structure of the atmosphere above the Marseille area are compared to the Doppler lidar data, for which the spatial resolution is comparable. This joint analysis underscores the consistency between the atmospheric boundary layer (ABL) observed by the Doppler lidar and that modelled by Méso-NH. The observations and simulations reveal the presence of a shallow sea breeze (SSB) superimposed on a deep sea breeze (DSB) above Marseille during daytime. Because of the step-like shape of the Marseille coastline, the SSB is organized in two branches of different directions, which converge above the city centre. The analysis of the 250-m wind fields shows evidence of the role of the local topography on the local dynamics. Indeed, the topography tends to reinforce the SSB while it weakens the DSB. The ABL is directly affected by the different sea-breeze circulations, while the urban effects appear to be negligible.

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

PubMed Central

Tsabar, Michael; Mason, Jennifer M.; Chan, Yuen-Ling; Bishop, Douglas K.; Haber, James E.

2015-01-01

Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments. PMID:26019181

Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli

PubMed Central

Qiu, Ji; Swartz, James R.; Georgiou, George

1998-01-01

The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 μg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins in E. coli without the need for in vitro refolding. PMID:9835579

γH2AX/53BP1 foci as a potential pre-treatment marker of HNSCC tumors radiosensitivity - preliminary methodological study and discussion

NASA Astrophysics Data System (ADS)

Falk, Martin; Horakova, Zuzana; Svobodova, Marketa; Masarik, Michal; Kopecna, Olga; Gumulec, Jaromir; Raudenska, Martina; Depes, Daniel; Bacikova, Alena; Falkova, Iva; Binkova, Hana

2017-09-01

In order to improve patients' post-treatment quality of life, a shift from surgery to non-surgical (chemo)radio-treatment is recognized in head and neck oncology. However, about half of HNSCC tumors are resistant to irradiation and an efficient marker of individual tumor radiosensitivity is still missing. We analyzed whether various parameters of DNA double strand break (DSB) repair determined in vitro can predict, prior to clinical treatment initiation, the radiosensitivity of tumors. We compared formation and decrease of γH2AX/53BP1 foci in 48 h after irradiating tumor cell primocultures with 2 Gy of γ-rays. To better understand complex tumor behavior, three different cell type primocultures - CD90-, CD90+, and a mixed culture of these cells - were isolated from 1 clinically radioresistant, 2 radiosensitive, and 4 undetermined HPV-HNSCC tumors and followed separately. While DSB repair was delayed and the number of persisting DSBs increased in the radiosensitive tumors, the results for the radioresistant tumor were similar to cultured normal human skin fibroblasts. Hence, DSB repair kinetics/efficiency may correlate with clinical response to radiotherapy for a subset of HNSCC tumors but the size (and therefore practical relevance) of this subset remains to be determined. The same is true for contribution of different cell type primocultures to tumor radioresistance.

SIRT6 stabilizes DNA-dependent Protein Kinase at chromatin for DNA double-strand break repair

PubMed Central

McCord, Ronald A.; Michishita, Eriko; Hong, Tao; Berber, Elisabeth; Boxer, Lisa D.; Kusumoto, Rika; Guan, Shenheng; Shi, Xiaobing; Gozani, Or; Burlingame, Alma L.; Bohr, Vilhelm A.; Chua, Katrin F.

2009-01-01

The Sir2 chromatin regulatory factor links maintenance of genomic stability to life span extension in yeast. The mammalian Sir2 family member SIRT6 has been proposed to have analogous functions, because SIRT6-deficiency leads to shortened life span and an aging-like degenerative phenotype in mice, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor with chromatin impacts on the efficiency of repair, and establish a link between chromatin regulation, DNA repair, and a mammalian Sir2 factor. PMID:20157594

Increased apoptosis and DNA double-strand breaks in the embryonic mouse brain in response to very low-dose X-rays but not 50 Hz magnetic fields.

PubMed

Saha, Shreya; Woodbine, Lisa; Haines, Jackie; Coster, Margaret; Ricket, Nicole; Barazzuol, Lara; Ainsbury, Elizabeth; Sienkiewicz, Zenon; Jeggo, Penny

2014-11-06

The use of X-rays for medical diagnosis is enhancing exposure to low radiation doses. Exposure to extremely low-frequency electromagnetic or magnetic fields is also increasing. Epidemiological studies show consistent associations of childhood leukaemia with exposure to magnetic fields but any causal relationship is unclear. A limitation in assessing the consequence of such exposure is the availability of sensitive assays. The embryonic neuronal stem and progenitor cell compartments are radiosensitive tissues. Using sensitive assays, we report a statistically significant increase in DNA double-strand break (DSB) formation and apoptosis in the embryonic neuronal stem cell compartment following in utero exposure to 10-200 mGy X-rays. Both endpoints show a linear response. We also show that DSB repair is delayed following exposure to doses below 50 mGy compared with 100 mGy. Thus, we demonstrate in vivo consequences of low-dose radiation. In contrast to these impacts, we did not observe any significant induction of DSBs or apoptosis following exposure to 50 Hz magnetic fields (100 or 300 µT). We conclude that any DSB induction by treatment with magnetic fields is lower than following exposure to 10 mGy X-rays. For comparison, certain procedures involving computed tomography scanning are equivalent to 1-5 mGy X-rays.

Local and sex-specific biases in crossover vs. noncrossover outcomes at meiotic recombination hot spots in mice.

PubMed

de Boer, Esther; Jasin, Maria; Keeney, Scott

2015-08-15

Meiotic recombination initiated by programmed double-strand breaks (DSBs) yields two types of interhomolog recombination products, crossovers and noncrossovers, but what determines whether a DSB will yield a crossover or noncrossover is not understood. In this study, we analyzed the influence of sex and chromosomal location on mammalian recombination outcomes by constructing fine-scale recombination maps in both males and females at two mouse hot spots located in different regions of the same chromosome. These include the most comprehensive maps of recombination hot spots in oocytes to date. One hot spot, located centrally on chromosome 1, behaved similarly in male and female meiosis: Crossovers and noncrossovers formed at comparable levels and ratios in both sexes. In contrast, at a distal hot spot, crossovers were recovered only in males even though noncrossovers were obtained at similar frequencies in both sexes. These findings reveal an example of extreme sex-specific bias in recombination outcome. We further found that estimates of relative DSB levels are surprisingly poor predictors of relative crossover frequencies between hot spots in males. Our results demonstrate that the outcome of mammalian meiotic recombination can be biased, that this bias can vary depending on location and cellular context, and that DSB frequency is not the only determinant of crossover frequency. © 2015 de Boer et al.; Published by Cold Spring Harbor Laboratory Press.

UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages

PubMed Central

Greinert, R.; Volkmer, B.; Henning, S.; Breitbart, E. W.; Greulich, K. O.; Cardoso, M. C.; Rapp, Alexander

2012-01-01

UVA (320–400 nm) represents the main spectral component of solar UV radiation, induces pre-mutagenic DNA lesions and is classified as Class I carcinogen. Recently, discussion arose whether UVA induces DNA double-strand breaks (dsbs). Only few reports link the induction of dsbs to UVA exposure and the underlying mechanisms are poorly understood. Using the Comet-assay and γH2AX as markers for dsb formation, we demonstrate the dose-dependent dsb induction by UVA in G1-synchronized human keratinocytes (HaCaT) and primary human skin fibroblasts. The number of γH2AX foci increases when a UVA dose is applied in fractions (split dose), with a 2-h recovery period between fractions. The presence of the anti-oxidant Naringin reduces dsb formation significantly. Using an FPG-modified Comet-assay as well as warm and cold repair incubation, we show that dsbs arise partially during repair of bi-stranded, oxidative, clustered DNA lesions. We also demonstrate that on stretched chromatin fibres, 8-oxo-G and abasic sites occur in clusters. This suggests a replication-independent formation of UVA-induced dsbs through clustered single-strand breaks via locally generated reactive oxygen species. Since UVA is the main component of solar UV exposure and is used for artificial UV exposure, our results shine new light on the aetiology of skin cancer. PMID:22941639

Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination

PubMed Central

Sugawara, Neal; Pâques, Frédéric; Colaiácovo, Mónica; Haber, James E.

1997-01-01

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles. When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3′ tail. PMID:9256462

Microdosimetry of DNA conformations: relation between direct effect of (60)Co gamma rays and topology of DNA geometrical models in the calculation of A-, B- and Z-DNA radiation-induced damage yields.

PubMed

Semsarha, Farid; Raisali, Gholamreza; Goliaei, Bahram; Khalafi, Hossein

2016-05-01

In order to obtain the energy deposition pattern of ionizing radiation in the nanometric scale of genetic material and to investigate the different sensitivities of the DNA conformations, direct effects of (60)Co gamma rays on the three A, B and Z conformations of DNA have been studied. For this purpose, single-strand breaks (SSB), double-strand breaks (DSB), base damage (BD), hit probabilities and three microdosimetry quantities (imparted energy, mean chord length and lineal energy) in the mentioned DNA conformations have been calculated and compared by using GEometry ANd Tracking 4 (Geant4) toolkit. The results show that A-, B- and Z-DNA conformations have the highest yields of DSB (1.2 Gy(-1) Gbp(-1)), SSB (25.2 Gy(-1) Gbp(-1)) and BD (4.81 Gy(-1) Gbp(-1)), respectively. Based on the investigation of direct effects of radiation, it can be concluded that the DSB yield is largely correlated to the topological characteristics of DNA models, although the SSB yield is not. Moreover, according to the comparative results of the present study, a reliable candidate parameter for describing the relationship between DNA damage yields and geometry of DNA models in the theoretical radiation biology research studies would be the mean chord length (4 V/S) of the models.

Meiotic double-strand breaks at the interface of chromosome movement, chromosome remodeling, and reductional division

PubMed Central

Storlazzi, Aurora; Tessé, Sophie; Gargano, Silvana; James, Françoise; Kleckner, Nancy; Zickler, Denise

2003-01-01

Chromosomal processes related to formation and function of meiotic chiasmata have been analyzed in Sordaria macrospora. Double-strand breaks (DSBs), programmed or γ-rays-induced, are found to promote four major events beyond recombination and accompanying synaptonemal complex formation: (1) juxtaposition of homologs from long-distance interactions to close presynaptic coalignment at midleptotene; (2) structural destabilization of chromosomes at leptotene/zygotene, including sister axis separation and fracturing, as revealed in a mutant altered in the conserved, axis-associated cohesin-related protein Spo76/Pds5p; (3) exit from the bouquet stage, with accompanying global chromosome movements, at zygotene/pachytene (bouquet stage exit is further found to be a cell-wide regulatory transition and DSB transesterase Spo11p is suggested to have a new noncatalytic role in this transition); (4) normal occurrence of both meiotic divisions, including normal sister separation. Functional interactions between DSBs and the spo76-1 mutation suggest that Spo76/Pds5p opposes local destabilization of axes at developing chiasma sites and raise the possibility of a regulatory mechanism that directly monitors the presence of chiasmata at metaphase I. Local chromosome remodeling at DSB sites appears to trigger an entire cascade of chromosome movements, morphogenetic changes, and regulatory effects that are superimposed upon a foundation of DSB-independent processes. PMID:14563680

Dietary sugars, serum and the biocide chlorhexidine digluconate modify the population and structural dynamics of mixed Candida albicans and Escherichia coli biofilms.

PubMed

Thein, Z M; Smaranayake, Y H; Smaranayake, L P

2007-11-01

Despite the increasing recognition of the role played by mixed species biofilms in health and disease, the behavior and factors modulating these biofilms remain elusive. We therefore compared the effect of serum, two dietary sugars (sucrose and galactose) and a biocide, chlorhexidine digluconate, on a dual species biofilm (DSB) of Candida albicans and Escherichia coli and, their single species biofilm (SSB) counterparts. Both modes of biofilm growth on polystyrene plastic surfaces were quantified using a viable cell count method and visualized using confocal scanning laser microscopy (CSLM). Present data indicate that co-culture of C. albicans with varying initial concentrations of E. coli leads to a significant inhibition of yeast growth (r=-0.964; p<0.001). Parallel ultrastructural studies using CSLM and a Live/Dead stain confirmed that E. coli growth rendered blastospores and hyphal yeasts non-viable in DSB. SSB of C. albicans showed pronounced growth when its growth surface was pretreated with serum and by sugar supplements in the incubating medium (p<0.05). Intriguingly, C. albicans in DSB was more resistant to the antiseptic effect of chlorhexidine digluconate. Taken together, the current data elucidate some features of the colonization resistance offered by bacteria in mixed bacterial/fungal habitats and how such phenomena may contribute to the development of fungal superinfection during antimicrobial therapy.

Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament

PubMed Central

Ma, Chu Jian; Gibb, Bryan; Kwon, YoungHo; Sung, Patrick; Greene, Eric C.

2017-01-01

Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly. PMID:27903895

Increased apoptosis and DNA double-strand breaks in the embryonic mouse brain in response to very low-dose X-rays but not 50 Hz magnetic fields

PubMed Central

Saha, Shreya; Woodbine, Lisa; Haines, Jackie; Coster, Margaret; Ricket, Nicole; Barazzuol, Lara; Ainsbury, Elizabeth; Sienkiewicz, Zenon; Jeggo, Penny

2014-01-01

The use of X-rays for medical diagnosis is enhancing exposure to low radiation doses. Exposure to extremely low-frequency electromagnetic or magnetic fields is also increasing. Epidemiological studies show consistent associations of childhood leukaemia with exposure to magnetic fields but any causal relationship is unclear. A limitation in assessing the consequence of such exposure is the availability of sensitive assays. The embryonic neuronal stem and progenitor cell compartments are radiosensitive tissues. Using sensitive assays, we report a statistically significant increase in DNA double-strand break (DSB) formation and apoptosis in the embryonic neuronal stem cell compartment following in utero exposure to 10–200 mGy X-rays. Both endpoints show a linear response. We also show that DSB repair is delayed following exposure to doses below 50 mGy compared with 100 mGy. Thus, we demonstrate in vivo consequences of low-dose radiation. In contrast to these impacts, we did not observe any significant induction of DSBs or apoptosis following exposure to 50 Hz magnetic fields (100 or 300 µT). We conclude that any DSB induction by treatment with magnetic fields is lower than following exposure to 10 mGy X-rays. For comparison, certain procedures involving computed tomography scanning are equivalent to 1–5 mGy X-rays. PMID:25209403

DNA repair goes hip-hop: SMARCA and CHD chromatin remodellers join the break dance.

PubMed

Rother, Magdalena B; van Attikum, Haico

2017-10-05

Proper signalling and repair of DNA double-strand breaks (DSB) is critical to prevent genome instability and diseases such as cancer. The packaging of DNA into chromatin, however, has evolved as a mere obstacle to these DSB responses. Posttranslational modifications and ATP-dependent chromatin remodelling help to overcome this barrier by modulating nucleosome structures and allow signalling and repair machineries access to DSBs in chromatin. Here we recap our current knowledge on how ATP-dependent SMARCA- and CHD-type chromatin remodellers alter chromatin structure during the signalling and repair of DSBs and discuss how their dysfunction impacts genome stability and human disease.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Authors.

Accuracy enhanced distance measurement system using double-sideband modulated frequency scanning interferometry

NASA Astrophysics Data System (ADS)

Chen, Xilun; Wang, Xiangchuan; Pan, Shilong

2017-03-01

An implementation of a distance measurement system using double-sideband with suppressed carrier modulation (DSB-SC) frequency scanning interferometry is proposed to reduce the variations in the optical path and improve the measurement accuracy. In this proposed system, the electro-optic DSB-SC is used to create dual-swept signals with opposite scanning directions. For each swept signal, the relative distance between the reference arm and the measuring arm is determined by the beat frequency of signals from two arms. By multiplying both beat signals, measurement errors caused by variations in the optical path can be greatly reduced. As an experimental demonstration, a vibration was introduced in the optical path length. The experimental results show that the variations can be suppressed for over 19.9 dB.

Involvement of DNA-PK(sub cs) in DSB Repair Following Fe-56 Ion Irradiation

NASA Technical Reports Server (NTRS)

O'Neill, Peter; Harper, Jane; Anderson, Jennifer a.; Cucinnota, Francis A.

2007-01-01

When cells are exposed to radiation, cellular lesions are induced in the DNA including double strand breaks (DSBs), single strand breaks and clustered DNA damage, which if not repaired with high fidelity may lead to detrimental biological consequences. Complex DSBs are induced by ionizing radiation and characterized by the presence of base lesions close to the break termini. They are believed to be one of the major causes of the biological effects of IR. The complexity of DSBs increases with the ionization density of the radiation and these complex DSBs are distinct from the damage induced by sparsely ionizing gamma-radiation. It has been hypothesized that complex DSBs produced by heavy ions in space pose problems to the DNA repair machinery. We have used imm uno-cyto-chemical staining of phosphorylated histone H2AX (gamma-H2AX) foci, as a marker of DSBs. We have investigated the formation and loss of gamma-H2AX foci and RAD51 foci (a protein involved in the homologous recombination pathway) in mammalian cells induced by low fluences of low-LET gamma-radiation and high-LET Fe-56 ions (1GeV/n, 151 keV/micron LET). M059J and M059K cells, which are deficient and proficient in DNA-PK(sub cs) activity respectively, were used to examine the role of DNA-PK(sub cs), a key protein in the non-homologous end joining (NHEJ) pathway of DSB repair, along with HF19 human fibroblasts. Followi ng irradiation with Fe-56 ions the rate of repair was slower in M059J cells compared with that in M059K, indicating a role for DNA-PK(sub cs) in the repair of DSB induced by Fe-56 ions. However a small percentage of DSBs induced are rejoined within 5 h although many DSBs still persist up to 24 h. When RAD51 was examined in M059J/K cells, RAD51 foci are visible 24 hours after irradiation in approximately 40% of M059J cells compared with <5% of M059K cells indicating that persistent DSBs or those formed at stalled replication forks recruit RAD51 in DNA-PK(sub cs) deficient cells. Following 1 Gy gamma-radiation the induction of gamma-H2AX foci is similar in M059J and M059K cells. However, the repair rate of DSBs is slower in M059J cells than in M059K as shown previously but faster than seen with DSB induced by 56Fe ions. Vanillin, an inhibitor of DNA-PK(sub cs), reduces significantly the rate of DSB repair in HF19 cells following 1 Gy gamma-radiation but at 0.25 Gy gamma-irradiation the rate of DSB repair is similar in the presence or absence vanillin, thus suggesting the repair of a sub-set of DSBs induced by low dose, low-LET radiation does not require DNA-PK(sub cs). This sub-set of DSBs is formed in lower yield with high LET radiation. T he complexity of DNA DSBs induced by HZE radiation will be discussed in terms of reduced repair efficiency and provide scope to model different sub-classes of DSBs as precursors that may lead to the detrimental health effects of HZE radiation.

Space Radiation Effects on Human Cells: Modeling DNA Breakage, DNA Damage Foci Distribution, Chromosomal Aberrations and Tissue Effects

NASA Technical Reports Server (NTRS)

Ponomarev, A. L.; Huff, J. L.; Cucinotta, F. A.

2011-01-01

Future long-tem space travel will face challenges from radiation concerns as the space environment poses health risk to humans in space from radiations with high biological efficiency and adverse post-flight long-term effects. Solar particles events may dramatically affect the crew performance, while Galactic Cosmic Rays will induce a chronic exposure to high-linear-energy-transfer (LET) particles. These types of radiation, not present on the ground level, can increase the probability of a fatal cancer later in astronaut life. No feasible shielding is possible from radiation in space, especially for the heavy ion component, as suggested solutions will require a dramatic increase in the mass of the mission. Our research group focuses on fundamental research and strategic analysis leading to better shielding design and to better understanding of the biological mechanisms of radiation damage. We present our recent effort to model DNA damage and tissue damage using computational models based on the physics of heavy ion radiation, DNA structure and DNA damage and repair in human cells. Our particular area of expertise include the clustered DNA damage from high-LET radiation, the visualization of DSBs (DNA double strand breaks) via DNA damage foci, image analysis and the statistics of the foci for different experimental situations, chromosomal aberration formation through DSB misrepair, the kinetics of DSB repair leading to a model-derived spectrum of chromosomal aberrations, and, finally, the simulation of human tissue and the pattern of apoptotic cell damage. This compendium of theoretical and experimental data sheds light on the complex nature of radiation interacting with human DNA, cells and tissues, which can lead to mutagenesis and carcinogenesis later in human life after the space mission.

Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends

NASA Technical Reports Server (NTRS)

Lobrich, M.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

1995-01-01

An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

l-Cysteine supplementation increases insulin sensitivity mediated by upregulation of GSH and adiponectin in high glucose treated 3T3-L1 adipocytes.

PubMed

Achari, Arunkumar E; Jain, Sushil K

2017-09-15

Diabetic patients have lower blood levels of l-cysteine (LC) and glutathione (GSH). This study examined the hypothesis that LC supplementation positively up regulates the effects of insulin on GSH and glucose metabolism in 3T3-L1 adipocyte model. 3T3L1 adipocytes were treated with LC (250 μM, 2 h) and/or insulin (15 or 30 nM, 2 h), and high glucose (HG, 25 mM, 20 h). Results showed that HG caused significant increase (95%) in ROS and reduction in the protein levels of DsbA-L (43%), adiponectin (64%), GCLC (20%), GCLM (21%), GSH (50%), and GLUT-4 (23%) in adipocytes. Furthermore, HG caused a reduction in total (35%) and HMW adiponectin (30%) secretion. Treatment with insulin alone significantly (p < 0.05) reduced ROS levels as well as increased DsbA-L, adiponectin, GCLC, GCLM, GSH, and GLUT-4 protein levels, glucose utilization, and improved total and HMW adiponectin secretion in HG treated adipocytes compared to HG alone. Interestingly, LC supplementation along with insulin caused greater reduction in ROS levels and significantly (p < 0.05) boosted the DsbA-L (41% vs LC, 29% vs Insulin), adiponectin (92% Vs LC, 84% Vs insulin) protein levels and total (32% Vs LC, 22% Vs insulin) and HMW adiponectin (75% Vs LC, 39% Vs insulin) secretion compared with the either insulin or LC alone in HG-treated cells. In addition, LC supplementation along with insulin increased GCLC (21% Vs LC, 14% insulin), GCLM (28% Vs LC, 16% insulin) and GSH (25% Vs LC and insulin) levels compared with the either insulin or LC alone in HG-treated cells. Furthermore, LC and insulin increases GLUT-4 protein expression (65% Vs LC, 18% Vs Insulin), glucose utilization (57% Vs LC, 27% Vs insulin) compared with the either insulin or LC alone in HG-treated cells. Similarly, LC supplementation increased insulin action significantly in cells maintained in medium contained control glucose. To explore the beneficial effect of LC is mediated by the upregulation of GCLC, we knocked down GCLC using siRNA in adipoctyes. There was a significant decrease in DsbA-L and GLUT-4 mRNA levels and GSH levels in GCLC knockdown adipocytes and LC supplementation up regulates GCLC, DsbA-L and GLUT-4 mRNA expression and GSH levels in GCLC knockdown cells. These results demonstrated that LC along with insulin increases GSH levels thereby improving adiponectin secretion and glucose utilization in adipocytes. This suggests that LC supplementation can increase insulin sensitivity and can be used as an adjuvant therapy for diabetes. Copyright © 2017. Published by Elsevier Inc.

Double-strand break repair and genetic recombination in topoisomerase and primase mutants of bacteriophage T4.

PubMed

Shcherbakov, Victor P; Kudryashova, Elena

2014-09-01

The effects of primase and topoisomerase II deficiency on the double-strand break (DSB) repair and genetic recombination in bacteriophage T4 were studied in vivo using focused recombination. Site-specific DSBs were induced by SegC endonuclease in the rIIB gene of one of the parents. The frequency/distance relationship was determined in crosses of the wild-type phage, topoisomerase II mutant amN116 (gene 39), and primase mutant E219 (gene 61). Ordinary two-factor (i×j) and three-factor (i k×j) crosses between point rII mutations were also performed. These data provide information about the frequency and distance distribution of the single-exchange (splice) and double-exchange (patch) events. In two-factor crosses ets1×i, the topoisomerase and primase mutants had similar recombinant frequencies in crosses at ets1-i distances longer than 1000 bp, comprising about 80% of the corresponding wild-type values. They, however, differ remarkably in crosses at shorter distances. In the primase mutant, the recombinant frequencies are similar to those in the wild-type crosses at distances less than 100 bp, being a bit diminished at longer distances. In two-factor crosses ets1×i of the topoisomerase mutant, the recombinant frequencies were reduced ten-fold at the shortest distances. In three-factor crosses a6 ets1×i, where we measure patch-related recombination, the primase mutant was quite proficient across the entire range of distances. The topoisomerase mutant crosses demonstrated virtually complete absence of rII(+) recombinants at distances up to 33 bp, with the frequencies increasing steadily at longer distances. The data were interpreted as follows. The primase mutant is fully recombination-proficient. An obvious difference from the wild-type state is some shortage of EndoVII function leading to prolonged existence of HJs and thus stretched out ds-branch migration. This is also true for the topoisomerase mutant. However, the latter is deficient in the ss-branch migration step of the DSB repair pathway and partially deficient in HJ initiation. In apparent contradiction to their effects on the DSB-induced site-specific recombination, the topoisomerase and primase mutants demonstrated about 3-8-fold increase in the recombinant frequencies in the ordinary crosses, with the recombination running exclusively via patches. This implies that most of the spontaneous recombination events are not initiated by dsDNA ends in these mutants. Copyright © 2014 Elsevier B.V. All rights reserved.

Dosimetric evaluation of radionuclides for VCAM-1-targeted radionuclide therapy of early brain metastases.

PubMed

Falzone, Nadia; Ackerman, Nicole L; Rosales, Liset de la Fuente; Bernal, Mario A; Liu, Xiaoxuan; Peeters, Sarah Gja; Soto, Manuel Sarmiento; Corroyer-Dulmont, Aurélien; Bernaudin, Myriam; Grimoin, Elisa; Touzani, Omar; Sibson, Nicola R; Vallis, Katherine A

2018-01-01

Brain metastases develop frequently in patients with breast cancer, and present a pressing therapeutic challenge. Expression of vascular cell adhesion molecule 1 (VCAM-1) is upregulated on brain endothelial cells during the early stages of metastasis and provides a target for the detection and treatment of early brain metastases. The aim of this study was to use a model of early brain metastasis to evaluate the efficacy of α-emitting radionuclides, 149 Tb, 211 At, 212 Pb, 213 Bi and 225 Ac; β-emitting radionuclides, 90 Y, 161 Tb and 177 Lu; and Auger electron (AE)-emitters 67 Ga, 89 Zr, 111 In and 124 I, for targeted radionuclide therapy (TRT). Histologic sections and two photon microscopy of mouse brain parenchyma were used to inform a cylindrical vessel geometry using the Geant4 general purpose Monte Carlo (MC) toolkit with the Geant4-DNA low energy physics models. Energy deposition was evaluated as a radial function and the resulting phase spaces were superimposed on a DNA model to estimate double-strand break (DSB) yields for representative β- and α-emitters, 177 Lu and 212 Pb. Relative biological effectiveness (RBE) values were determined by only evaluating DNA damage due to physical interactions. 177 Lu produced 2.69 ± 0.08 DSB per GbpGy, without significant variation from the lumen of the vessel to a radius of 100 µm. The DSB yield of 212 Pb included two local maxima produced by the 6.1 MeV and 8.8 MeV α-emissions from decay products, 212 Bi and 212 Po, with yields of 7.64 ± 0.12 and 9.15 ± 0.24 per GbpGy, respectively. Given its higher DSB yield 212 Pb may be more effective for short range targeting of early micrometastatic lesions than 177 Lu. MC simulation of a model of early brain metastases provides invaluable insight into the potential efficacy of α-, β- and AE-emitting radionuclides for TRT. 212 Pb, which has the attributes of a theranostic radionuclide since it can be used for SPECT imaging, showed a favorable dose profile and RBE.

Atomic Simulation of Complex DNA DSBs and the Interactions with the Ku70/80 Heterodimer

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2011-01-01

DNA double strand breaks (DSBs) induced by ionizing radiation (IR) usually contain modified bases such as 8-oxo-7,8-dihydroguanine (8-oxoG) and thymine glycol, apurinic/apyrimidinic (AP) sites, 2-deoxyribonolactone, or single-strand breaks (SSBs). The presence of such lesions in close proximity to the DSB terminus makes the DNA nicks more difficult to repair and rejoin than endogenously induced simple DSBs, and as such a major determinant of the biological effects of high linear energy transfer (LET) radiation as encountered in space travel. In this study we conducted molecular dynamics simulations on a series of DNA duplexes with various complex lesions of 8-oxoG and AP sites, in an effort to investigate the effects of such lesions to the structural integrity and stability of DNA after insulted by IR. We also simulated the interaction of such complex DSBs with the Ku70/80 heterodimer, the first protein in mammalian cells to embark the non-homologous end joining (NHEJ) DNA repair pathway. The results indicate, compared to DNA with simple DSBs, the complex lesions can enhance the hydrogen bonds opening rate at the DNA terminus, and increase the mobility of the whole duplex, thus they present more deleterious effects to the genome integrity if not captured and repaired promptly in cells. Simulations also demonstrate the binding of Ku drastically reduces structural disruption and flexibility caused by the complex lesions, and the interactions of Ku with complex DSBs have a different potential energy landscape from the bound structure with simple DSB. In all complex DSBs systems, the binding of DSB terminus with Ku70 is softened while the binding of the middle duplex with Ku80 is tightened. This energy shift may help the Ku protein to secure at the DSB terminus for a longer time, so that other end processing factors or repair pathways can proceed at the lesions before NHEJ repair process starts. These atomic simulations may provide valuable new insight into the selective action of repair proteins on damaged DNA.

Characterization of a Novel Type of HIV-1 Particle Assembly Inhibitor Using a Quantitative Luciferase-Vpr Packaging-Based Assay

PubMed Central

Errazuriz, Elisabeth; Coric, Pascale; Souquet, Florence; Turcaud, Serge; Boulanger, Pierre; Bouaziz, Serge; Hong, Saw See

2011-01-01

The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity. PMID:22073298

Regulation of DNA repair mechanism in human glioma xenograft cells both in vitro and in vivo in nude mice.

PubMed

Ponnala, Shivani; Veeravalli, Krishna Kumar; Chetty, Chandramu; Dinh, Dzung H; Rao, Jasti S

2011-01-01

Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells. Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls. Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point.

Regulation of DNA Repair Mechanism in Human Glioma Xenograft Cells both In Vitro and In Vivo in Nude Mice

PubMed Central

Ponnala, Shivani; Veeravalli, Krishna Kumar; Chetty, Chandramu; Dinh, Dzung H.; Rao, Jasti S.

2011-01-01

Background Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells. Methodology/Principal Findings Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls. Conclusion/Significance Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point. PMID:22022560

Application of Fragment Based Drug Discovery to Membrane Proteins: Biophysical Identification of Ligands of the Integral Membrane Enzyme DsbB

PubMed Central

Früh, Virginie; Zhou, Yunpeng; Chen, Dan; Loch, Caroline; Eiso, AB; Grinkova, Yelena N.; Verheij, Herman; Sligar, Stephen G; Bushweller, John H.; Siegal, Gregg

2014-01-01

Summary Membrane proteins are important pharmaceutical targets, but they pose significant challenges for fragment based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. The E. coli inner membrane protein DsbB was solubilized in detergent micelles and lipid bilayer nanodiscs. The solubilized protein was immobilized with retention of functionality and used to screen 1,071 drug fragments for binding using Target Immobilized NMR Screening. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 μM. The ability to insert a broad array of membrane proteins into nanodiscs, combined with the efficiency of TINS, demonstrates the feasibility of finding fragments targeting membrane proteins. PMID:20797617

Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes.

PubMed

Hilton, Benjamin A; Liu, Ji; Cartwright, Brian M; Liu, Yiyong; Breitman, Maya; Wang, Youjie; Jones, Rowdy; Tang, Hui; Rusinol, Antonio; Musich, Phillip R; Zou, Yue

2017-09-01

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is caused by a point mutation in the LMNA gene, resulting in production of a truncated farnesylated-prelamin A protein (progerin). We previously reported that XPA mislocalized to the progerin-induced DNA double-strand break (DSB) sites, blocking DSB repair, which led to DSB accumulation, DNA damage responses, and early replication arrest in HGPS. In this study, the XPA mislocalization to DSBs occurred at stalled or collapsed replication forks, concurrent with a significant loss of PCNA at the forks, whereas PCNA efficiently bound to progerin. This PCNA sequestration likely exposed ds-ssDNA junctions at replication forks for XPA binding. Depletion of XPA or progerin each significantly restored PCNA at replication forks. Our results suggest that although PCNA is much more competitive than XPA in binding replication forks, PCNA sequestration by progerin may shift the equilibrium to favor XPA binding. Furthermore, we demonstrated that progerin-induced apoptosis could be rescued by XPA, suggesting that XPA-replication fork binding may prevent apoptosis in HGPS cells. Our results propose a mechanism for progerin-induced genome instability and accelerated replicative senescence in HGPS.-Hilton, B. A., Liu, J., Cartwright, B. M., Liu, Y., Breitman, M., Wang, Y., Jones, R., Tang, H., Rusinol, A., Musich, P. R., Zou, Y. Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes. © FASEB.

Characterization of glioma stem cells through multiple stem cell markers and their specific sensitization to double-strand break-inducing agents by pharmacological inhibition of ataxia telangiectasia mutated protein.

PubMed

Raso, Alessandro; Vecchio, Donatella; Cappelli, Enrico; Ropolo, Monica; Poggi, Alessandro; Nozza, Paolo; Biassoni, Roberto; Mascelli, Samantha; Capra, Valeria; Kalfas, Fotios; Severi, Paolo; Frosina, Guido

2012-09-01

Previous studies have shown that tumor-driving glioma stem cells (GSC) may promote radio-resistance by constitutive activation of the DNA damage response started by the ataxia telangiectasia mutated (ATM) protein. We have investigated whether GSC may be specifically sensitized to ionizing radiation by inhibiting the DNA damage response. Two grade IV glioma cell lines (BORRU and DR177) were characterized for a number of immunocytochemical, karyotypic, proliferative and differentiative parameters. In particular, the expression of a panel of nine stem cell markers was quantified by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. Overall, BORRU and DR177 displayed pronounced and poor stem phenotypes, respectively. In order to improve the therapeutic efficacy of radiation on GSC, the cells were preincubated with a nontoxic concentration of the ATM inhibitors KU-55933 and KU-60019 and then irradiated. BORRU cells were sensitized to radiation and radio-mimetic chemicals by ATM inhibitors whereas DR177 were protected under the same conditions. No sensitization was observed after cell differentiation or to drugs unable to induce double-strand breaks (DSB), indicating that ATM inhibitors specifically sensitize glioma cells possessing stem phenotype to DSB-inducing agents. In conclusion, pharmacological inhibition of ATM may specifically sensitize GSC to DSB-inducing agents while sparing nonstem cells. © 2012 The Authors; Brain Pathology © 2012 International Society of Neuropathology.

AtMMS21, an SMC5/6 complex subunit, is involved in stem cell niche maintenance and DNA damage responses in Arabidopsis roots.

PubMed

Xu, Panglian; Yuan, Dongke; Liu, Ming; Li, Chunxin; Liu, Yiyang; Zhang, Shengchun; Yao, Nan; Yang, Chengwei

2013-04-01

Plants maintain stem cells in meristems to sustain lifelong growth; these stem cells must have effective DNA damage responses to prevent mutations that can propagate to large parts of the plant. However, the molecular links between stem cell functions and DNA damage responses remain largely unexplored. Here, we report that the small ubiquitin-related modifier E3 ligase AtMMS21 (for methyl methanesulfonate sensitivity gene21) acts to maintain the root stem cell niche by mediating DNA damage responses in Arabidopsis (Arabidopsis thaliana). Mutation of AtMMS21 causes defects in the root stem cell niche during embryogenesis and postembryonic stages. AtMMS21 is essential for the proper expression of stem cell niche-defining transcription factors. Moreover, mms21-1 mutants are hypersensitive to DNA-damaging agents, have a constitutively increased DNA damage response, and have more DNA double-strand breaks (DSBs) in the roots. Also, mms21-1 mutants exhibit spontaneous cell death within the root stem cell niche, and treatment with DSB-inducing agents increases this cell death, suggesting that AtMMS21 is required to prevent DSB-induced stem cell death. We further show that AtMMS21 functions as a subunit of the STRUCTURAL MAINTENANCE OF CHROMOSOMES5/6 complex, an evolutionarily conserved chromosomal ATPase required for DNA repair. These data reveal that AtMMS21 acts in DSB amelioration and stem cell niche maintenance during Arabidopsis root development.

53BP1 promotes microhomology-mediated end-joining in G1-phase cells

PubMed Central

Xiong, Xiahui; Du, Zhanwen; Wang, Ying; Feng, Zhihui; Fan, Pan; Yan, Chunhong; Willers, Henning; Zhang, Junran

2015-01-01

Alternative non-homologous end joining (alt-NHEJ) was originally identified as a backup repair mechanism in the absence of classical NHEJ (c-NHEJ) factors but recent studies have demonstrated that alt-NHEJ is active even when c-NHEJ as well as homologous recombination is available. The functions of 53BP1 in NHEJ processes are not well understood. Here, we report that 53BP1 promotes DNA double-strand break (DSB) repair and genomic stability not only in c-NHEJ-proficient but also -deficient human G1-phase cells. Using an array of repair substrates we show that these effects of 53BP1 are correlated with a promotion of microhomology-mediated end-joining (MMEJ), a subtype of alt-NHEJ, in G1-phase. Consistent with a specific role in MMEJ we confirm that 53BP1 status does not affect c-NHEJ. 53BP1 supports sequence deletion during MMEJ consistent with a putative role in facilitating end-resection. Interestingly, promotion of MMEJ by 53BP1 in G1-phase cells is only observed in the presence of functional BRCA1. Depletion of both 53BP1 and BRCA1 increases repair needing microhomology usage and augments loss of DNA sequence, suggesting that MMEJ is a highly regulated DSB repair process. Together, these findings significantly expand our understanding of the cell-cycle-dependent roles of 53BP1 in DSB repair. PMID:25586219

Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters

PubMed Central

Sedletska, Yuliya; Radicella, J. Pablo; Sage, Evelyne

2013-01-01

Unresolved repair of clustered DNA lesions can lead to the formation of deleterious double strand breaks (DSB) or to mutation induction. Here, we investigated the outcome of clusters composed of base lesions for which base excision repair enzymes have different kinetics of excision/incision. We designed multiply damaged sites (MDS) composed of a rapidly excised uracil (U) and two oxidized bases, 5-hydroxyuracil (hU) and 8-oxoguanine (oG), excised more slowly. Plasmids harboring these U-oG/hU MDS-carrying duplexes were introduced into Escherichia coli cells either wild type or deficient for DNA n-glycosylases. Induction of DSB was estimated from plasmid survival and mutagenesis determined by sequencing of surviving clones. We show that a large majority of MDS is converted to DSB, whereas almost all surviving clones are mutated at hU. We demonstrate that mutagenesis at hU is correlated with excision of the U placed on the opposite strand. We propose that excision of U by Ung initiates the loss of U-oG-carrying strand, resulting in enhanced mutagenesis at the lesion present on the opposite strand. Our results highlight the importance of the kinetics of excision by base excision repair DNA n-glycosylases in the processing and fate of MDS and provide evidence for the role of strand loss/replication fork collapse during the processing of MDS on their mutational consequences. PMID:23945941

REV7 counteracts DNA double-strand break resection and impacts PARP inhibition

PubMed Central

Xu, Guotai; Yuan, Jingsong; Mistrik, Martin; Bouwman, Peter; Bartkova, Jirina; Gogola, Ewa; Warmerdam, Daniël; Barazas, Marco; Jaspers, Janneke E.; Watanabe, Kenji; Pieterse, Mark; Kersbergen, Ariena; Sol, Wendy; Celie, Patrick H. N.; Schouten, Philip C.; van den Broek, Bram; Salman, Ahmed; Nieuwland, Marja; de Rink, Iris; de Ronde, Jorma; Jalink, Kees; Boulton, Simon J.; Chen, Junjie; van Gent, Dik C.; Bartek, Jiri; Jonkers, Jos; Borst, Piet; Rottenberg, Sven

2015-01-01

Summary Error-free repair of DNA double-strand breaks (DSB) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway1. In the absence of BRCA1-mediated HR, administration of PARP inhibitors induces synthetic lethality of tumor cells of patients with breast or ovarian cancers2,3. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration4. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases5. In particular, little is known about BRCA1-independent restoration of HR. Here, we show that loss of REV7 (also known as MAD2L2) re-establishes CtIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and appears to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance6. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining (NHEJ) during immunoglobulin class switch recombination. Our results reveal an unexpected critical function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells. PMID:25799992

Interactions of the SAP Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Carra, Claudio; Huff, Janice; Pluth, Janice M.; Cucinotta, Francis A.

2007-01-01

NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku70/80 complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The SAP domain of Ku70 (residues 556-609), contains an a helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the SAP/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the SAP domain of Ku70 and a 10 base pairs DNA duplex. Large-scale conformational changes were observed and some putative binding modes were suggested based on energetic analysis. These modes are consistent with previous experimental investigations. In addition, the results indicate that cooperation of SAP with other domains of Ku70/80 is necessary to explain the high affinity of binding as observed in experiments.

Homozygous deficiency of ubiquitin-ligase ring-finger protein RNF168 mimics the radiosensitivity syndrome of ataxia-telangiectasia

PubMed Central

Devgan, S S; Sanal, O; Doil, C; Nakamura, K; Nahas, S A; Pettijohn, K; Bartek, J; Lukas, C; Lukas, J; Gatti, R A

2011-01-01

Maintaining genomic integrity is critical to avoid life-threatening disorders, such as premature aging, neurodegeneration and cancer. A multiprotein cascade operates at sites of DNA double-strand breaks (DSBs) to recognize, signal and repair damage. RNF168 (ring-finger nuclear factor) contributes to this emerging pathway of several E3 ubiquitin ligases that perform sequential ubiquitylations on damaged chromosomes, chromatin modifications essential for aggregation of repair complexes at the DSB sites. Here, we report the clinical and cellular phenotypes associated with a newly identified homozygous nonsense mutation in the RNF168 gene of a patient with a syndrome mimicking ataxia-telangiectasia. The mutation eliminated both of RNF168's ubiquitin-binding motifs, thus blocking progression of the ubiquitylation cascade and retention of repair proteins including tumor suppressors 53BP1 and BRCA1 at DSB sites, consistent with the observed defective DNA damage checkpoints/repair and pronounced radiosensitivity. Rapid screening for RNF168 pathway deficiency was achieved by scoring patients' lymphoblastoid cells for irradiation-induced nuclear foci containing 53BP1, a robust assay we propose for future diagnostic applications. The formation of radiation-induced DSB repair foci was rescued by ectopic expression of wild-type RNF168 in patient's cells, further causally linking the RNF168 mutation with the pathology. Clinically, this novel syndrome featured ataxia, telangiectasia, elevated alphafetoprotein, immunodeficiency, microcephaly and pulmonary failure and has implications for the differential diagnosis of autosomal recessive ataxias. PMID:21394101

Etoposide Induces Nuclear Re-Localisation of AID

PubMed Central

Lambert, Laurens J.; Walker, Simon; Feltham, Jack; Lee, Heather J.; Reik, Wolf; Houseley, Jonathan

2013-01-01

During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is a highly mutagenic protein that is maintained in the cytoplasm at steady state, however AID is shuttled across the nuclear membrane and the protein transiently present in the nucleus appears sufficient for targeted alteration of immunoglobulin loci. AID has been implicated in epigenetic reprogramming in primordial germ cells and cell fusions and in induced pluripotent stem cells (iPS cells), however AID expression in non-B cells is very low. We hypothesised that epigenetic reprogramming would require a pathway that instigates prolonged nuclear residence of AID. Here we show that AID is completely re-localised to the nucleus during drug withdrawal following etoposide treatment, in the period in which double strand breaks (DSBs) are repaired. Re-localisation occurs 2-6 hours after etoposide treatment, and AID remains in the nucleus for 10 or more hours, during which time cells remain live and motile. Re-localisation is cell-cycle dependent and is only observed in G2. Analysis of DSB dynamics shows that AID is re-localised in response to etoposide treatment, however re-localisation occurs substantially after DSB formation and the levels of re-localisation do not correlate with γH2AX levels. We conclude that DSB formation initiates a slow-acting pathway which allows stable long-term nuclear localisation of AID, and that such a pathway may enable AID-induced DNA demethylation during epigenetic reprogramming. PMID:24324754

Sensitization to radiation and alkylating agents by inhibitors of poly(ADP-ribose) polymerase is enhanced in cells deficient in DNA double-strand break repair.

PubMed

Löser, Dana A; Shibata, Atsushi; Shibata, Akiko K; Woodbine, Lisa J; Jeggo, Penny A; Chalmers, Anthony J

2010-06-01

As single agents, chemical inhibitors of poly(ADP-ribose) polymerase (PARP) are nontoxic and have clinical efficacy against BRCA1- and BRCA2-deficient tumors. PARP inhibitors also enhance the cytotoxicity of ionizing radiation and alkylating agents but will only improve clinical outcomes if tumor sensitization exceeds effects on normal tissues. It is unclear how tumor DNA repair proficiency affects the degree of sensitization. We have previously shown that the radiosensitizing effect of PARP inhibition requires DNA replication and will therefore affect rapidly proliferating tumors more than normal tissues. Because many tumors exhibit defective DNA repair, we investigated the impact of double-strand break (DSB) repair integrity on the sensitizing effects of the PARP inhibitor olaparib. Sensitization to ionizing radiation and the alkylating agent methylmethane sulfonate was enhanced in DSB repair-deficient cells. In Artemis(-/-) and ATM(-/-) mouse embryo fibroblasts, sensitization was replication dependent and associated with defective repair of replication-associated damage. Radiosensitization of Ligase IV(-/-) mouse embryo fibroblasts was independent of DNA replication and is explained by inhibition of "alternative" end joining. After methylmethane sulfonate treatment, PARP inhibition promoted replication-independent accumulation of DSB, repair of which required Ligase IV. Our findings predict that the sensitizing effects of PARP inhibitors will be more pronounced in rapidly dividing and/or DNA repair defective tumors than normal tissues and show their potential to enhance the therapeutic ratio achieved by conventional DNA-damaging agents.

Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament.

PubMed

Ma, Chu Jian; Gibb, Bryan; Kwon, YoungHo; Sung, Patrick; Greene, Eric C

2017-01-25

Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

I-SceI-Induced Gene Replacement at a Natural Locus in Embryonic Stem Cells

PubMed Central

Cohen-Tannoudji, Michel; Robine, Sylvie; Choulika, André; Pinto, Daniel; El Marjou, Fatima; Babinet, Charles; Louvard, Daniel; Jaisser, Frédéric

1998-01-01

Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH2-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 × 10−6) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest. PMID:9488460

Non-DSB clustered DNA lesions. Does theory colocalize with the experiment?

NASA Astrophysics Data System (ADS)

Nikitaki, Zacharenia; Nikolov, Vladimir; Mavragani, Ifigeneia V.; Plante, Ianik; Emfietzoglou, Dimitris; Iliakis, George; Georgakilas, Alexandros G.

2016-11-01

Ionizing radiation results in various kinds of DNA lesions such as double strand breaks (DSBs) and other non-DSB base lesions. These lesions may be formed in close proximity (i.e., within a few nanometers) resulting in clustered types of DNA lesions. These damage clusters are considered the fingerprint of ionizing radiation, notably charged particles of high linear energy transfer (LET). Accumulating theoretical and experimental evidence suggests that the induction of these clustered lesions appears under various irradiation conditions but also as a result of high levels of oxidative stress. The biological significance of these clustered DNA lesions pertains to the inability of cells to process them efficiently compared to isolated DNA lesions. The results in the case of unsuccessful or erroneous repair can vary from mutations up to chromosomal instability. In this mini review, we discuss of several Monte Carlo simulations codes and experimental evidence regarding the induction and repair of radiation-induced non-DSB complex DNA lesions. We also critically present the most widely used methodologies (i.e., gel electrophoresis and fluorescence microscopy [in situ colocalization assays]). Based on the comparison of different approaches, we provide examples and suggestions for the improved detection of these lesions in situ. Based on the current status of knowledge, we conclude that there is a great need for improvement of the detection techniques at the cellular or tissue level, which will provide valuable information for understanding the mechanisms used by the cell to process clustered DNA lesions.

Effects of camptothecin or TOP1 overexpression on genetic stability in Saccharomyces cerevisiae.

PubMed

Sloan, Roketa; Huang, Shar-Yin Naomi; Pommier, Yves; Jinks-Robertson, Sue

2017-11-01

Topoisomerase I (Top1) removes DNA torsional stress by nicking and resealing one strand of DNA, and is essential in higher eukaryotes. The enzyme is frequently overproduced in tumors and is the sole target of the chemotherapeutic drug camptothecin (CPT) and its clinical derivatives. CPT stabilizes the covalent Top1-DNA cleavage intermediate, which leads to toxic double-strand breaks (DSBs) when encountered by a replication fork. In the current study, we examined genetic instability associated with CPT treatment or with Top1 overexpression in the yeast Saccharomyces cerevisiae. Two types of instability were monitored: Top1-dependent deletions in haploid strains, which do not require processing into a DSB, and instability at the repetitive ribosomal DNA (rDNA) locus in diploid strains, which reflects DSB formation. Three 2-bp deletion hotspots were examined and mutations at each were elevated either when a wild-type strain was treated with CPT or when TOP1 was overexpressed, with the mutation frequency correlating with the level of TOP1 overexpression. Under both conditions, deletions at novel positions were enriched. rDNA stability was examined by measuring loss-of-heterozygosity and as was observed previously upon CPT treatment of a wild-type strain, Top1 overexpression destabilized rDNA. We conclude that too much, as well as too little of Top1 is detrimental to eukaryotic genomes, and that CPT has destabilizing effects that extend beyond those associated with DSB formation. Copyright © 2017 Elsevier B.V. All rights reserved.

Disposal site quality team final report

DOT National Transportation Integrated Search

2001-09-01

The disposal site quality team was formed in July 2000 to address Caltrans (Department) and Federal Highway Administration (FHWA) policies on disposal, staging, and borrow areas (DSB), including plant sites, contractor yards, and access roads. Caltra...

Investigating Microbial Activity in Diazotrophic Methane Seep Sediment via Transcript Analysis and Single-Cell FISH-NanoSIMS

NASA Astrophysics Data System (ADS)

Dekas, A. E.; Connon, S. A.; Chadwick, G.; Orphan, V. J.

2012-12-01

Methane seep microbial ecosystems are phylogenetically diverse and physiologically complex, and require culture-independent techniques to accurately investigate metabolic activity. In the present study we combine an RNA analysis of four key microbial genes with FISH-NanoSIMS analysis of single cells to determine the diversity of nitrogen fixing microorganisms (diazotrophs) present at a deep-sea methane-seeping site, as well as investigate the methane-dependency of a variety of community members. Recently, methane-dependent nitrogen fixation was observed in Mound 12 Costa Rica sediments, and was spatially correlated with the abundance of aggregates of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacterial symbionts (SRB). Combined with the detection of 15N uptake from 15N2 in these aggregates, this suggested that the ANME-SRB aggregates are the primary diazotrophs in seep sediment. However, the diversity of dinitrogenase reductase (nifH) sequences recovered from several deep-sea locales, including Mound 12, suggests a greater diversity of diazotrophs in marine sediment. To investigate the activity of these potential diazotrophs in Mound 12 sediment, we investigated a suite of RNA transcripts in 15N2 incubations in both the presence and absence of methane: nifH, bacterial 16S rRNA, methyl coenzyme M reductase A (mcrA), and adenosine-5'-phosposulfate reductase alpha subunit (aprA). No nifH transcripts were recovered in incubations without methane, consistent with previous measurements lacking 15N2 uptake in the same sediments. The activity of the bacterial community in general, assessed by variable transcription, was also greatly affected by the presence or absence of methane. Single-cell fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS) was employed to confirm diazotrophic activity (15N2 uptake) and protein synthesis (15NH4+ uptake) of particular species implicated as ecologically important by the pattern of transcripts recovered in the mesocosm experiments. This analysis revealed 15N enrichment in free-living (i.e. non-ANME associated) members of the Desulfobulbaceae in 15N2 incubations with methane, while free-living Desulfosarcina/Desulfococcus cells, as well as nearly 40 unidentified DAPI-stained cells, were not 15N enriched. However, further NanoSIMS analyses of DSB in a variety of incubation conditions suggests that this enrichment may be due to N sharing between the ANME and DSB while in tight physical association, and then subsequent dissociation, rather than nitrogen fixation by the DSB. If true, this is an excellent example of the potential pitfalls of single cell stable isotope labeling experiments, and potential false positives due to the recycling of labeled material between (even transiently) closely associated symbionts. This work highlights both the utility of transcript analysis as a hypothesis-generator for direct analyses of microbial activity via stable isotope labeling, as well as the need to contextualize labeling experiments with investigations of microbial community structure.

Human MLH1 suppresses the insertion of telomeric sequences at intra-chromosomal sites in telomerase-expressing cells

PubMed Central

Jia, Pingping; Chastain, Megan; Zou, Ying; Her, Chengtao

2017-01-01

Abstract Aberrant formation of interstitial telomeric sequences (ITSs) promotes genome instabilities. However, it is unclear how aberrant ITS formation is suppressed in human cells. Here, we report that MLH1, a key protein involved in mismatch repair (MMR), suppresses telomeric sequence insertion (TSI) at intra-chromosomal regions. The frequency of TSI can be elevated by double-strand break (DSB) inducer and abolished by ATM/ATR inhibition. Suppression of TSI requires MLH1 recruitment to DSBs, indicating that MLH1's role in DSB response/repair is important for suppressing TSI. Moreover, TSI requires telomerase activity but is independent of the functional status of p53 and Rb. Lastly, we show that TSI is associated with chromosome instabilities including chromosome loss, micronuclei formation and chromosome breakage that are further elevated by replication stress. Our studies uncover a novel link between MLH1, telomerase, telomere and genome stability. PMID:28180301

Doppler-multipath tolerant voice communication

NASA Astrophysics Data System (ADS)

Harris, R. M.

Line of sight communication between high performance aircraft has been found to be subject to a peculiar form of multipath radio wave propagation - Doppler multipath. It degrades analogue voice reception on the standard fit ultrahigh frequency radio, producing low frequency random noise and warbling. Various modifications were carried out on the aircraft's communications system, but the problem remained. All the evidence points to a natural phenomenon. The reported observations are corroborated by theoretical studies and laboratory simulations of multipath radio wave propagation between two points moving relative to a diffusely scattering reflector. Theoretical predictions of Rician fading have explained the disruption of speech transmitted using conventional dsb(am) modulation. This also indicated suppressing the carrier as a radical cure. Double sideband suppressed carrier radios have been developed for airborne evaluation in comparison with standard dsb(am). The air to air flying trials proved the superior performance of the suppressed carrier system under conditions of Doppler multipath.

DNA Damage Signals and Space Radiation Risk

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.

2011-01-01

Space radiation is comprised of high-energy and charge (HZE) nuclei and protons. The initial DNA damage from HZE nuclei is qualitatively different from X-rays or gamma rays due to the clustering of damage sites which increases their complexity. Clustering of DNA damage occurs on several scales. First there is clustering of single strand breaks (SSB), double strand breaks (DSB), and base damage within a few to several hundred base pairs (bp). A second form of damage clustering occurs on the scale of a few kbp where several DSB?s may be induced by single HZE nuclei. These forms of damage clusters do not occur at low to moderate doses of X-rays or gamma rays thus presenting new challenges to DNA repair systems. We review current knowledge of differences that occur in DNA repair pathways for different types of radiation and possible relationships to mutations, chromosomal aberrations and cancer risks.

An Integrated 520-600 GHz Sub-Harmonic Mixer and Tripler Combination Based on GaAs MMIC Membrane Planar Schottky Diodes

NASA Technical Reports Server (NTRS)

Thomas, B.; Gill, J.; Maestrini, A.; Lee, C.; Lin, R.; Sin, S.; Peralta, A.; Mehdi, I.

2011-01-01

We present here the design, development and test of an integrated sub-millimeter front-end featuring a 520-600 GHz sub-harmonic mixer and a 260-300 GHz frequency tripler in a single cavity. Both devices used GaAs MMIC membrane planar Schottky diode technology. The sub-harmonic mixer/tripler circuit has been tested using conventional machined as well as silicon micro-machined blocks. Measurement results on the metal block give best DSB mixer noise temperature of 2360 K and conversion losses of 7.7 dB at 520 GHz. Preliminary results on the silicon micro-machined blocks give a DSB mixer noise temperature of 4860 K and conversion losses of 12.16 dB at 540 GHz. The LO input power required to pump the integrated tripler/sub-harmonic mixer for both packages is between 30 and 50 mW

An Integrated 520-600 GHz Sub-Harmonic Mixer and Tripler Combination Based on GaAs MMIC Membrane Planar Schottky Diodes

NASA Technical Reports Server (NTRS)

Thomas, B.; Gill, J.; Maestrini, A.; Lee, C.; Lin, R.; Sin, S.; Peralta, A.; Mehdi, I.

2010-01-01

We present here the design, development and test of an integrated sub-millimeter front-end featuring a 520-600 GHz sub-harmonic mixer and a 260-300 GHz frequency tripler in a single cavity. Both devices used GaAs MMIC membrane planar Schottky diode technology. The sub-harmonic mixer/tripler circuit has been tested using conventional machined as well as silicon micro-machined blocks. Measurement results on the metal block give best DSB mixer noise temperature of 2360 K and conversion losses of 7.7 dB at 520 GHz. Preliminary results on the silicon micro-machined blocks give a DSB mixer noise temperature of 4860 K and conversion losses of 12.16 dB at 540 GHz. The LO input power required to pump the integrated tripler/sub-harmonic mixer for both packages is between 30 and 50 mW.

A low noise 230 GHz heterodyne receiver employing .25 sq micron area Nb/AlO(x)/Nb tunnel junctions

NASA Technical Reports Server (NTRS)

Kooi, Jacob W.; Chan, M.; Phillips, T. G.; Bumble, B.; Leduc, H. G.

1992-01-01

Recent results for a full height rectangular waveguide mixer with an integrated IF matching network are reported. Two 0.25 sq micron Nb/AlO(x)/Nb superconducting insulating superconducting (SIS) tunnel junctions with a current density of about 8500 A/sq cm and omega RC of about 2.5 at 230 GHz have been tested. Detailed measurements of the receiver noise have been made from 200-290 GHz for both junctions at 4.2 K. The lowest receiver noise temperatures were recorded at 239 GHz, measuring 48 K DSB at 4.2 K and 40 K DSB at 2.1 K. The 230 GHz receiver incorporates a one octave wide integrated low pass filter and matching network which transforms the pumped IF junction impedance to 50 ohms over a wide range of impedances.

Multiband DSB-SC modulated radio over IsOWC link with coherent homodyne detection

NASA Astrophysics Data System (ADS)

Kang, Zong; Zhu, Jiang

2018-02-01

In this paper, we present a multiband double sideband-suppressed carrier (DSB-SC) modulated radio over intersatellite optical wireless communication (IsOWC) link with coherent homodyne detection. The proposed system can provide the transparent transport of multiband radio frequency (RF) signals with higher linearity and better receiver sensitivity than the intensity modulated with direct detection (IM/DD) scheme. The full system model and the exactly analytical expression of signal to noise and distortion ratio (SNDR) are derived considering the third-order intermodulation product and amplifier spontaneous emission (ASE) noise. The finite extinction ratio (ER) of Mach-Zehnder Modulator (MZM) and the saturation property of erbium doped fiber amplifier (EDFA) are also considered. Numerical results of SNDR with various numbers of subchannels and ERs are given. Results indicate that the optimal modulation index exists to maximize the SNDR and the power of local oscillator (LO) carrier should be within an appropriate range.

Ultra-wideband microwave photonic link based on single-sideband modulation

NASA Astrophysics Data System (ADS)

Li, Jingnan; Wang, Yunxin; Wang, Dayong; Zhou, Tao; Zhong, Xin; Xu, Jiahao; Yang, Dengcai; Rong, Lu

2017-10-01

Comparing with the conventional double-sideband (DSB) modulation in communication system, single-sideband (SSB) modulation only demands half bandwidth of DSB in transmission. Two common ways are employed to implement SSB modulation by using optical filter (OF) or electrical 90° phase shift, respectively. However, the bandwidth of above methods is limited by characteristics of current OF and electrical phase shift. To overcome this problem, an ultra-wideband microwave photonic link based on SSB modulation is proposed and demonstrated. The radio frequency (RF) signal modulates a single-drive dual-parallel Mach-Zehnder modulator, and the SSB modulation is realized by combining an electrical 90° hybrid coupler and an optical bandpass filter. The experimental results indicate that the system can achieve SSB modulation for RF signal from 2 to 40 GHz. The proposed microwave photonic link provides an ultra-wideband approach based on SSB modulation for radio-over-fiber system.

In vitro isolation and molecular characterization of an Ehrlichia canis strain from São Paulo, Brazil

PubMed Central

Aguiar, Daniel M.; Hagiwara, Mitika K.; Labruna, Marcelo B.

2008-01-01

An Ehrlichia canis isolate was obtained from an naturally infected dog exhibiting clinical signs of ehrlichiosis in São Paulo Municipality, state of São Paulo, Brazil. The isolate was characterized by PCR and DNA sequencing of portions of the ehrlichial genes dsb, 16SrRNA, and p28. Partial dsb and 16S rRNA sequences were identical to three and five other E. canis strains, respectively, from different countries and continents (including North America, Africa, Asia and Europe). Conversely, the p28 partial sequence for this E. canis (São Paulo) differed by 1, 2, and 2 nucleotides from the corresponding sequences of the E. canis strains Jake (from USA), Oklahoma (USA), and VHE (Venezuela), respectively. The results in this study indicate that E. canis is the only recognized Ehrlichia species infecting dogs in Brazil. PMID:24031251

Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system.

PubMed

Loh, Qiuting; Leong, Siew Wen; Tye, Gee Jun; Choong, Yee Siew; Lim, Theam Soon

2015-05-15

The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display. Copyright © 2015 Elsevier Inc. All rights reserved.

APOBEC3 cytidine deaminases in double-strand DNA break repair and cancer promotion.

PubMed

Nowarski, Roni; Kotler, Moshe

2013-06-15

High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR. ©2013 AACR.

DNA Double-Strand Break Repair as Determinant of Cellular Radiosensitivity to Killing and Target in Radiation Therapy

PubMed Central

Mladenov, Emil; Magin, Simon; Soni, Aashish; Iliakis, George

2013-01-01

Radiation therapy plays an important role in the management of a wide range of cancers. Besides innovations in the physical application of radiation dose, radiation therapy is likely to benefit from novel approaches exploiting differences in radiation response between normal and tumor cells. While ionizing radiation induces a variety of DNA lesions, including base damages and single-strand breaks, the DNA double-strand break (DSB) is widely considered as the lesion responsible not only for the aimed cell killing of tumor cells, but also for the general genomic instability that leads to the development of secondary cancers among normal cells. Homologous recombination repair (HRR), non-homologous end-joining (NHEJ), and alternative NHEJ, operating as a backup, are the major pathways utilized by cells for the processing of DSBs. Therefore, their function represents a major mechanism of radiation resistance in tumor cells. HRR is also required to overcome replication stress – a potent contributor to genomic instability that fuels cancer development. HRR and alternative NHEJ show strong cell-cycle dependency and are likely to benefit from radiation therapy mediated redistribution of tumor cells throughout the cell-cycle. Moreover, the synthetic lethality phenotype documented between HRR deficiency and PARP inhibition has opened new avenues for targeted therapies. These observations make HRR a particularly intriguing target for treatments aiming to improve the efficacy of radiation therapy. Here, we briefly describe the major pathways of DSB repair and review their possible contribution to cancer cell radioresistance. Finally, we discuss promising alternatives for targeting DSB repair to improve radiation therapy and cancer treatment. PMID:23675572

DNA damage induced by the direct effect of radiation

NASA Astrophysics Data System (ADS)

Yokoya, A.; Shikazono, N.; Fujii, K.; Urushibara, A.; Akamatsu, K.; Watanabe, R.

2008-10-01

We have studied the nature of DNA damage induced by the direct effect of radiation. The yields of single- (SSB) and double-strand breaks (DSB), base lesions and clustered damage were measured using the agarose gel electrophoresis method after exposing to various kinds of radiations to a simple model DNA molecule, fully hydrated closed-circular plasmid DNA (pUC18). The yield of SSB does not show significant dependence on linear energy transfer (LET) values. On the other hand, the yields of base lesions revealed by enzymatic probes, endonuclease III (Nth) and formamidopyrimidine DNA glycosylase (Fpg), which excise base lesions and leave a nick at the damage site, strongly depend on LET values. Soft X-ray photon (150 kVp) irradiation gives a maximum yield of the base lesions detected by the enzymatic probes as SSB and clustered damage, which is composed of one base lesion and proximate other base lesions or SSBs. The clustered damage is visualized as an enzymatically induced DSB. The yields of the enzymatically additional damages strikingly decrease with increasing levels of LET. These results suggest that in higher LET regions, the repair enzymes used as probes are compromised because of the dense damage clustering. The studies using simple plasmid DNA as a irradiation sample, however, have a technical difficulty to detect multiple SSBs in a plasmid DNA. To detect the additional SSBs induced in opposite strand of the first SSB, we have also developed a novel technique of DNA-denaturation assay. This allows us to detect multiply induced SSBs in both strand of DNA, but not induced DSB.

VDE-initiated intein homing in Saccharomyces cerevisiae proceeds in a meiotic recombination-like manner.

PubMed

Fukuda, Tomoyuki; Nogami, Satoru; Ohya, Yoshikazu

2003-07-01

Inteins and group I introns found in prokaryotic and eukaryotic organisms occasionally behave as mobile genetic elements. During meiosis of the yeast Saccharomyces cerevisiae, the site-specific endonuclease encoded by VMA1 intein, VDE, triggers a single double-strand break (DSB) at an inteinless allele, leading to VMA1 intein homing. Besides the accumulating information on the in vitro activity of VDE, very little has been known about the molecular mechanism of intein homing in yeast nucleus. We developed an assay to detect the product of VMA1 intein homing in yeast genome. We analysed mutant phenotypes of RecA homologs, Rad51p and Dmc1p, and their interacting proteins, Rad54p and Tid1p, and found that they all play critical roles in intein inheritance. The absence of DSB end processing proteins, Sae2p and those in the Mre11-Rad50-Xrs2 complex, also causes partial reduction in homing efficiency. As with meiotic recombination, crossover events are frequently observed during intein homing. We also observed that the absence of premeiotic DNA replication caused by hydroxyurea (HU) or clb5delta clb6delta mutation reduces VDE-mediated DSBs. The repairing system working in intein homing shares molecular machinery with meiotic recombination induced by Spo11p. Moreover, like Spo11p-induced DNA cleavage, premeiotic DNA replication is a prerequisite for a VDE-induced DSB. VMA1 intein thus utilizes several host factors involved in meiotic and recombinational processes to spread its genetic information and guarantee its progeny through establishment of a parasitic relationship with the organism.

Spectrum of complex DNA damages depends on the incident radiation

NASA Astrophysics Data System (ADS)

Hada, M.; Sutherland, B.

Ionizing radiation induces clustered DNA damages in DNA-two or more abasic sites oxidized bases and strand breaks on opposite DNA strands within a few helical turns Clustered damages are considered to be difficult to repair and therefore potentially lethal and mutagenic damages Although induction of single strand breaks and isolated lesions has been studied extensively little is known of factors affecting induction of clusters other than double strand breaks DSB The aim of the present study was to determine whether the type of incident radiation could affect yield or spectra of specific clusters Genomic T7 DNA a simple 40 kbp linear blunt-ended molecule was irradiated in non-scavenging buffer conditions with Fe 970 MeV n Ti 980 MeV n C 293 MeV n Si 586 MeV n ions or protons 1 GeV n at the NASA Space Radiation Laboratory or with 100 kVp X-rays Irradiated DNA was treated with homogeneous Fpg or Nfo proteins or without enzyme treatment for DSB quantitation then electrophoresed in neutral agarose gels DSB Fpg-OxyPurine clusters and Nfo-Abasic clusters were quantified by number average length analysis The results show that the yields of all these complex damages depend on the incident radiation Although LETs are similar protons induced twice as many DSBs than did X-rays Further the spectrum of damage also depends on the radiation The yield damage Mbp Gy of all damages decreased with increasing linear energy transfer LET of the radiation The relative frequencies of DSBs to Abasic- and OxyBase clusters were higher

Next generation multi-scale biophysical characterization of high precision cancer particle radiotherapy using clinical proton, helium-, carbon- and oxygen ion beams

PubMed Central

Niklas, Martin; Zimmermann, Ferdinand; Chaudhri, Naved; Krunic, Damir; Tessonnier, Thomas; Ferrari, Alfredo; Parodi, Katia; Jäkel, Oliver; Debus, Jürgen; Haberer, Thomas; Abdollahi, Amir

2016-01-01

The growing number of particle therapy facilities worldwide landmarks a novel era of precision oncology. Implementation of robust biophysical readouts is urgently needed to assess the efficacy of different radiation qualities. This is the first report on biophysical evaluation of Monte Carlo simulated predictive models of prescribed dose for four particle qualities i.e., proton, helium-, carbon- or oxygen ions using raster-scanning technology and clinical therapy settings at HIT. A high level of agreement was found between the in silico simulations, the physical dosimetry and the clonogenic tumor cell survival. The cell fluorescence ion track hybrid detector (Cell-Fit-HD) technology was employed to detect particle traverse per cell nucleus. Across a panel of radiobiological surrogates studied such as late ROS accumulation and apoptosis (caspase 3/7 activation), the relative biological effectiveness (RBE) chiefly correlated with the radiation species-specific spatio-temporal pattern of DNA double strand break (DSB) formation and repair kinetic. The size and the number of residual nuclear γ-H2AX foci increased as a function of linear energy transfer (LET) and RBE, reminiscent of enhanced DNA-damage complexity and accumulation of non-repairable DSB. These data confirm the high relevance of complex DSB formation as a central determinant of cell fate and reliable biological surrogates for cell survival/RBE. The multi-scale simulation, physical and radiobiological characterization of novel clinical quality beams presented here constitutes a first step towards development of high precision biologically individualized radiotherapy. PMID:27494855

Forkhead-Associated Domain of Yeast Xrs2, a Homolog of Human Nbs1, Promotes Nonhomologous End Joining Through Interaction With a Ligase IV Partner Protein, Lif1

PubMed Central

Matsuzaki, Kenichiro; Shinohara, Akira; Shinohara, Miki

2008-01-01

DNA double-strand breaks (DSB) are repaired through two different pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). Yeast Xrs2, a homolog of human Nbs1, is a component of the Mre11-Rad50-Xrs2 (MRX) complex required for both HR and NHEJ. Previous studies showed that the N-terminal forkhead-associated (FHA) domain of Xrs2/Nbs1 in yeast is not involved in HR, but is likely to be in NHEJ. In this study, we showed that the FHA domain of Xrs2 plays a critical role in efficient DSB repair by NHEJ. The FHA domain of Xrs2 specifically interacts with Lif1, a component of the ligase IV complex, Dnl4-Nej1-Lif1 (DNL). Lif1, which is phosphorylated in vivo, contains two Xrs2-binding regions. Serine 383 of Lif1 plays an important role in the interaction with Xrs2 as well as in NHEJ. Interestingly, the phospho-mimetic substitutions of serine 383 enhance the NHEJ activity of Lif1. Our results suggest that the phosphorylation of Lif1 at serine 383 is recognized by the Xrs2 FHA domain, which in turn may promote recruitment of the DNL complex to DSB for NHEJ. The interaction between Xrs2 and Lif1 through the FHA domain is conserved in humans; the FHA domain Nbs1 interacts with Xrcc4, a Lif1 homolog of human. PMID:18458108

The excitation and distribution of CO (J = 6-5) emission in the Orion Nebula

NASA Technical Reports Server (NTRS)

Buhl, D.; Chin, G.; Fetterman, H. R.; Koepf, G. A.; Peck, D. D.

1982-01-01

Observations of the 691-GHz (J = 6-5) transition of CO in the BN/KL region of Orion obtained in February 1981, at the IR Telescope Facility at Mauna Kea are reported. The system employs a heterodyne receiver with an overall noise temperature of 3900 K DSB at 432 microns, 64 5-MHz IF-filter-bank channels, and a chopping secondary with 120-arcsec excursion, and has 35-arcsec resolution. Sample data are presented graphically and analyzed using a rate equation and a kinetic model. A 35 x 45-arcsec core with hot broad plateau emission (antenna temperature 180 + or - 36 K) surrounded by an area of strong narrow-line 120-K emission is found. It is suggested that the plateau emission originates in a wide thin sheet after a shock wave, with H2 density at least 10 to the 6th/cu cm and gas kinetic temperature greater than 500 K.

Genomic Evidence of Chemotrophic Metabolisms in Deep-Dwelling Chloroflexi Conferred by Ancient Horizontal Gene Transfer Events

NASA Astrophysics Data System (ADS)

Momper, L. M.; Magnabosco, C.; Amend, J.; Osburn, M. R.; Fournier, G. P.

2017-12-01

The marine and terrestrial subsurface biospheres represent quite likely the largest reservoirs for life on Earth, directly impacting surface processes and global cycles throughout Earth's history. In the deep subsurface biosphere (DSB) organic carbon and energy are often extremely scarce. However, archaea and bacteria are able to persist in the DSB to at least 3.5 km below surface [1]. Understanding how they persist, and by what metabolisms they subsist, are key questions in this biosphere. To address these questions we investigated 5 global DSB environments: one legacy mine in South Dakota, USA, 3 mines in South Africa and marine fluids circulating beneath the Juan de Fuca Ridge. Boreholes within these mines provided access to fluids buried beneath the earth's surface and sampled depths down to 3.1 km. Geochemical data were collected concomitantly with DNA for metagenomic sequencing. We examined genomes of the ancient and deeply branching Chloroflexi for metabolic capabilities and interrogated the geochemical drivers behind those metabolisms with in situ thermodynamic modeling of reaction energetics. In total, 23 Chloroflexi genomes were identified and analyzed from the 5 subsurface sites. Genes for nitrate reduction (nar) and sulfite reduction (dsr) were found in many of the South Africa Chloroflexi but were absent from genomes collected in South Dakota. Indeed, nitrate reduction was among the most energetically favorable reactions in South African fluids (10-14 kJ cell-1 sec -1 per mol of reactant) and sulfur reduction with Fe2+ or H2 was also exergonic [2]. Conversely, genes for nitrite and nitrous oxide reduction (nrf, nir and nos) were found in genomes collected in South Dakota and Juan de Fuca, but not South Africa. We examined the origin of genes conferring these metabolisms in the Chloroflexi genomes. We discovered evidence for horizontal gene transfer (HGT) for all of these putative metabolisms. Retention of these genes in Chloroflexi lineages indicates HGT may have conferred an advantageous metabolism in DSB environments. We are using molecular dating techniques to constrain the timing of these HGT events on geologic timescales. [1] Baker J. B. et al. (2003) Environ Microbiol., 5, 267-277. [2] Magnabosco C. et al. (2016) ISME J, 10(3), 730-741.

Tumors overexpressing RNF168 show altered DNA repair and responses to genotoxic treatments, genomic instability and resistance to proteotoxic stress.

PubMed

Chroma, K; Mistrik, M; Moudry, P; Gursky, J; Liptay, M; Strauss, R; Skrott, Z; Vrtel, R; Bartkova, J; Kramara, J; Bartek, J

2017-04-27

Chromatin DNA damage response (DDR) is orchestrated by the E3 ubiquitin ligase ring finger protein 168 (RNF168), resulting in ubiquitin-dependent recruitment of DDR factors and tumor suppressors breast cancer 1 (BRCA1) and p53 binding protein 1 (53BP1). This ubiquitin signaling regulates pathway choice for repair of DNA double-strand breaks (DSB), toxic lesions whose frequency increases during tumorigenesis. Recruitment of 53BP1 curbs DNA end resection, thereby limiting homologous recombination (HR) and directing DSB repair toward error-prone non-homologous end joining (NHEJ). Under cancer-associated ubiquitin starvation conditions reflecting endogenous or treatment-evoked proteotoxic stress, the ubiquitin-dependent accrual of 53BP1 and BRCA1 at the DNA damage sites is attenuated or lost. Challenging this current paradigm, here we identified diverse human cancer cell lines that display 53BP1 recruitment to DSB sites even under proteasome inhibitor-induced proteotoxic stress, that is, under substantial depletion of free ubiquitin. We show that central to this unexpected phenotype is overabundance of RNF168 that enables more efficient exploitation of the residual-free ubiquitin. Cells with elevated RNF168 are more resistant to combined treatment by ionizing radiation and proteasome inhibition, suggesting that such aberrant RNF168-mediated signaling might reflect adaptation to chronic proteotoxic and genotoxic stresses experienced by tumor cells. Moreover, the overabundant RNF168 and the ensuing unorthodox recruitment patterns of 53BP1, RIF1 and REV7 (monitored on laser micro-irradiation-induced DNA damage) shift the DSB repair balance from HR toward NHEJ, a scenario accompanied by enhanced chromosomal instability/micronuclei formation and sensitivity under replication stress-inducing treatments with camptothecin or poly(ADP-ribose) polymerase (PARP) inhibitor. Overall, our data suggest that the deregulated RNF168/53BP1 pathway could promote tumorigenesis by selecting for a more robust, better stress-adapted cancer cell phenotype, through altered DNA repair, fueling genomic instability and tumor heterogeneity. Apart from providing insights into cancer (patho)biology, the elevated RNF168, documented here also by immunohistochemistry on human clinical tumor specimens, may impact responses to standard-of-care and some emerging targeted cancer therapies.

[Sciatic nerve block "out-of-plane" distal to the bifurcation: effective and safe].

PubMed

Geiser, T; Apel, J; Vicent, O; Büttner, J

2017-03-01

Ultrasound guided distal sciatic nerve block (DSB) at bifurcation level shows fast onset and provides excellent success rates. However, its safe performance might be difficult for the unexperienced physician. Just slightly distal to the bifurcation, the tibial nerve (TN) and common fibular nerve (CFN) can be shown clearly separated from each other. Therefore, we investigated if a block done here would provide similar quality results compared to the DSB proximally to the division, with a potentially lower risk of nerve damage. In this randomized, prospective trial, 56 patients per group received either a DSB distal to the bifurcation "out-of-plane" (dist.) or proximally "in-plane" (prox.) with 30 ml of Mepivacaine 1% each. Success was tested by a blinded examiner after 15 and 30 min respectively (sensory and motor block of TN and CFN: 0 = none, 2 = complete, change of skin temperature). Videos of the blocks were inspected by an independent expert retrospectively with regard to the spread of the local anesthetic (LA) and accidental intraneural injection. Cumulative single nerve measurements and temperature changes revealed significant shorter onset and better efficacy (dist/prox: 15 min: 3.13 ± 1.86/1.82 ± 1.62; 30 min: 5.73 ± 1.92/3.21 ± 1.88; T 15 min : 30.3 ± 3.48/28.0 ± 3.67, T 30 min . 33.0 ± 2.46/30.6 ± 3.86; MV/SD; ANOVA; p < 0.01) combined with a higher rate of subparaneural spread in the dist. group (41/51 vs.12/53; χ2; p < 0,01). Procedure times were similar. There were no complications in either group. The subparaneural spread of the LA turned out to be crucial for better results in the distal group. The steep angle using the out-of-plane approach favors needle penetration through the paraneural sheath. The distance between the branches allows the safe application of the LA, so an effective block can be done with just one injection. DSB slightly distal to the bifurcation, in an out-of-plane technique between the TN and CFN, can be done fast, effectively and safe.

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage.

PubMed

Mallik, Sarita; Popodi, Ellen M; Hanson, Andrew J; Foster, Patricia L

2015-09-01

Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. DNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Effects of geometrical structure on spatial distribution of thermal energy in two-dimensional triangular lattices

NASA Astrophysics Data System (ADS)

Liu, Yong-Yang; Xu, Yu-Liang; Liu, Zhong-Qiang; Li, Jing; Wang, Chun-Yang; Kong, Xiang-Mu

2018-07-01

Employing the correlation matrix technique, the spatial distribution of thermal energy in two-dimensional triangular lattices in equilibrium, interacting with linear springs, is studied. It is found that the spatial distribution of thermal energy varies with the included angle of the springs. In addition, the average thermal energy of the longer springs is lower. Springs with different included angle and length will lead to an inhomogeneous spatial distribution of thermal energy. This suggests that the spatial distribution of thermal energy is affected by the geometrical structure of the system: the more asymmetric the geometrical structure of the system is, the more inhomogeneous is the spatial distribution of thermal energy.

BioSentinel: Mission Development of a Radiation Biosensor to Gauge DNA Damage and Repair Beyond Low Earth Orbit on a 6U Nanosatellite.

NASA Technical Reports Server (NTRS)

Lewis, Brian; Hanel, Robert; Bhattacharya, Sharmila; Ricco, Antonion J.; Agasid, Elwood; Reiss-Bubenheim, Debra; Straume, Tore; Parra, Macerena; Boone, Travis; Santa Maria, Sergio;

Spatial distribution of traffic in a cellular mobile data network

NASA Astrophysics Data System (ADS)

Linnartz, J. P. M. G.

1987-02-01

The use of integral transforms of the probability density function for the received power to analyze the relation between the spatial distributions of offered and throughout packet traffic in a mobile radio network with Rayleigh fading channels and ALOHA multiple access was assessed. A method to obtain the spatial distribution of throughput traffic from a prescribed spatial distribution of offered traffic is presented. Incoherent and coherent addition of interference signals is considered. The channel behavior for heavy traffic loads is studied. In both the incoherent and coherent case, the spatial distribution of offered traffic required to ensure a prescribed spatially uniform throughput is synthesized numerically.

Spatial patterns of distribution and abundance of Harrisia portoricensis, an endangered Caribbean cactus

Treesearch

J. Rojas-Sandoval; E. J. Melendez-Ackerman; NO-VALUE

2013-01-01

Aims The spatial distribution of biotic and abiotic factors may play a dominant role in determining the distribution and abundance of plants in arid and semiarid environments. In this study, we evaluated how spatial patterns of microhabitat variables and the degree of spatial dependence of these variables influence the distribution and abundance of the endangered...

[Spatial distribution pattern of Chilo suppressalis analyzed by classical method and geostatistics].

PubMed

Yuan, Zheming; Fu, Wei; Li, Fangyi

2004-04-01

Two original samples of Chilo suppressalis and their grid, random and sequence samples were analyzed by classical method and geostatistics to characterize the spatial distribution pattern of C. suppressalis. The limitations of spatial distribution analysis with classical method, especially influenced by the original position of grid, were summarized rather completely. On the contrary, geostatistics characterized well the spatial distribution pattern, congregation intensity and spatial heterogeneity of C. suppressalis. According to geostatistics, the population was up to Poisson distribution in low density. As for higher density population, its distribution was up to aggregative, and the aggregation intensity and dependence range were 0.1056 and 193 cm, respectively. Spatial heterogeneity was also found in the higher density population. Its spatial correlativity in line direction was more closely than that in row direction, and the dependence ranges in line and row direction were 115 and 264 cm, respectively.

Importance of spatial autocorrelation in modeling bird distributions at a continental scale

USGS Publications Warehouse

Bahn, V.; O'Connor, R.J.; Krohn, W.B.

2006-01-01

Spatial autocorrelation in species' distributions has been recognized as inflating the probability of a type I error in hypotheses tests, causing biases in variable selection, and violating the assumption of independence of error terms in models such as correlation or regression. However, it remains unclear whether these problems occur at all spatial resolutions and extents, and under which conditions spatially explicit modeling techniques are superior. Our goal was to determine whether spatial models were superior at large extents and across many different species. In addition, we investigated the importance of purely spatial effects in distribution patterns relative to the variation that could be explained through environmental conditions. We studied distribution patterns of 108 bird species in the conterminous United States using ten years of data from the Breeding Bird Survey. We compared the performance of spatially explicit regression models with non-spatial regression models using Akaike's information criterion. In addition, we partitioned the variance in species distributions into an environmental, a pure spatial and a shared component. The spatially-explicit conditional autoregressive regression models strongly outperformed the ordinary least squares regression models. In addition, partialling out the spatial component underlying the species' distributions showed that an average of 17% of the explained variation could be attributed to purely spatial effects independent of the spatial autocorrelation induced by the underlying environmental variables. We concluded that location in the range and neighborhood play an important role in the distribution of species. Spatially explicit models are expected to yield better predictions especially for mobile species such as birds, even in coarse-grained models with a large extent. ?? Ecography.

Identification of two metallothionein genes and their roles in stress responses of Musca domestica toward hyperthermy and cadmium tolerance.

PubMed

Tang, Ting; Huang, Da-wei; Zhang, Di; Wu, Yin-jian; Murphy, Robert W; Liu, Feng-song

2011-10-01

Stress proteins such as metallothioneins (MTs) play a key role in cellular protection against environmental stressors. In nature, insects such as houseflies (Musca domestica) are commonly exposed to multiple stressors including heavy metals (e.g. Cadmium, Cd) and high temperatures. In this paper, we identify two novel MT genes from the cDNAs of M. domestica, MdMT1 and MdMT2, which putatively encode 40 and 42 amino acid residues respectively. Expression of the two MTs' mRNAs, which are examined in the fat body, gut, hemocyte, and the epidermis. From our study, we saw that the expression of MdMT1 and MdMT2 are enhanced by Cd and thermal stress. Levels of expression are highest at 10 mM Cd(2+) within a 24-h period, and expressions increase significantly with exposure to 10 mM Cd for 12h. Levels of the mRNAs are up-regulated after heat shock and that of MdMT2 reaches its maximum peak faster than MdMT1. Both of the MT genes might be involved in a transient systemic tolerance response to stressors and they may play important roles in heavy metal and high temperature tolerance in the housefly. To detect whether or not the MTs bind heavy metals, the target genes are cloned into the prokaryotic expression vector pET-DsbA to obtain fusion protein expressed in Escherichia coli BL21 (DE3). Recombinant DsbA-MdMT1 significantly increases tolerance of the host bacteria to Cd(2+), but DsbA-MdMT2 is absent. These differential characteristics will facilitate future investigations into the physiological functions of MTs. Copyright © 2011 Elsevier Inc. All rights reserved.

Glycogen synthase kinase 3beta inhibition enhances repair of DNA double-strand breaks in irradiated hippocampal neurons.

PubMed

Yang, Eddy S; Nowsheen, Somaira; Wang, Tong; Thotala, Dinesh K; Xia, Fen

2011-05-01

Prevention of cranial radiation-induced morbidity following the treatment of primary and metastatic brain cancers, including long-term neurocognitive deficiencies, remains challenging. Previously, we have shown that inhibition of glycogen synthase kinase 3β (GSK3β) results in protection of hippocampal neurons from radiation (IR)-induced apoptosis and attenuation of neurocognitive dysfunction resulting from cranial IR. In this study, we examined whether regulation of the repair of IR-induced DNA damage is one of the mechanisms involved in the radioprotective effects of neurons by inhibition of GSK3β. Specifically, this study showed that inhibition of GSK3β accelerated double strand-break (DSB) repair efficiency in irradiated mouse hippocampal neurons, as assessed by the neutral comet assay. This coincided with attenuation of IR-induced γ-H2AX foci, a well characterized in situ marker of DSBs. To confirm the effect of GSK3 activity on the efficacy of DSB repair, we further demonstrated that biochemical or genetic inhibition of GSK3 activity resulted in enhanced capacity in nonhomologous end-joining-mediated repair of DSBs in hippocampal neurons. Importantly, none of these effects were observed in malignant glioma cells. Taken together, these results suggested that enhanced repair of IR-induced DNA damage may be a novel mechanism by which inhibition of GSK3β specifically protects hippocampal neurons from IR-induced apoptosis. Furthermore, these findings warrant future investigations of the molecular mechanisms underlying the role of GSK3β in the DSB repair of normal neurons and the potential clinical application of neuroprotection with GSK3β inhibitors during cranial IR.

In vivo sensitivity of the embryonic and adult neural stem cell compartments to low-dose radiation.

PubMed

Barazzuol, Lara; Jeggo, Penny A

2016-08-01

The embryonic brain is radiation-sensitive, with cognitive deficits being observed after exposure to low radiation doses. Exposure of neonates to radiation can cause intracranial carcinogenesis. To gain insight into the basis underlying these outcomes, we examined the response of the embryonic, neonatal and adult brain to low-dose radiation, focusing on the neural stem cell compartments. This review summarizes our recent findings. At E13.5-14.5 the embryonic neocortex encompasses rapidly proliferating stem and progenitor cells. Exploiting mice with a hypomorphic mutation in DNA ligase IV (Lig4(Y288C) ), we found a high level of DNA double-strand breaks (DSBs) at E14.5, which we attribute to the rapid proliferation. We observed endogenous apoptosis in Lig4(Y288C) embryos and in WT embryos following exposure to low radiation doses. An examination of DSB levels and apoptosis in adult neural stem cell compartments, the subventricular zone (SVZ) and the subgranular zone (SGZ) revealed low DSB levels in Lig4(Y288C) mice, comparable with the levels in differentiated neuronal tissues. We conclude that the adult SVZ does not incur high levels of DNA breakage, but sensitively activates apoptosis; apoptosis was less sensitively activated in the SGZ, and differentiated neuronal tissues did not activate apoptosis. P5/P15 mice showed intermediate DSB levels, suggesting that DSBs generated in the embryo can be transmitted to neonates and undergo slow repair. Interestingly, this analysis revealed a stage of high endogenous apoptosis in the neonatal SVZ. Collectively, these studies reveal that the adult neural stem cell compartment, like the embryonic counterpart, can sensitively activate apoptosis. © The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Poly(ADP-Ribose) Polymerase-1 and DNA-Dependent Protein Kinase Have Equivalent Roles in Double Strand Break Repair Following Ionizing Radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, Jody; Smith, Graeme; Curtin, Nicola J., E-mail: n.j.curtin@ncl.ac.u

2009-12-01

Purpose: Radiation-induced DNA double strand breaks (DSBs) are predominantly repaired by nonhomologous end joining (NHEJ), involving DNA-dependent protein kinase (DNA-PK). Poly(ADP-ribose) polymerase-1 (PARP-1), well characterized for its role in single strand break repair, may also facilitate DSB repair. We investigated the activation of these enzymes by differing DNA ends and their interaction in the cellular response to ionizing radiation (IR). Methods and Materials: The effect of PARP and DNA-PK inhibitors (KU-0058684 and NU7441) on repair of IR-induced DSBs was investigated in DNA-PK and PARP-1 proficient and deficient cells by measuring gammaH2AX foci and neutral comets. Complementary in vitro enzyme kineticsmore » assays demonstrated the affinities of DNA-PK and PARP-1 for DSBs with varying DNA termini. Results: DNA-PK and PARP-1 both promoted the fast phase of resolution of IR-induced DSBs in cells. Inactivation of both enzymes was not additive, suggesting that PARP-1 and DNA-PK cooperate within the same pathway to promote DSB repair. The affinities of the two enzymes for oligonucleotides with blunt, 3' GGG or 5' GGG overhanging termini were similar and overlapping (K{sub dapp} = 2.6-6.4nM for DNA-PK; 1.7-4.5nM for PARP-1). DNA-PK showed a slightly greater affinity for overhanging DNA and was significantly more efficient when activated by a 5' GGG overhang. PARP-1 had a preference for blunt-ended DNA and required a separate factor for efficient stimulation by a 5' GGG overhang. Conclusion: DNA-PK and PARP-1 are both required in a pathway facilitating the fast phase of DNA DSB repair.« less

Interactions of the C-terminal Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Huff, Janice; Pluth, Janice M.; Cucinotta, Francis A.

2007-01-01

NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku(sub 70/80) complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The C-terminal domain of Ku70 (Ku70c, residues 559-609), contains an helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the Ku70c/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the subunit Ku70c and a 14 base pairs DNA duplex, whose starting structures are designed to be variable so as to mimic their different binding modes. By analyzing conformational changes and energetic properties of the complex during MD simulations, we found that interactions are preferred at DNA ends, and within the major groove, which is consistent with previous experimental investigations. In addition, the results indicate that cooperation of Ku70c with other subunits of Ku(sub 70/80) is necessary to explain the high affinity of binding as observed in experiments.

Computational Study on Full-length Human Ku70 with Double Stranded DNA: Dynamics, Interactions and Functional Implications

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2009-01-01

The Ku70/80 heterodimer is the first repair protein in the initial binding of double-strand break (DSB) ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. In this study we constructed a full-length human Ku70 structure based on its crystal structure, and performed 20 ns conventional molecular dynamic (CMD) simulations on this protein and several other complexes with short DNA duplexes of different sequences. The trajectories of these simulations indicated that, without the topological support of Ku80, the residues in the bridge and C-terminal arm of Ku70 are more flexible than other experimentally identified domains. We studied the two missing loops in the crystal structure and predicted that they are also very flexible. Simulations revealed that they make an important contribution to the Ku70 interaction with DNA. Dislocation of the previously studied SAP domain was observed in several systems, implying its role in DNA binding. Targeted molecular dynamic (TMD) simulation was also performed for one system with a far-away 14bp DNA duplex. The TMD trajectory and energetic analysis disclosed detailed interactions of the DNA-binding residues during the DNA dislocation, and revealed a possible conformational transition for a DSB end when encountering Ku70 in solution. Compared to experimentally based analysis, this study identified more detailed interactions between DNA and Ku70. Free energy analysis indicated Ku70 alone is able to bind DNA with relatively high affinity, with consistent contributions from various domains of Ku70 in different systems. The functional implications of these domains in the processes of Ku heterodimerization and DNA damage recognition and repair can be characterized in detail based upon this analysis.

A chloroplast membrane protein LTO1/AtVKOR involving in redox regulation and ROS homeostasis.

PubMed

Lu, Ying; Wang, Hua-Rong; Li, Han; Cui, Hao-Ran; Feng, Yue-Guang; Wang, Xiao-Yun

2013-09-01

The role of LTO1/ At VKOR-DsbA in ROS homeostasis and in redox regulation of cysteine-containing proteins in chloroplast was studied in lto1 - 2 mutant, and a potential target of LTO1 was captured. A chloroplast membrane protein LTO1/AtVKOR-DsbA encoded by the gene At4g35760 was recently found to be an oxidoreductase and involved in assembly of PSII. Here, the growth of a mutant lto1-2 line of Arabidopsis was found to be severely stunted and transgenic complementation ultimately demonstrated the phenotype changes were due to this gene. A proteomic experiment identified 23 proteins presenting a differential abundance in lto1-2 compared with wild-type plants, including components in PSII and proteins scavenging active oxygen. Three scavengers of active oxygen, L-ascorbate peroxidase 1, peroxisomal catalase 2, dehydroascorbate reductase 1, are reduced in lto1-2 plants, corresponding to high levels of accumulation of reactive oxygen species (ROS). The photosynthetic activities of PSII and the quantity of core protein D1 decreased significantly in lto1-2. Further investigation showed the synthesis of D1 was not affected in mutants both at transcription and translation levels. The soluble DsbA-like domain of LTO1 was found to have reduction, oxidation and isomerization activities, and could promote the formation of disulfide bonds in a lumenal protein, FKBP13. A potential target of LTO1 was captured which was involving in chlorophyll degradation and photooxidative stress response. Experimental results imply that LTO1 plays important roles in redox regulation, ROS homeostasis and maintenance of PSII.

To Nick or Not to Nick: Comparison of I-SceI Single- and Double-Strand Break-Induced Recombination in Yeast and Human Cells

PubMed Central

Katz, Samantha S.; Gimble, Frederick S.; Storici, Francesca

2014-01-01

Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB), or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI) to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293) cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy. PMID:24558436

Double-strand break repair processes drive evolution of the mitochondrial genome in Arabidopsis.

PubMed

Davila, Jaime I; Arrieta-Montiel, Maria P; Wamboldt, Yashitola; Cao, Jun; Hagmann, Joerg; Shedge, Vikas; Xu, Ying-Zhi; Weigel, Detlef; Mackenzie, Sally A

2011-09-27

The mitochondrial genome of higher plants is unusually dynamic, with recombination and nonhomologous end-joining (NHEJ) activities producing variability in size and organization. Plant mitochondrial DNA also generally displays much lower nucleotide substitution rates than mammalian or yeast systems. Arabidopsis displays these features and expedites characterization of the mitochondrial recombination surveillance gene MSH1 (MutS 1 homolog), lending itself to detailed study of de novo mitochondrial genome activity. In the present study, we investigated the underlying basis for unusual plant features as they contribute to rapid mitochondrial genome evolution. We obtained evidence of double-strand break (DSB) repair, including NHEJ, sequence deletions and mitochondrial asymmetric recombination activity in Arabidopsis wild-type and msh1 mutants on the basis of data generated by Illumina deep sequencing and confirmed by DNA gel blot analysis. On a larger scale, with mitochondrial comparisons across 72 Arabidopsis ecotypes, similar evidence of DSB repair activity differentiated ecotypes. Forty-seven repeat pairs were active in DNA exchange in the msh1 mutant. Recombination sites showed asymmetrical DNA exchange within lengths of 50- to 556-bp sharing sequence identity as low as 85%. De novo asymmetrical recombination involved heteroduplex formation, gene conversion and mismatch repair activities. Substoichiometric shifting by asymmetrical exchange created the appearance of rapid sequence gain and loss in association with particular repeat classes. Extensive mitochondrial genomic variation within a single plant species derives largely from DSB activity and its repair. Observed gene conversion and mismatch repair activity contribute to the low nucleotide substitution rates seen in these genomes. On a phenotypic level, these patterns of rearrangement likely contribute to the reproductive versatility of higher plants.

Low-dose carbon ion irradiation effects on DNA damage and oxidative stress in the mouse testis

NASA Astrophysics Data System (ADS)

Liu, Yang; Long, Jing; Zhang, Luwei; Zhang, Hong; Liu, Bin; Zhao, Weiping; Wu, Zhehua

2011-01-01

To investigate the effects of low-dose carbon ion irradiation on reproductive system of mice, the testes of outbred Kunming strain mice were whole-body irradiated with 0, 0.05, 0.1, 0.5 and 1 Gy, respectively. We measured DNA double-strand breaks (DNA DSBs) and oxidative stress parameters including malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and testis weight and sperm count at 12 h, 21 d and 35 d after irradiation in mouse testis. At 12 h postirradiation, a significant increase in DNA DSB level but no pronounced alterations in MDA content or SOD activity were observed in 0.5 and 1 Gy groups compared with the control group. At 21 d postirradiation, there was a significant reduction in sperm count and distinct enhancements of DSB level and MDA content in 0.5 and 1 Gy groups in comparison with control. At 35 d postirradiation, the levels of DNA DSBs and MDA, and SOD activity returned to the baseline except for the MDA content in 1 Gy (P < 0.05), while extreme falls of sperm count were still observed in 0.5 (P < 0.01) and 1 Gy (P < 0.01) groups. For the 0.05 or 0.1 Gy group, no differences were found in DNA DSB level and MDA content between control and at 12 h, 21 d and 35 d after irradiation, indicating that lower doses of carbon ion irradiation have no significant influence on spermatogenesis processes. In this study, male germ cells irradiated with over 0.5 Gy of carbon ions are difficult to repair completely marked by the sperm count. Furthermore, these data suggest that the deleterious effects may be chronic or delayed in reproductive system after whole-body exposure to acute high-dose carbon ions.

Altered Gene Expressions and Cytogenetic Repair Efficiency in Cells with Suppressed Expression of XPA after Proton Exposure

NASA Technical Reports Server (NTRS)

Zhang, Ye; Rohde, Larry H.; Gridley, Daila S.; Mehta, Satish K.; Pierson, Duane L.; Wu, Honglu

2009-01-01

Cellular responses to damages from ionizing radiation (IR) exposure are influenced not only by the genes involved in DNA double strand break (DSB) repair, but also by non- DSB repair genes. We demonstrated previously that suppressed expression of several non-DSB repair genes, such as XPA, elevated IR-induced cytogenetic damages. In the present study, we exposed human fibroblasts that were treated with control or XPA targeting siRNA to 250 MeV protons (0 to 4 Gy), and analyzed chromosome aberrations and expressions of genes involved in DNA repair. As expected, after proton irradiation, cells with suppressed expression of XPA showed a significantly elevated frequency of chromosome aberrations compared with control siRNA treated (CS) cells. Protons caused more severe DNA damages in XPA knock-down cells, as 36% cells contained multiple aberrations compared to 25% in CS cells after 4Gy proton irradiation. Comparison of gene expressions using the real-time PCR array technique revealed that expressions of p53 and its regulated genes in irradiated XPA suppressed cells were altered similarly as in CS cells, suggesting that the impairment of IR induced DNA repair in XPA suppressed cells is p53-independent. Except for XPA, which was more than 2 fold down regulated in XPA suppressed cells, several other DNA damage sensing and repair genes (GTSE1, RBBP8, RAD51, UNG and XRCC2) were shown a more than 1.5 fold difference between XPA knock-down cells and CS cells after proton exposure. The possible involvement of these genes in the impairment of DNA repair in XPA suppressed cells will be further investigated.

Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.

PubMed

Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji

2016-06-01

The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds. © The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Optical transmission radiation damage and recovery stimulation of DSB: Ce3+ inorganic scintillation material

NASA Astrophysics Data System (ADS)

Borisevich, A.; Dormenev, V.; Korjik, M.; Kozlov, D.; Mechinsky, V.; Novotny, R. W.

2015-02-01

Recently, a new scintillation material DSB: Ce3+ was announced. It can be produced in a form of glass or nano-structured glass ceramics with application of standard glass production technology with successive thermal annealing. When doped with Ce3+, material can be applied as scintillator. Light yield of scintillation is near 100 phe/MeV. Un-doped material has a wide optical window from 4.5eV and can be applied to detect Cherenkov light. Temperature dependence of the light yield LY(T) is 0.05% which is 40 times less than in case of PWO. It can be used for detectors tolerant to a temperature variation between -20° to +20°C. Several samples with dimensions of 15x15x7 mm3 have been tested for damage effects on the optical transmission under irradiation with γ-quanta. It was found that the induced absorption in the scintillation range depends on the doping concentration and varies in range of 0.5-7 m-1. Spontaneous recovery of induced absorption has fast initial component. Up to 25% of the damaged transmission is recuperated in 6 hours. Afterwards it remains practically constant if the samples are kept in the dark. However, induced absorption is reduced by a factor of 2 by annealing at 50°C and completely removed in a short time when annealing at 100°C. A significant acceleration of the induced absorption recovery is observed by illumination with visible and IR light. This effect is observed for the first time in a Ce-doped scintillation material. It indicates, that radiation induced absorption in DSB: Ce scintillation material can be retained at the acceptable level by stimulation with light in a strong irradiation environment of collider experiments.

Inhibition of DNA-dependent protein kinase catalytic subunit by small molecule inhibitor NU7026 sensitizes human leukemic K562 cells to benzene metabolite-induced apoptosis.

PubMed

You, Hao; Kong, Meng-meng; Wang, Li-ping; Xiao, Xiao; Liao, Han-lin; Bi, Zhuo-yue; Yan, Hong; Wang, Hong; Wang, Chun-hong; Ma, Qiang; Liu, Yan-qun; Bi, Yong-yi

2013-02-01

Benzene is an established leukotoxin and leukemogen in humans. We have previously reported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to mediate the cellular response to DNA double strand break (DSB) caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026), as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phosphorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apoptosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.

Relative stabilities of IgG1 and IgG4 Fab domains: Influence of the light–heavy interchain disulfide bond architecture

PubMed Central

Heads, James T; Adams, Ralph; D'Hooghe, Lena E; Page, Matt J T; Humphreys, David P; Popplewell, Andrew G; Lawson, Alastair D; Henry, Alistair J

2012-01-01

The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 CH1 and IgG4 CH1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L–H) bond, did not affect thermal stability. Introducing the IgG1 type L–H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L–H interchain DSB with the IgG4 type L–H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 CH1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format. PMID:22761163

The yeast Fun30 and human SMARCAD1 chromatin remodelers promote DNA end resection

PubMed Central

Costelloe, Thomas; Louge, Raphaël; Tomimatsu, Nozomi; Mukherjee, Bipasha; Martini, Emmanuelle; Khadaroo, Basheer; Dubois, Kenny; Wiegant, Wouter W.; Thierry, Agnès; Burma, Sandeep; van Attikum, Haico; Llorente, Bertrand

2012-01-01

Several homology-dependent pathways can repair potentially lethal DNA double-strand breaks (DSBs). The first step common to all homologous recombination reactions is the 5′-3′ degradation of DSB ends that yields 3′ single-stranded DNA (ssDNA) required for loading of checkpoint and recombination proteins. The Mre11-Rad50-Xrs2/NBS1 complex and Sae2/CtIP initiate end resection while long-range resection depends on the exonuclease Exo1 or the helicase-topoisomerase complex Sgs1-Top3-Rmi1 with the endonuclease Dna21-6. DSBs occur in the context of chromatin, but how the resection machinery navigates through nucleosomal DNA is a process that is not well understood7. Here, we show that the yeast S. cerevisiae Fun30 protein and its human counterpart SMARCAD18, two poorly characterized ATP-dependent chromatin remodelers of the Snf2 ATPase family, are novel factors that are directly involved in the DSB response. Fun30 physically associates with DSB ends and directly promotes both Exo1- and Sgs1-dependent end resection through a mechanism involving its ATPase activity. The function of Fun30 in resection facilitates repair of camptothecin (CPT)-induced DNA lesions, and it becomes dispensable when Exo1 is ectopically overexpressed. Interestingly, SMARCAD1 is also recruited to DSBs and the kinetics of recruitment is similar to that of Exo1. Loss of SMARCAD1 impairs end resection, recombinational DNA repair and renders cells hypersensitive to DNA damage resulting from CPT or PARP inhibitor treatments. These findings unveil an evolutionarily conserved role for the Fun30 and SMARCAD1 chromatin remodelers in controlling end resection, homologous recombination and genome stability in the context of chromatin. PMID:22960744

Ultrasonically assisted hydrothermal synthesis of activated carbon-HKUST-1-MOF hybrid for efficient simultaneous ultrasound-assisted removal of ternary organic dyes and antibacterial investigation: Taguchi optimization.

PubMed

Azad, F Nasiri; Ghaedi, M; Dashtian, K; Hajati, S; Pezeshkpour, V

2016-07-01

Activated carbon (AC) composite with HKUST-1 metal organic framework (AC-HKUST-1 MOF) was prepared by ultrasonically assisted hydrothermal method and characterized by FTIR, SEM and XRD analysis and laterally was applied for the simultaneous ultrasound-assisted removal of crystal violet (CV), disulfine blue (DSB) and quinoline yellow (QY) dyes in their ternary solution. In addition, this material, was screened in vitro for their antibacterial actively against Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PAO1) bacteria. In dyes removal process, the effects of important variables such as initial concentration of dyes, adsorbent mass, pH and sonication time on adsorption process optimized by Taguchi approach. Optimum values of 4, 0.02 g, 4 min, 10 mg L(-1) were obtained for pH, AC-HKUST-1 MOF mass, sonication time and the concentration of each dye, respectively. At the optimized condition, the removal percentages of CV, DSB and QY were found to be 99.76%, 91.10%, and 90.75%, respectively, with desirability of 0.989. Kinetics of adsorption processes follow pseudo-second-order model. The Langmuir model as best method with high applicability for representation of experimental data, while maximum mono layer adsorption capacity for CV, DSB and QY on AC-HKUST-1 estimated to be 133.33, 129.87 and 65.37 mg g(-1) which significantly were higher than HKUST-1 as sole material with Qm to equate 59.45, 57.14 and 38.80 mg g(-1), respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Single- and double-strand break formation in DNA irradiated in aqueous solution: dependence on dose and OH radical scavenger concentration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Siddiqi, M.A.; Bothe, E.

The yields of single- and double-strand breaks (SSB and DSB) in calf thymus DNA, after /sup 60/Co gamma irradiation in dilute aqueous solution, have been determined via molecular weight measurements using a low-angle laser light scattering technique. The irradiations were administered to N/sub 2/O-containing solutions of DNA in the absence and presence of oxygen and with different concentrations of the OH radical scavengers phenol, tertiary butanol, and methanol. OH radicals were found to produce SSB linearly with dose with a G value of 55 nmol J-1 and 54 nmol J-1 in deoxygenated and oxygenated solutions, respectively. DSB were formed accordingmore » to a linear-quadratic dose relationship and the G value of linearly formed DSB were GDSB alpha(r.t.) = 3.5 nmol J-1 in deoxygenated and 3.2 nmol J-1 in oxygenated solution. The ratio of GSSB/GDSB alpha(r.t.) = gamma of 19 +/- 6 was independent of the scavenger concentration in the case of tertiary butanol and methanol-containing solutions. GDSB alpha(r.t.) is interpreted to result from a radical site transferred from a sugar moiety of the cleaved strand to the complementary intact strand. This process of radical transfer and subsequent cleavage of the second strand occurs with a probability of about 6 +/- 2% in the presence of oxygen at all scavenger concentrations studied. These data on scavenging capacity on GDSB alpha(r.t.) suggest that the double-strand breakage produced via radical transfer remains higher than that resulting from direct effect, up to scavenging capacities of about 10(9) s-1.« less

DNA Double-Strand Breaks Coupled with PARP1 and HNRNPA2B1 Binding Sites Flank Coordinately Expressed Domains in Human Chromosomes

PubMed Central

Fedoseeva, Daria M.; Sosin, Dmitri V.; Grachev, Sergei A.; Serebraykova, Marina V.; Romanenko, Svetlana A.; Vorobieva, Nadezhda V.; Kravatsky, Yuri V.

2013-01-01

Genome instability plays a key role in multiple biological processes and diseases, including cancer. Genome-wide mapping of DNA double-strand breaks (DSBs) is important for understanding both chromosomal architecture and specific chromosomal regions at DSBs. We developed a method for precise genome-wide mapping of blunt-ended DSBs in human chromosomes, and observed non-random fragmentation and DSB hot spots. These hot spots are scattered along chromosomes and delimit protected 50–250 kb DNA domains. We found that about 30% of the domains (denoted forum domains) possess coordinately expressed genes and that PARP1 and HNRNPA2B1 specifically bind DNA sequences at the forum domain termini. Thus, our data suggest a novel type of gene regulation: a coordinated transcription or silencing of gene clusters delimited by DSB hot spots as well as PARP1 and HNRNPa2B1 binding sites. PMID:23593027

Phenotypic diversification by enhanced genome restructuring after induction of multiple DNA double-strand breaks.

PubMed

Muramoto, Nobuhiko; Oda, Arisa; Tanaka, Hidenori; Nakamura, Takahiro; Kugou, Kazuto; Suda, Kazuki; Kobayashi, Aki; Yoneda, Shiori; Ikeuchi, Akinori; Sugimoto, Hiroki; Kondo, Satoshi; Ohto, Chikara; Shibata, Takehiko; Mitsukawa, Norihiro; Ohta, Kunihiro

2018-05-18

DNA double-strand break (DSB)-mediated genome rearrangements are assumed to provide diverse raw genetic materials enabling accelerated adaptive evolution; however, it remains unclear about the consequences of massive simultaneous DSB formation in cells and their resulting phenotypic impact. Here, we establish an artificial genome-restructuring technology by conditionally introducing multiple genomic DSBs in vivo using a temperature-dependent endonuclease TaqI. Application in yeast and Arabidopsis thaliana generates strains with phenotypes, including improved ethanol production from xylose at higher temperature and increased plant biomass, that are stably inherited to offspring after multiple passages. High-throughput genome resequencing revealed that these strains harbor diverse rearrangements, including copy number variations, translocations in retrotransposons, and direct end-joinings at TaqI-cleavage sites. Furthermore, large-scale rearrangements occur frequently in diploid yeasts (28.1%) and tetraploid plants (46.3%), whereas haploid yeasts and diploid plants undergo minimal rearrangement. This genome-restructuring system (TAQing system) will enable rapid genome breeding and aid genome-evolution studies.

Modeling non-homologous end joining.

PubMed

Li, Yongfeng; Cucinotta, Francis A

2011-08-21

Non-homologous end joining (NHEJ) is an important DNA repair pathway for DNA double-strand breaks. Several proteins, including Ku, DNA-PKcs, Artemis, XRCC4/Ligase IV and XLF, are involved in the NHEJ for the DNA damage detection, DNA free end processing and ligation. The classical model of NHEJ is a sequential model in which DNA-PKcs is first recruited by the Ku bound DNA prior to any other repair proteins. Recent experimental study (McElhinny et al., 2000; Costantini et al., 2007; Mari et al., 2006; Yano and Chen, 2008) suggested that the recruitment ordering is not crucial. In this work, by proposing a mathematical model in terms of biochemical reaction network and performing stability and related analysis, we demonstrate theoretically that if DSB repair pathway independent of DNA-PKcs exists, then the classical sequential model and new two-phase model are essentially indistinguishable in the sense that DSB can be repaired thoroughly in both models when the repair proteins are sufficient. Published by Elsevier Ltd.

Phenothiazine Inhibitors of TLKs Affect Double-Strand Break Repair and DNA Damage Response Recovery and Potentiate Tumor Killing with Radiomimetic Therapy

PubMed Central

Ronald, Sharon; Awate, Sanket; Rath, Abhijit; Carroll, Jennifer; Galiano, Floyd; Dwyer, Donard; Kleiner-Hancock, Heather; Mathis, J. Michael; Vigod, Simone

2013-01-01

The Tousled-like kinases (TLKs) are involved in chromatin assembly, DNA repair, and transcription. Two TLK genes exist in humans, and their expression is often dysregulated in cancer. TLKs phosphorylate Asf1 and Rad9, regulating double-strand break (DSB) repair and the DNA damage response (DDR). TLKs maintain genomic stability and are important therapeutic intervention targets. We identified specific inhibitors of TLKs from several compound libraries, some of which belong to the family of phenothiazine antipsychotics. The inhibitors prevented the TLK-mediated phosphorylation of Rad9(S328) and impaired checkpoint recovery and DSB repair. The inhibitor thioridazine (THD) potentiated tumor killing with chemotherapy and also had activity alone. Staining for γ-H2AX revealed few positive cells in untreated tumors, but large numbers in mice treated with low doxorubicin or THD alone, possibly the result of the accumulation of DSBs that are not promptly repaired as they may occur in the harsh tumor growth environment. PMID:23946870

Dose-Rate Effects in Breaking DNA Strands by Short Pulses of Extreme Ultraviolet Radiation.

PubMed

Vyšín, Luděk; Burian, Tomáš; Ukraintsev, Egor; Davídková, Marie; Grisham, Michael E; Heinbuch, Scott; Rocca, Jorge J; Juha, Libor

2018-05-01

In this study, we examined dose-rate effects on strand break formation in plasmid DNA induced by pulsed extreme ultraviolet (XUV) radiation. Dose delivered to the target molecule was controlled by attenuating the incident photon flux using aluminum filters as well as by changing the DNA/buffer-salt ratio in the irradiated sample. Irradiated samples were examined using agarose gel electrophoresis. Yields of single- and double-strand breaks (SSBs and DSBs) were determined as a function of the incident photon fluence. In addition, electrophoresis also revealed DNA cross-linking. Damaged DNA was inspected by means of atomic force microscopy (AFM). Both SSB and DSB yields decreased with dose rate increase. Quantum yields of SSBs at the highest photon fluence were comparable to yields of DSBs found after synchrotron irradiation. The average SSB/DSB ratio decreased only slightly at elevated dose rates. In conclusion, complex and/or clustered damages other than cross-links do not appear to be induced under the radiation conditions applied in this study.

Altered Hematopoiesis in Mice Lacking DNA Polymerase μ Is Due to Inefficient Double-Strand Break Repair

PubMed Central

Lucas, Daniel; Escudero, Beatriz; Ligos, José Manuel; Segovia, Jose Carlos; Estrada, Juan Camilo; Terrados, Gloria; Blanco, Luis; Samper, Enrique; Bernad, Antonio

2009-01-01

Polymerase mu (Polμ) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polμ deficiency results in impaired Vκ-Jκ recombination and altered somatic hypermutation and centroblast development. In Polμ−/− mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body γ-irradiation revealed that Polμ also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polμ function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues. PMID:19229323

Histone Variant Regulates DNA Repair via Chromatin Condensation | Center for Cancer Research

Cancer.gov

Activating the appropriate DNA repair pathway is essential for maintaining the stability of the genome after a break in both strands of DNA. How a pathway is selected, however, is not well understood. Since these double strand breaks (DSBs) occur while DNA is packaged as chromatin, changes in its organization are necessary for repair to take place. Numerous alterations have been associated with DSBs, including modifications of histone tails and exchange of histone variants, some increasing chromatin accessibility, others reducing it. In fact, distinct domains flanking a single DSB have been observed that are bound by opposing repair pathway proteins 53BP1and BRCA1, which promote non-homologous end joining (NHEJ) and homologous recombination (HR), respectively. To investigate whether DSB-proximal chromatin reorganization affects repair pathway selection, Philipp Oberdoerffer, Ph.D., of CCR’s Laboratory of Receptor Biology and Gene Expression, and his colleagues performed a high-throughput RNA interference (RNAi) screen for chromatin-related genes that modulate HR.

γH2AX foci formation in the absence of DNA damage: mitotic H2AX phosphorylation is mediated by the DNA-PKcs/CHK2 pathway.

PubMed

Tu, Wen-Zhi; Li, Bing; Huang, Bo; Wang, Yu; Liu, Xiao-Dan; Guan, Hua; Zhang, Shi-Meng; Tang, Yan; Rang, Wei-Qing; Zhou, Ping-Kun

2013-11-01

Phosphorylated H2AX is considered to be a biomarker for DNA double-strand breaks (DSB), but recent evidence suggests that γH2AX does not always indicate the presence of DSB. Here we demonstrate the bimodal dynamic of H2AX phosphorylation induced by ionizing radiation, with the second peak appearing when G2/M arrest is induced. An increased level of γH2AX occurred in mitotic cells, and this increase was attenuated by DNA-PKcs inactivation or Chk2 depletion, but not by ATM inhibition. The phosphorylation-mimic CHK2-T68D abrogated the attenuation of mitotic γH2AX induced by DNA-PKcs inactivation. Thus, the DNA-PKcs/CHK2 pathway mediates the mitotic phosphorylation of H2AX in the absence of DNA damage. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Nanoparticulate carbon black in cigarette smoke induces DNA cleavage and Th17-mediated emphysema.

PubMed

You, Ran; Lu, Wen; Shan, Ming; Berlin, Jacob M; Samuel, Errol Lg; Marcano, Daniela C; Sun, Zhengzong; Sikkema, William Ka; Yuan, Xiaoyi; Song, Lizhen; Hendrix, Amanda Y; Tour, James M; Corry, David B; Kheradmand, Farrah

2015-10-05

Chronic inhalation of cigarette smoke is the major cause of sterile inflammation and pulmonary emphysema. The effect of carbon black (CB), a universal constituent of smoke derived from the incomplete combustion of organic material, in smokers and non-smokers is less known. In this study, we show that insoluble nanoparticulate carbon black (nCB) accumulates in human myeloid dendritic cells (mDCs) from emphysematous lung and in CD11c(+) lung antigen presenting cells (APC) of mice exposed to smoke. Likewise, nCB intranasal administration induced emphysema in mouse lungs. Delivered by smoking or intranasally, nCB persisted indefinitely in mouse lung, activated lung APCs, and promoted T helper 17 cell differentiation through double-stranded DNA break (DSB) and ASC-mediated inflammasome assembly in phagocytes. Increasing the polarity or size of CB mitigated many adverse effects. Thus, nCB causes sterile inflammation, DSB, and emphysema and explains adverse health outcomes seen in smokers while implicating the dangers of nCB exposure in non-smokers.

Protein Determinants of Meiotic DNA Break Hotspots

PubMed Central

Fowler, Kyle R.; Gutiérrez-Velasco, Susana

2013-01-01

SUMMARY Meiotic recombination, crucial for proper chromosome segregation and genome evolution, is initiated by programmed DNA double-strand breaks (DSBs) in yeasts and likely all sexually reproducing species. In fission yeast, DSBs occur up to hundreds of times more frequently at special sites, called hotspots, than in other regions of the genome. What distinguishes hotspots from cold regions is an unsolved problem, although transcription factors determine some hotspots. We report the discovery that three coiled-coil proteins – Rec25, Rec27, and Mug20 – bind essentially all hotspots with unprecedented specificity even without DSB formation. These small proteins are components of linear elements, are related to synaptonemal complex proteins, and are essential for nearly all DSBs at most hotspots. Our results indicate these hotspot determinants activate or stabilize the DSB-forming protein Rec12 (Spo11 homolog) rather than promote its binding to hotspots. We propose a new paradigm for hotspot determination and crossover control by linear element proteins. PMID:23395004

The CAF-1 and Hir Histone Chaperones Associate with Sites of Meiotic Double-Strand Breaks in Budding Yeast

PubMed Central

Brachet, Elsa; Béneut, Claire; Serrentino, Maria-Elisabetta; Borde, Valérie

2015-01-01

In the meiotic prophase, programmed DNA double-strand breaks (DSB) are introduced along chromosomes to promote homolog pairing and recombination. Although meiotic DSBs usually occur in nucleosome-depleted, accessible regions of chromatin, their repair by homologous recombination takes place in a nucleosomal environment. Nucleosomes may represent an obstacle for the recombination machinery and their timely eviction and reincorporation into chromatin may influence the outcome of recombination, for instance by stabilizing recombination intermediates. Here we show in budding yeast that nucleosomes flanking a meiotic DSB are transiently lost during recombination, and that specific histone H3 chaperones, CAF-1 and Hir, are mobilized at meiotic DSBs. However, the absence of these chaperones has no effect on meiotic recombination, suggesting that timely histone reincorporation following their eviction has no influence on the recombination outcome, or that redundant pathways are activated. This study is the first example of the involvement of histone H3 chaperones at naturally occurring, developmentally programmed DNA double-strand breaks. PMID:25938567

Report of the Defense Science Board Task Force on National Aero-Space Plane (NASP) Program

NASA Technical Reports Server (NTRS)

1992-01-01

Six years ago, the Defense Science Board (DSB) initiated a review of the concept, technical basis, program content, and missions of the National Aerospace Plane (NASP) program. The report was completed in Sep. 1988, and the recommendations contributed to strengthening the technical efforts in the NASP program. Since then, substantial technological progress has been made in the technology development phase (Phase 2) of the program. Phase 2 of the program is currently scheduled to end in late Fiscal Year 1993, with a decision whether to proceed to the experimental flight vehicle phase (Phase 3) to be made at that time. This decision will be a very significant one for the Department of Defense (DoD) and the National Aeronautics and Space Administration (NASA). In February of this year, the DSB was chartered to revisit the NASP program to assess the degree to which the many technical challenges of the program have been resolved, or are likely to be resolved by the end of Phase 2.

A low noise 665 GHz SIS quasi-particle waveguide receiver

NASA Technical Reports Server (NTRS)

Kooi, J. W.; Walker, C. K.; Leduc, H. G.; Hunter, T. R.; Benford, D. J.; Phillips, T. G.

1993-01-01

Recent results on a 565-690 GHz SIS heterodyne receiver employing a 0.36 micron(sup 2) Nb/AlOx/Nb SIS tunnel junction with high quality circular non-contacting back short and E-plane tuners in a full height wave guide mount are reported. No resonant tuning structures were incorporated in the junction design at this time, even though such structures are expected to help the performance of the receiver. The receiver operates to at least the gap frequency of Niobium, approximately 680 GHz. Typical receiver noise temperatures from 565-690 GHz range from 160K to 230K with a best value of 185K DSB at 648 GHz. With the mixer cooled from 4.3K to 2K the measured receiver noise temperatures decreased by approximately 15 percent, giving roughly 180K DSB from 660 to 680 GHz. The receiver has a full 1 GHz IF pass band and was successfully installed at the Caltech Submillimeter Observatory in Hawaii.

Spatial analysis of cities using Renyi entropy and fractal parameters

NASA Astrophysics Data System (ADS)

Chen, Yanguang; Feng, Jian

2017-12-01

The spatial distributions of cities fall into two groups: one is the simple distribution with characteristic scale (e.g. exponential distribution), and the other is the complex distribution without characteristic scale (e.g. power-law distribution). The latter belongs to scale-free distributions, which can be modeled with fractal geometry. However, fractal dimension is not suitable for the former distribution. In contrast, spatial entropy can be used to measure any types of urban distributions. This paper is devoted to generalizing multifractal parameters by means of dual relation between Euclidean and fractal geometries. The main method is mathematical derivation and empirical analysis, and the theoretical foundation is the discovery that the normalized fractal dimension is equal to the normalized entropy. Based on this finding, a set of useful spatial indexes termed dummy multifractal parameters are defined for geographical analysis. These indexes can be employed to describe both the simple distributions and complex distributions. The dummy multifractal indexes are applied to the population density distribution of Hangzhou city, China. The calculation results reveal the feature of spatio-temporal evolution of Hangzhou's urban morphology. This study indicates that fractal dimension and spatial entropy can be combined to produce a new methodology for spatial analysis of city development.

The Potential for Spatial Distribution Indices to Signal Thresholds in Marine Fish Biomass

PubMed Central

Reuchlin-Hugenholtz, Emilie

2015-01-01

The frequently observed positive relationship between fish population abundance and spatial distribution suggests that changes in distribution can be indicative of trends in abundance. If contractions in spatial distribution precede declines in spawning stock biomass (SSB), spatial distribution reference points could complement the SSB reference points that are commonly used in marine conservation biology and fisheries management. When relevant spatial distribution information is integrated into fisheries management and recovery plans, risks and uncertainties associated with a plan based solely on the SSB criterion would be reduced. To assess the added value of spatial distribution data, we examine the relationship between SSB and four metrics of spatial distribution intended to reflect changes in population range, concentration, and density for 10 demersal populations (9 species) inhabiting the Scotian Shelf, Northwest Atlantic. Our primary purpose is to assess their potential to serve as indices of SSB, using fisheries independent survey data. We find that metrics of density offer the best correlate of spawner biomass. A decline in the frequency of encountering high density areas is associated with, and in a few cases preceded by, rapid declines in SSB in 6 of 10 populations. Density-based indices have considerable potential to serve both as an indicator of SSB and as spatially based reference points in fisheries management. PMID:25789624

Design and implementation of a distributed large-scale spatial database system based on J2EE

NASA Astrophysics Data System (ADS)

Gong, Jianya; Chen, Nengcheng; Zhu, Xinyan; Zhang, Xia

2003-03-01

With the increasing maturity of distributed object technology, CORBA, .NET and EJB are universally used in traditional IT field. However, theories and practices of distributed spatial database need farther improvement in virtue of contradictions between large scale spatial data and limited network bandwidth or between transitory session and long transaction processing. Differences and trends among of CORBA, .NET and EJB are discussed in details, afterwards the concept, architecture and characteristic of distributed large-scale seamless spatial database system based on J2EE is provided, which contains GIS client application, web server, GIS application server and spatial data server. Moreover the design and implementation of components of GIS client application based on JavaBeans, the GIS engine based on servlet, the GIS Application server based on GIS enterprise JavaBeans(contains session bean and entity bean) are explained.Besides, the experiments of relation of spatial data and response time under different conditions are conducted, which proves that distributed spatial database system based on J2EE can be used to manage, distribute and share large scale spatial data on Internet. Lastly, a distributed large-scale seamless image database based on Internet is presented.

Analysis of skin tissues spatial fluorescence distribution by the Monte Carlo simulation

NASA Astrophysics Data System (ADS)

Y Churmakov, D.; Meglinski, I. V.; Piletsky, S. A.; Greenhalgh, D. A.

2003-07-01

A novel Monte Carlo technique of simulation of spatial fluorescence distribution within the human skin is presented. The computational model of skin takes into account the spatial distribution of fluorophores, which would arise due to the structure of collagen fibres, compared to the epidermis and stratum corneum where the distribution of fluorophores is assumed to be homogeneous. The results of simulation suggest that distribution of auto-fluorescence is significantly suppressed in the near-infrared spectral region, whereas the spatial distribution of fluorescence sources within a sensor layer embedded in the epidermis is localized at an `effective' depth.

Integrating satellite actual evapotranspiration patterns into distributed model parametrization and evaluation for a mesoscale catchment

NASA Astrophysics Data System (ADS)

Demirel, M. C.; Mai, J.; Stisen, S.; Mendiguren González, G.; Koch, J.; Samaniego, L. E.

2016-12-01

Distributed hydrologic models are traditionally calibrated and evaluated against observations of streamflow. Spatially distributed remote sensing observations offer a great opportunity to enhance spatial model calibration schemes. For that it is important to identify the model parameters that can change spatial patterns before the satellite based hydrologic model calibration. Our study is based on two main pillars: first we use spatial sensitivity analysis to identify the key parameters controlling the spatial distribution of actual evapotranspiration (AET). Second, we investigate the potential benefits of incorporating spatial patterns from MODIS data to calibrate the mesoscale Hydrologic Model (mHM). This distributed model is selected as it allows for a change in the spatial distribution of key soil parameters through the calibration of pedo-transfer function parameters and includes options for using fully distributed daily Leaf Area Index (LAI) directly as input. In addition the simulated AET can be estimated at the spatial resolution suitable for comparison to the spatial patterns observed using MODIS data. We introduce a new dynamic scaling function employing remotely sensed vegetation to downscale coarse reference evapotranspiration. In total, 17 parameters of 47 mHM parameters are identified using both sequential screening and Latin hypercube one-at-a-time sampling methods. The spatial patterns are found to be sensitive to the vegetation parameters whereas streamflow dynamics are sensitive to the PTF parameters. The results of multi-objective model calibration show that calibration of mHM against observed streamflow does not reduce the spatial errors in AET while they improve only the streamflow simulations. We will further examine the results of model calibration using only multi spatial objective functions measuring the association between observed AET and simulated AET maps and another case including spatial and streamflow metrics together.

Combinatorial DNA Damage Pairing Model Based on X-Ray-Induced Foci Predicts the Dose and LET Dependence of Cell Death in Human Breast Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vadhavkar, Nikhil; Pham, Christopher; Georgescu, Walter

In contrast to the classic view of static DNA double-strand breaks (DSBs) being repaired at the site of damage, we hypothesize that DSBs move and merge with each other over large distances (m). As X-ray dose increases, the probability of having DSB clusters increases as does the probability of misrepair and cell death. Experimental work characterizing the X-ray dose dependence of radiation-induced foci (RIF) in nonmalignant human mammary epithelial cells (MCF10A) is used here to validate a DSB clustering model. We then use the principles of the local effect model (LEM) to predict the yield of DSBs at the submicronmore » level. Two mechanisms for DSB clustering, namely random coalescence of DSBs versus active movement of DSBs into repair domains are compared and tested. Simulations that best predicted both RIF dose dependence and cell survival after X-ray irradiation favored the repair domain hypothesis, suggesting the nucleus is divided into an array of regularly spaced repair domains of ~;;1.55 m sides. Applying the same approach to high-linear energy transfer (LET) ion tracks, we are able to predict experimental RIF/m along tracks with an overall relative error of 12percent, for LET ranging between 30 350 keV/m and for three different ions. Finally, cell death was predicted by assuming an exponential dependence on the total number of DSBs and of all possible combinations of paired DSBs within each simulated RIF. Relative biological effectiveness (RBE) predictions for cell survival of MCF10A exposed to high-LET showed an LET dependence that matches previous experimental results for similar cell types. Overall, this work suggests that microdosimetric properties of ion tracks at the submicron level are sufficient to explain both RIF data and survival curves for any LET, similarly to the LEM assumption. Conversely, high-LET death mechanism does not have to infer linear-quadratic dose formalism as done in the LEM. In addition, the size of repair domains derived in our model are based on experimental RIF and are three times larger than the hypothetical LEM voxel used to fit survival curves. Our model is therefore an alternative to previous approaches that provides a testable biological mechanism (i.e., RIF). In addition, we propose that DSB pairing will help develop more accurate alternatives to the linear cancer risk model (LNT) currently used for regulating exposure to very low levels of ionizing radiation.« less

[Spatial distribution pattern of Pontania dolichura larvae and sampling technique].

PubMed

Zhang, Feng; Chen, Zhijie; Zhang, Shulian; Zhao, Huiyan

2006-03-01

In this paper, the spatial distribution pattern of Pontania dolichura larvae was analyzed with Taylor's power law, Iwao's distribution function, and six aggregation indexes. The results showed that the spatial distribution pattern of P. dolichura larvae was of aggregated, and the basic component of the distribution was individual colony, with the aggregation intensity increased with density. On branches, the aggregation was caused by the adult behavior of laying eggs and the spatial position of leaves, while on leaves, the aggregation was caused by the spatial position of news leaves in spring when m < 2.37, and by the spatial position of news leaves in spring and the behavior of eclosion and laying eggs when m > 2.37. By using the parameters alpha and beta in Iwao's m * -m regression equation, the optimal and sequential sampling numbers were determined.

Software Acquisition Improvement in the Aeronautical Systems Center

DTIC Science & Technology

2008-09-01

software fielded, a variety of different methods were suggested by the interviewees. These included blocks, suites and other tailored processes developed...12 Selection of Research Method ...DoD look to the commercial market to buy tools, methods , environments, and application software, instead of custom-built software (DSB: 1987). These

DSB Task Force on Cyber Supply Chain

DTIC Science & Technology

2017-02-06

27 3.4 Cybersecurity for Commercial and Open Source Components...Communications and Intelligence ASD(L&MR): Assistant Secretary of Defense for Logistics and Materiel Readiness ASD(R&E): Assistant Secretary of Defense...system BSIMM: Building Security in Maturity Model C4ISR: command, control, communications, computers, intelligence , surveillance and

Performance Evaluation of the Honeywell GG1308 Miniature Ring Laser Gyroscope

DTIC Science & Technology

1993-01-01

information. The final display line provides the current DSB configuration status. An external strobe was established between the Contraves motion...components and systems. The core of the facility is a Contraves -Goerz Model 57CD 2-axis motion simulator capable of highly precise position, rate and

An Air Force Strategy for the Long Haul

DTIC Science & Technology

2009-01-01

neuver ( MVM ) that suggests a very heavy vertical takeoff and landing (VTOL) plat- form. It is clear from a recent Defense Science Board (DSB...refueling tanker) KC-X Future aerial refueling tanker LEO Low earth orbit MQ-1 Predator (unmanned aerial vehicle) MQ-9 Reaper (unmanned aerial vehicle) MVM

47 CFR 73.758 - System specifications for digitally modulated emissions in the HF broadcasting service.

Code of Federal Regulations, 2010 CFR

2010-10-01

... this paragraph. (4) Modulation. Quadrature amplitude modulation (QAM) with orthogonal frequency... frequency broadcasting (HFBC) band, provided the protection afforded to the analog emissions is at least as... used for either DSB or SSB emissions. (c) Emission characteristics—(1) Bandwidth and center frequency...

Inner membrane fusion mediates spatial distribution of axonal mitochondria

PubMed Central

Yu, Yiyi; Lee, Hao-Chih; Chen, Kuan-Chieh; Suhan, Joseph; Qiu, Minhua; Ba, Qinle; Yang, Ge

2016-01-01

In eukaryotic cells, mitochondria form a dynamic interconnected network to respond to changing needs at different subcellular locations. A fundamental yet unanswered question regarding this network is whether, and if so how, local fusion and fission of individual mitochondria affect their global distribution. To address this question, we developed high-resolution computational image analysis techniques to examine the relations between mitochondrial fusion/fission and spatial distribution within the axon of Drosophila larval neurons. We found that stationary and moving mitochondria underwent fusion and fission regularly but followed different spatial distribution patterns and exhibited different morphology. Disruption of inner membrane fusion by knockdown of dOpa1, Drosophila Optic Atrophy 1, not only increased the spatial density of stationary and moving mitochondria but also changed their spatial distributions and morphology differentially. Knockdown of dOpa1 also impaired axonal transport of mitochondria. But the changed spatial distributions of mitochondria resulted primarily from disruption of inner membrane fusion because knockdown of Milton, a mitochondrial kinesin-1 adapter, caused similar transport velocity impairment but different spatial distributions. Together, our data reveals that stationary mitochondria within the axon interconnect with moving mitochondria through fusion and fission and that local inner membrane fusion between individual mitochondria mediates their global distribution. PMID:26742817

Effects of radiation quality and oxygen on clustered DNA lesions and cell death.

PubMed

Stewart, Robert D; Yu, Victor K; Georgakilas, Alexandros G; Koumenis, Constantinos; Park, Joo Han; Carlson, David J

2011-11-01

Radiation quality and cellular oxygen concentration have a substantial impact on DNA damage, reproductive cell death and, ultimately, the potential efficacy of radiation therapy for the treatment of cancer. To better understand and quantify the effects of radiation quality and oxygen on the induction of clustered DNA lesions, we have now extended the Monte Carlo Damage Simulation (MCDS) to account for reductions in the initial lesion yield arising from enhanced chemical repair of DNA radicals under hypoxic conditions. The kinetic energy range and types of particles considered in the MCDS have also been expanded to include charged particles up to and including (56)Fe ions. The induction of individual and clustered DNA lesions for arbitrary mixtures of different types of radiation can now be directly simulated. For low-linear energy transfer (LET) radiations, cells irradiated under normoxic conditions sustain about 2.9 times as many double-strand breaks (DSBs) as cells irradiated under anoxic conditions. New experiments performed by us demonstrate similar trends in the yields of non-DSB (Fpg and Endo III) clusters in HeLa cells irradiated by γ rays under aerobic and hypoxic conditions. The good agreement among measured and predicted DSBs, Fpg and Endo III cluster yields suggests that, for the first time, it may be possible to determine nucleotide-level maps of the multitude of different types of clustered DNA lesions formed in cells under reduced oxygen conditions. As particle LET increases, the MCDS predicts that the ratio of DSBs formed under normoxic to hypoxic conditions by the same type of radiation decreases monotonically toward unity. However, the relative biological effectiveness (RBE) of higher-LET radiations compared to (60)Co γ rays (0.24 keV/μm) tends to increase with decreasing oxygen concentration. The predicted RBE of a 1 MeV proton (26.9 keV/μm) relative to (60)Co γ rays for DSB induction increases from 1.9 to 2.3 as oxygen concentration decreases from 100% to 0%. For a 12 MeV (12)C ion (681 keV/μm), the 'predicted RBE for DSB induction increases from 3.4 (100% O(2)) to 9.8 (0% O(2)). Estimates of linear-quadratic (LQ) cell survival model parameters (α and β) are closely correlated to the Monte Carlo-predicted trends in DSB induction for a wide range of particle types, energies and oxygen concentrations. The analysis suggests α is, as a first approximation, proportional to the initial number of DSBs per cell, and β is proportional to the square of the initial number of DSBs per cell. Although the reported studies provide some evidence supporting the hypothesis that DSBs are a biologically critical form of clustered DNA lesion, the induction of Fpg and Endo III clusters in HeLa cells irradiated by γ rays exhibits similar trends with oxygen concentration. Other types of non-DSB cluster may still play an important role in reproductive cell death. The MCDS captures many of the essential trends in the formation of clustered DNA lesions by ionizing radiation and provides useful information to probe the multiscale effects and interactions of ionizing radiation in cells and tissues. Information from Monte Carlo simulations of cluster induction may also prove useful for efforts to better exploit radiation quality and reduce the impact of tumor hypoxia in proton and carbon-ion radiation therapy.

Metabolic Flexibility as a Major Predictor of Spatial Distribution in Microbial Communities

PubMed Central

Carbonero, Franck; Oakley, Brian B.; Purdy, Kevin J.

2014-01-01

A better understand the ecology of microbes and their role in the global ecosystem could be achieved if traditional ecological theories can be applied to microbes. In ecology organisms are defined as specialists or generalists according to the breadth of their niche. Spatial distribution is often used as a proxy measure of niche breadth; generalists have broad niches and a wide spatial distribution and specialists a narrow niche and spatial distribution. Previous studies suggest that microbial distribution patterns are contrary to this idea; a microbial generalist genus (Desulfobulbus) has a limited spatial distribution while a specialist genus (Methanosaeta) has a cosmopolitan distribution. Therefore, we hypothesise that this counter-intuitive distribution within generalist and specialist microbial genera is a common microbial characteristic. Using molecular fingerprinting the distribution of four microbial genera, two generalists, Desulfobulbus and the methanogenic archaea Methanosarcina, and two specialists, Methanosaeta and the sulfate-reducing bacteria Desulfobacter were analysed in sediment samples from along a UK estuary. Detected genotypes of both generalist genera showed a distinct spatial distribution, significantly correlated with geographic distance between sites. Genotypes of both specialist genera showed no significant differential spatial distribution. These data support the hypothesis that the spatial distribution of specialist and generalist microbes does not match that seen with specialist and generalist large organisms. It may be that generalist microbes, while having a wider potential niche, are constrained, possibly by intrageneric competition, to exploit only a small part of that potential niche while specialists, with far fewer constraints to their niche, are more capable of filling their potential niche more effectively, perhaps by avoiding intrageneric competition. We suggest that these counter-intuitive distribution patterns may be a common feature of microbes in general and represent a distinct microbial principle in ecology, which is a real challenge if we are to develop a truly inclusive ecology. PMID:24465487

The spatial distribution of cropland carbon transfer in Jilin province during 2014

NASA Astrophysics Data System (ADS)

Cai, Xintong; Meng, Jian; Li, Qiuhui; Gao, Shuang; Zhu, Xianjin

2018-01-01

Cropland carbon transfer (CCT, gC yr-1) is an important component in the carbon budget of terrestrial ecosystems. Analyzing the value of CCT and its spatial variation would provide a data basis for assessing the regional carbon balance. Based on the data from Jilin statistical yearbook 2015, we investigated the spatial variation of CCT in Jilin province during 2014. Results suggest that the CCT of Jilin province was 30.83 TgC, which exhibited a decreasing trend from the centre to the border but the west side was higher than the east. The magnitude of carbon transfer per area (MCT), which showed a similar spatial distribution with CCT, was the dominating component of CCT spatial distribution. The spatial distribution of MCT was jointly affected by that of the ratio of planting area to regional area (RPR) and carbon transfer per planting area (CTP), where RPR and CTP contributed 65.55% and 34.5% of MCT spatial distribution, respectively. Therefore, CCT in Jilin province spatially varied, which made it highly needed to consider the difference in CCT among regions when we assessing the regional carbon budget.

Novel programmable microwave photonic filter with arbitrary filtering shape and linear phase.

PubMed

Zhu, Xiaoqi; Chen, Feiya; Peng, Huanfa; Chen, Zhangyuan

2017-04-17

We propose and demonstrate a novel optical frequency comb (OFC) based microwave photonic filter which is able to realize arbitrary filtering shape with linear phase response. The shape of filter response is software programmable using finite impulse response (FIR) filter design method. By shaping the OFC spectrum using a programmable waveshaper, we can realize designed amplitude of FIR taps. Positive and negative sign of FIR taps are achieved by balanced photo-detection. The double sideband (DSB) modulation and symmetric distribution of filter taps are used to maintain the linear phase condition. In the experiment, we realize a fully programmable filter in the range from DC to 13.88 GHz. Four basic types of filters (lowpass, highpass, bandpass and bandstop) with different bandwidths, cut-off frequencies and central frequencies are generated. Also a triple-passband filter is realized in our experiment. To the best of our knowledge, it is the first demonstration of a programmable multiple passband MPF with linear phase response. The experiment shows good agreement with the theoretical result.

Relations between Spatial Distribution, Social Affiliations and Dominance Hierarchy in a Semi-Free Mandrill Population

PubMed Central

Naud, Alexandre; Chailleux, Eloise; Kestens, Yan; Bret, Céline; Desjardins, Dominic; Petit, Odile; Ngoubangoye, Barthélémy; Sueur, Cédric

2016-01-01

Although there exist advantages to group-living in comparison to a solitary lifestyle, costs and gains of group-living may be unequally distributed among group members. Predation risk, vigilance levels and food intake may be unevenly distributed across group spatial geometry and certain within-group spatial positions may be more or less advantageous depending on the spatial distribution of these factors. In species characterized with dominance hierarchy, high-ranking individuals are commonly observed in advantageous spatial position. However, in complex social systems, individuals can develop affiliative relationships that may balance the effect of dominance relationships in individual's spatial distribution. The objective of the present study is to investigate how the group spatial distribution of a semi-free ranging colony of Mandrills relates to its social organization. Using spatial observations in an area surrounding the feeding zone, we tested the three following hypothesis: (1) does dominance hierarchy explain being observed in proximity or far from a food patch? (2) Do affiliative associations also explain being observed in proximity or far from a food patch? (3) Do the differences in rank in the group hierarchy explain being co-observed in proximity of a food patch? Our results showed that high-ranking individuals were more observed in proximity of the feeding zone while low-ranking individuals were more observed at the boundaries of the observation area. Furthermore, we observed that affiliative relationships were also associated with individual spatial distributions and explain more of the total variance of the spatial distribution in comparison with dominance hierarchy. Finally, we found that individuals observed at a same moment in proximity of the feeding zone were more likely to be distant in the hierarchy while controlling for maternal kinship, age and sex similarity. This study brings some elements about how affiliative networks and dominance hierarchy are related to spatial positions in primates. PMID:27199845

Relations between Spatial Distribution, Social Affiliations and Dominance Hierarchy in a Semi-Free Mandrill Population.

PubMed

Naud, Alexandre; Chailleux, Eloise; Kestens, Yan; Bret, Céline; Desjardins, Dominic; Petit, Odile; Ngoubangoye, Barthélémy; Sueur, Cédric

2016-01-01

Although there exist advantages to group-living in comparison to a solitary lifestyle, costs and gains of group-living may be unequally distributed among group members. Predation risk, vigilance levels and food intake may be unevenly distributed across group spatial geometry and certain within-group spatial positions may be more or less advantageous depending on the spatial distribution of these factors. In species characterized with dominance hierarchy, high-ranking individuals are commonly observed in advantageous spatial position. However, in complex social systems, individuals can develop affiliative relationships that may balance the effect of dominance relationships in individual's spatial distribution. The objective of the present study is to investigate how the group spatial distribution of a semi-free ranging colony of Mandrills relates to its social organization. Using spatial observations in an area surrounding the feeding zone, we tested the three following hypothesis: (1) does dominance hierarchy explain being observed in proximity or far from a food patch? (2) Do affiliative associations also explain being observed in proximity or far from a food patch? (3) Do the differences in rank in the group hierarchy explain being co-observed in proximity of a food patch? Our results showed that high-ranking individuals were more observed in proximity of the feeding zone while low-ranking individuals were more observed at the boundaries of the observation area. Furthermore, we observed that affiliative relationships were also associated with individual spatial distributions and explain more of the total variance of the spatial distribution in comparison with dominance hierarchy. Finally, we found that individuals observed at a same moment in proximity of the feeding zone were more likely to be distant in the hierarchy while controlling for maternal kinship, age and sex similarity. This study brings some elements about how affiliative networks and dominance hierarchy are related to spatial positions in primates.

Considering the Spatial Layout Information of Bag of Features (BoF) Framework for Image Classification.

PubMed

Mu, Guangyu; Liu, Ying; Wang, Limin

2015-01-01

The spatial pooling method such as spatial pyramid matching (SPM) is very crucial in the bag of features model used in image classification. SPM partitions the image into a set of regular grids and assumes that the spatial layout of all visual words obey the uniform distribution over these regular grids. However, in practice, we consider that different visual words should obey different spatial layout distributions. To improve SPM, we develop a novel spatial pooling method, namely spatial distribution pooling (SDP). The proposed SDP method uses an extension model of Gauss mixture model to estimate the spatial layout distributions of the visual vocabulary. For each visual word type, SDP can generate a set of flexible grids rather than the regular grids from the traditional SPM. Furthermore, we can compute the grid weights for visual word tokens according to their spatial coordinates. The experimental results demonstrate that SDP outperforms the traditional spatial pooling methods, and is competitive with the state-of-the-art classification accuracy on several challenging image datasets.

Spatial coding-based approach for partitioning big spatial data in Hadoop

NASA Astrophysics Data System (ADS)

Yao, Xiaochuang; Mokbel, Mohamed F.; Alarabi, Louai; Eldawy, Ahmed; Yang, Jianyu; Yun, Wenju; Li, Lin; Ye, Sijing; Zhu, Dehai

2017-09-01

Spatial data partitioning (SDP) plays a powerful role in distributed storage and parallel computing for spatial data. However, due to skew distribution of spatial data and varying volume of spatial vector objects, it leads to a significant challenge to ensure both optimal performance of spatial operation and data balance in the cluster. To tackle this problem, we proposed a spatial coding-based approach for partitioning big spatial data in Hadoop. This approach, firstly, compressed the whole big spatial data based on spatial coding matrix to create a sensing information set (SIS), including spatial code, size, count and other information. SIS was then employed to build spatial partitioning matrix, which was used to spilt all spatial objects into different partitions in the cluster finally. Based on our approach, the neighbouring spatial objects can be partitioned into the same block. At the same time, it also can minimize the data skew in Hadoop distributed file system (HDFS). The presented approach with a case study in this paper is compared against random sampling based partitioning, with three measurement standards, namely, the spatial index quality, data skew in HDFS, and range query performance. The experimental results show that our method based on spatial coding technique can improve the query performance of big spatial data, as well as the data balance in HDFS. We implemented and deployed this approach in Hadoop, and it is also able to support efficiently any other distributed big spatial data systems.

Wave actions and topography determine the small-scale spatial distribution of newly settled Asari clams Ruditapes philippinarum on a tidal flat

NASA Astrophysics Data System (ADS)

Nambu, Ryogen; Saito, Hajime; Tanaka, Yoshio; Higano, Junya; Kuwahara, Hisami

2012-03-01

There are many studies on spatial distributions of Asari clam Ruditapes philippinarum adults on tidal flats but few have dealt with spatial distributions of newly settled Asari clam (<0.3 mm shell length, indicative of settlement patterns) in relation to physical/topographical conditions on tidal flats. We examined small-scale spatial distributions of newly settled individuals on the Matsunase tidal flat, central Japan, during the low spring tides on two days 29th-30th June 2007, together with the shear stress from waves and currents on the flat. The characteristics of spatial distribution of newly settled Asari clam markedly varied depending on both of hydrodynamic and topographical conditions on the tidal flat. Using generalized linear models (GLMs), factors responsible for affecting newly settled Asari clam density and its spatial distribution were distinguished between sampling days, with "crest" sites always having a negative influence each on the density and the distribution on both sampling days. The continuously recorded data for the wave-current flows at the "crest" site on the tidal flat showed that newly settled Asari clam, as well as bottom sediment particles, at the "crest" site to be easily displaced. Small-scale spatial distributions of newly settled Asari clam changed with more advanced benthic stages in relation to the wave shear stress.

Spatial distributions of dose enhancement around a gold nanoparticle at several depths of proton Bragg peak

NASA Astrophysics Data System (ADS)

Kwon, Jihun; Sutherland, Kenneth; Hashimoto, Takayuki; Shirato, Hiroki; Date, Hiroyuki

2016-10-01

Gold nanoparticles (GNPs) have been recognized as a promising candidate for a radiation sensitizer. A proton beam incident on a GNP can produce secondary electrons, resulting in an enhancement of the dose around the GNP. However, little is known about the spatial distribution of dose enhancement around the GNP, especially in the direction along the incident proton. The purpose of this study is to determine the spatial distribution of dose enhancement by taking the incident direction into account. Two steps of calculation were conducted using the Geant4 Monte Carlo simulation toolkit. First, the energy spectra of 100 and 195 MeV protons colliding with a GNP were calculated at the Bragg peak and three other depths around the peak in liquid water. Second, the GNP was bombarded by protons with the obtained energy spectra. Radial dose distributions were computed along the incident beam direction. The spatial distributions of the dose enhancement factor (DEF) and subtracted dose (Dsub) were then evaluated. The spatial DEF distributions showed hot spots in the distal radial region from the proton beam axis. The spatial Dsub distribution isotropically spread out around the GNP. Low energy protons caused higher and wider dose enhancement. The macroscopic dose enhancement in clinical applications was also evaluated. The results suggest that the consideration of the spatial distribution of GNPs in treatment planning will maximize the potential of GNPs.

Spatial Distribution of Surface Soil Moisture in a Small Forested Catchment

EPA Science Inventory

Predicting the spatial distribution of soil moisture is an important hydrological question. We measured the spatial distribution of surface soil moisture (upper 6 cm) using an Amplitude Domain Reflectometry sensor at the plot scale (2 × 2 m) and small catchment scale (0.84 ha) in...

[Analysis of influence on spatial distribution of fishing ground for Antarctic krill fishery in the northern South Shetland Islands based on GWR model].

PubMed

Chen, Lyu Feng; Zhu, Guo Ping

2018-03-01

Based on Antarctic krill fishery and marine environmental data collected by scientific observers, using geographically weighted regression (GWR) model, we analyzed the effects of the factors with spatial attributes, i.e., depth of krill swarm (DKS) and distance from fishing position to shore (DTS), and sea surface temperature (SST), on the spatial distribution of fishing ground in the northern South Shetland Islands. The results showed that there was no significant aggregation in spatial distribution of catch per unit fishing effort (CPUE). Spatial autocorrelations (positive) among three factors were observed in 2010 and 2013, but were not in 2012 and 2016. Results from GWR model showed that the extent for the impacts on spatial distribution of CPUEs varied among those three factors, following the order DKS>SST>DTS. Compared to the DKS and DTS, the impact of SST on the spatial distribution of CPUEs presented adverse trend in the eastern and western parts of the South Shetland Islands. Negative correlations occurred for the spatial effects of DKS and DTS on distribution of CPUEs, though with inter-annual and regional variation. Our results provide metho-dological reference for researches on the underlying mechanism for fishing ground formation for Antarctic krill fishery.

Real-time distribution of pelagic fish: combining hydroacoustics, GIS and spatial modelling at a fine spatial scale.

PubMed

Muška, Milan; Tušer, Michal; Frouzová, Jaroslava; Mrkvička, Tomáš; Ricard, Daniel; Seďa, Jaromír; Morelli, Federico; Kubečka, Jan

2018-03-29

Understanding spatial distribution of organisms in heterogeneous environment remains one of the chief issues in ecology. Spatial organization of freshwater fish was investigated predominantly on large-scale, neglecting important local conditions and ecological processes. However, small-scale processes are of an essential importance for individual habitat preferences and hence structuring trophic cascades and species coexistence. In this work, we analysed the real-time spatial distribution of pelagic freshwater fish in the Římov Reservoir (Czechia) observed by hydroacoustics in relation to important environmental predictors during 48 hours at 3-h interval. Effect of diurnal cycle was revealed of highest significance in all spatial models with inverse trends between fish distribution and predictors in day and night in general. Our findings highlighted daytime pelagic fish distribution as highly aggregated, with general fish preferences for central, deep and highly illuminated areas, whereas nighttime distribution was more disperse and fish preferred nearshore steep sloped areas with higher depth. This turnover suggests prominent movements of significant part of fish assemblage between pelagic and nearshore areas on a diel basis. In conclusion, hydroacoustics, GIS and spatial modelling proved as valuable tool for predicting local fish distribution and elucidate its drivers, which has far reaching implications for understanding freshwater ecosystem functioning.

Delineating Facies Spatial Distribution by Integrating Ensemble Data Assimilation and Indicator Geostatistics with Level Set Transformation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hammond, Glenn Edward; Song, Xuehang; Ye, Ming

A new approach is developed to delineate the spatial distribution of discrete facies (geological units that have unique distributions of hydraulic, physical, and/or chemical properties) conditioned not only on direct data (measurements directly related to facies properties, e.g., grain size distribution obtained from borehole samples) but also on indirect data (observations indirectly related to facies distribution, e.g., hydraulic head and tracer concentration). Our method integrates for the first time ensemble data assimilation with traditional transition probability-based geostatistics. The concept of level set is introduced to build shape parameterization that allows transformation between discrete facies indicators and continuous random variables. Themore » spatial structure of different facies is simulated by indicator models using conditioning points selected adaptively during the iterative process of data assimilation. To evaluate the new method, a two-dimensional semi-synthetic example is designed to estimate the spatial distribution and permeability of two distinct facies from transient head data induced by pumping tests. The example demonstrates that our new method adequately captures the spatial pattern of facies distribution by imposing spatial continuity through conditioning points. The new method also reproduces the overall response in hydraulic head field with better accuracy compared to data assimilation with no constraints on spatial continuity on facies.« less

Methodology to study the three-dimensional spatial distribution of prostate cancer and their dependence on clinical parameters

PubMed Central

Rojas, Kristians Diaz; Montero, Maria L.; Yao, Jorge; Messing, Edward; Fazili, Anees; Joseph, Jean; Ou, Yangming; Rubens, Deborah J.; Parker, Kevin J.; Davatzikos, Christos; Castaneda, Benjamin

2015-01-01

Abstract. A methodology to study the relationship between clinical variables [e.g., prostate specific antigen (PSA) or Gleason score] and cancer spatial distribution is described. Three-dimensional (3-D) models of 216 glands are reconstructed from digital images of whole mount histopathological slices. The models are deformed into one prostate model selected as an atlas using a combination of rigid, affine, and B-spline deformable registration techniques. Spatial cancer distribution is assessed by counting the number of tumor occurrences among all glands in a given position of the 3-D registered atlas. Finally, a difference between proportions is used to compare different spatial distributions. As a proof of concept, we compare spatial distributions from patients with PSA greater and less than 5  ng/ml and from patients older and younger than 60 years. Results suggest that prostate cancer has a significant difference in the right zone of the prostate between populations with PSA greater and less than 5  ng/ml. Age does not have any impact in the spatial distribution of the disease. The proposed methodology can help to comprehend prostate cancer by understanding its spatial distribution and how it changes according to clinical parameters. Finally, this methodology can be easily adapted to other organs and pathologies. PMID:26236756

The PARTRAC code: Status and recent developments

NASA Astrophysics Data System (ADS)

Friedland, Werner; Kundrat, Pavel

Biophysical modeling is of particular value for predictions of radiation effects due to manned space missions. PARTRAC is an established tool for Monte Carlo-based simulations of radiation track structures, damage induction in cellular DNA and its repair [1]. Dedicated modules describe interactions of ionizing particles with the traversed medium, the production and reactions of reactive species, and score DNA damage determined by overlapping track structures with multi-scale chromatin models. The DNA repair module describes the repair of DNA double-strand breaks (DSB) via the non-homologous end-joining pathway; the code explicitly simulates the spatial mobility of individual DNA ends in parallel with their processing by major repair enzymes [2]. To simulate the yields and kinetics of radiation-induced chromosome aberrations, the repair module has been extended by tracking the information on the chromosome origin of ligated fragments as well as the presence of centromeres [3]. PARTRAC calculations have been benchmarked against experimental data on various biological endpoints induced by photon and ion irradiation. The calculated DNA fragment distributions after photon and ion irradiation reproduce corresponding experimental data and their dose- and LET-dependence. However, in particular for high-LET radiation many short DNA fragments are predicted below the detection limits of the measurements, so that the experiments significantly underestimate DSB yields by high-LET radiation [4]. The DNA repair module correctly describes the LET-dependent repair kinetics after (60) Co gamma-rays and different N-ion radiation qualities [2]. First calculations on the induction of chromosome aberrations have overestimated the absolute yields of dicentrics, but correctly reproduced their relative dose-dependence and the difference between gamma- and alpha particle irradiation [3]. Recent developments of the PARTRAC code include a model of hetero- vs euchromatin structures to enable accounting for variations in DNA damage yields, complexity and repair between these regions. Second, the applicability of the code to low-energy ions has been extended to full stopping by using a modified Barkas scaling of proton cross sections for ions heavier than helium. Third, ongoing studies aim at hitherto unprecedented benchmarking of the code against experiments with sub-muµm focused bunches of low-LET ions mimicking single high-LET ion tracks [5] which separate effects of damage clustering on a sub-mum scale from DNA damage complexity on a nanometer scale. Fourth, motivated by implications for the involvement of mitochondria in intercellular signaling and radiation-induced bystander effects, ongoing work extends the range of PARTRAC DNA models to radiation effects on mitochondrial DNA. The contribution will discuss the PARTRAC modules, benchmarks to experimental data, recent and ongoing developments of the code, with special attention to its implications and potential applications in radiation protection and space research. Acknowledgement. This work was partially funded by the EU (Contract FP7-249689 ‘DoReMi’). References 1. Friedland et al., Mutat. Res. 711, 28 (2011) 2. Friedland et al., Int. J. Radiat. Biol. 88, 129 (2012) 3. Friedland et al., Mutat. Res. 756, 213 (2013) 4. Alloni et al., Radiat. Res. 179, 690 (2013) 5. Schmid et al., Phys. Med. Biol. 57, 5889 (2012)

Spatio-temporal patterns of key exploited marine species in the Northwestern Mediterranean Sea.

PubMed

Morfin, Marie; Fromentin, Jean-Marc; Jadaud, Angélique; Bez, Nicolas

2012-01-01

This study analyzes the temporal variability/stability of the spatial distributions of key exploited species in the Gulf of Lions (Northwestern Mediterranean Sea). To do so, we analyzed data from the MEDITS bottom-trawl scientific surveys from 1994 to 2010 at 66 fixed stations and selected 12 key exploited species. We proposed a geostatistical approach to handle zero-inflated and non-stationary distributions and to test for the temporal stability of the spatial structures. Empirical Orthogonal Functions and other descriptors were then applied to investigate the temporal persistence and the characteristics of the spatial patterns. The spatial structure of the distribution (i.e. the pattern of spatial autocorrelation) of the 12 key species studied remained highly stable over the time period sampled. The spatial distributions of all species obtained through kriging also appeared to be stable over time, while each species displayed a specific spatial distribution. Furthermore, adults were generally more densely concentrated than juveniles and occupied areas included in the distribution of juveniles. Despite the strong persistence of spatial distributions, we also observed that the area occupied by each species was correlated to its abundance: the more abundant the species, the larger the occupation area. Such a result tends to support MacCall's basin theory, according to which density-dependence responses would drive the expansion of those 12 key species in the Gulf of Lions. Further analyses showed that these species never saturated their habitats, suggesting that they are below their carrying capacity; an assumption in agreement with the overexploitation of several of these species. Finally, the stability of their spatial distributions over time and their potential ability to diffuse outside their main habitats give support to Marine Protected Areas as a potential pertinent management tool.

Report of the Defense Science Board Task Force on Computer Applications to Training and Wargaming

DTIC Science & Technology

1988-05-01

forms of experimental learning - are often dismissed as infeasible, distracting , dysfunctional, or more appropriate to training than to education...McWilliams will be the DSB Secretariat representative. It is not anticipated that your Inquiry will need to go into any T articular matters within the

BRCA2 and RAD51 promote double-strand break formation and cell death in response to gemcitabine.

PubMed

Jones, Rebecca M; Kotsantis, Panagiotis; Stewart, Grant S; Groth, Petra; Petermann, Eva

2014-10-01

Replication inhibitors cause replication fork stalling and double-strand breaks (DSB) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitize cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here, we investigate why, paradoxically, mutations in HR genes protect cells from killing by gemcitabine. Using DNA replication and DNA damage assays in mammalian cells, we show that even short gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression, and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at gemcitabine-stalled forks are converted into DSBs and thus contribute to gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumors. ©2014 American Association for Cancer Research.

DNA Replication Origins in Immunoglobulin Switch Regions Regulate Class Switch Recombination in an R-Loop-Dependent Manner.

PubMed

Wiedemann, Eva-Maria; Peycheva, Mihaela; Pavri, Rushad

2016-12-13

Class switch recombination (CSR) at the immunoglobulin heavy chain (IgH) locus generates antibody isotypes. CSR depends on double-strand breaks (DSBs) induced by activation-induced cytidine deaminase (AID). Although DSB formation and repair machineries are active in G1 phase, efficient CSR is dependent on cell proliferation and S phase entry; however, the underlying mechanisms are obscure. Here, we show that efficient CSR requires the replicative helicase, the Mcm complex. Mcm proteins are enriched at IgH switch regions during CSR, leading to assembly of facultative replication origins that require Mcm helicase function for productive CSR. Assembly of CSR-associated origins is facilitated by R loops and promotes the physical proximity (synapsis) of recombining switch regions, which is reduced by R loop inhibition or Mcm complex depletion. Thus, R loops contribute to replication origin specification that promotes DSB resolution in CSR. This suggests a mechanism for the dependence of CSR on S phase and cell division. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny.

PubMed

Vahidi Ferdousi, Leyla; Rocheteau, Pierre; Chayot, Romain; Montagne, Benjamin; Chaker, Zayna; Flamant, Patricia; Tajbakhsh, Shahragim; Ricchetti, Miria

2014-11-01

The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs. Copyright © 2014. Published by Elsevier B.V.

CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons.

PubMed

Billon, Pierre; Bryant, Eric E; Joseph, Sarah A; Nambiar, Tarun S; Hayward, Samuel B; Rothstein, Rodney; Ciccia, Alberto

2017-09-21

Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP. Copyright © 2017 Elsevier Inc. All rights reserved.

Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair

DOE PAGES

Andres, Sara N.; Appel, C. Denise; Westmoreland, James W.; ...

2015-01-12

Ctp1 (also known as CtIP or Sae2) collaborates with Mre11-Rad50-Nbs1 to initiate repair of DNA double-strand breaks (DSBs), but its functions remain enigmatic. In this paper, we report that tetrameric Schizosaccharomyces pombe Ctp1 contains multivalent DNA-binding and DNA-bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer, we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal 'RHR' DNA-interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA-bridging activity in vitro, and both the THDD and RHR are required for efficientmore » DSB repair in S. pombe. Finally, our results establish non-nucleolytic roles of Ctp1 in binding and coordination of DSB-repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CtIP-linked Seckel and Jawad syndromes.« less

Clustered DNA damages induced by high and low LET radiation, including heavy ions

NASA Technical Reports Server (NTRS)

Sutherland, B. M.; Bennett, P. V.; Schenk, H.; Sidorkina, O.; Laval, J.; Trunk, J.; Monteleone, D.; Sutherland, J.; Lowenstein, D. I. (Principal Investigator)

2001-01-01

Clustered DNA damages--here defined as two or more lesions (strand breaks, oxidized purines, oxidized pyrimidines or abasic sites) within a few helical turns--have been postulated as difficult to repair accurately, and thus highly significant biological lesions. Further, attempted repair of clusters may produce double strand breaks (DSBs). However, until recently, there was no way to measure ionizing radiation-induced clustered damages, except DSB. We recently described an approach for measuring classes of clustered damages (oxidized purine clusters, oxidized pyrimidine clusters, abasic clusters, along with DSB). We showed that ionizing radiation (gamma rays and Fe ions, 1 GeV/amu) does induce such clusters in genomic DNA in solution and in human cells. These studies also showed that each damage cluster results from one radiation hit (and its track), thus indicating that they can be induced by very low doses of radiation, i.e. two independent hits are not required for cluster induction. Further, among all complex damages, double strand breaks comprise--at most-- 20%, with the other clustered damages being at least 80%.

Targeting of the Fun30 nucleosome remodeller by the Dpb11 scaffold facilitates cell cycle-regulated DNA end resection

PubMed Central

Bantele, Susanne CS; Ferreira, Pedro; Gritenaite, Dalia; Boos, Dominik; Pfander, Boris

2017-01-01

DNA double strand breaks (DSBs) can be repaired by either recombination-based or direct ligation-based mechanisms. Pathway choice is made at the level of DNA end resection, a nucleolytic processing step, which primes DSBs for repair by recombination. Resection is thus under cell cycle control, but additionally regulated by chromatin and nucleosome remodellers. Here, we show that both layers of control converge in the regulation of resection by the evolutionarily conserved Fun30/SMARCAD1 remodeller. Budding yeast Fun30 and human SMARCAD1 are cell cycle-regulated by interaction with the DSB-localized scaffold protein Dpb11/TOPBP1, respectively. In yeast, this protein assembly additionally comprises the 9-1-1 damage sensor, is involved in localizing Fun30 to damaged chromatin, and thus is required for efficient long-range resection of DSBs. Notably, artificial targeting of Fun30 to DSBs is sufficient to bypass the cell cycle regulation of long-range resection, indicating that chromatin remodelling during resection is underlying DSB repair pathway choice. DOI: http://dx.doi.org/10.7554/eLife.21687.001 PMID:28063255

Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity

PubMed Central

Oeck, S.; Al-Refae, K.; Riffkin, H.; Wiel, G.; Handrick, R.; Klein, D.; Iliakis, G.; Jendrossek, V.

2017-01-01

The survival kinase Akt has clinical relevance to radioresistance. However, its contributions to the DNA damage response, DNA double strand break (DSB) repair and apoptosis remain poorly defined and often contradictory. We used a genetic approach to explore the consequences of genetic alterations of Akt1 for the cellular radiation response. While two activation-associated mutants with prominent nuclear access, the phospho-mimicking Akt1-TDSD and the clinically relevant PH-domain mutation Akt1-E17K, accelerated DSB repair and improved survival of irradiated Tramp-C1 murine prostate cancer cells and Akt1-knockout murine embryonic fibroblasts in vitro, the classical constitutively active membrane-targeted myrAkt1 mutant had the opposite effects. Interestingly, DNA-PKcs directly phosphorylated Akt1 at S473 in an in vitro kinase assay but not vice-versa. Pharmacological inhibition of DNA-PKcs or Akt restored radiosensitivity in tumour cells expressing Akt1-E17K or Akt1-TDSD. In conclusion, Akt1-mediated radioresistance depends on its activation state and nuclear localization and is accessible to pharmacologic inhibition. PMID:28209968

Novel genotype of Ehrlichia canis detected in samples of human blood bank donors in Costa Rica.

PubMed

Bouza-Mora, Laura; Dolz, Gaby; Solórzano-Morales, Antony; Romero-Zuñiga, Juan José; Salazar-Sánchez, Lizbeth; Labruna, Marcelo B; Aguiar, Daniel M

2017-01-01

This study focuses on the detection and identification of DNA and antibodies to Ehrlichia spp. in samples of blood bank donors in Costa Rica using molecular and serological techniques. Presence of Ehrlichia canis was determined in 10 (3.6%) out of 280 blood samples using polymerase chain reaction (PCR) targeting the ehrlichial dsb conserved gene. Analysis of the ehrlichial trp36 polymorphic gene in these 10 samples revealed substantial polymorphism among the E. canis genotypes, including divergent tandem repeat sequences. Nucleotide sequences of dsb and trp36 amplicons revealed a novel genotype of E. canis in blood bank donors from Costa Rica. Indirect immunofluorescence assay (IFA) detected antibodies in 35 (35%) of 100 serum samples evaluated. Thirty samples showed low endpoint titers (64-256) to E. canis, whereas five sera yielded high endpoint titers (1024-8192); these five samples were also E. canis-PCR positive. These findings represent the first report of the presence of E. canis in humans in Central America. Copyright © 2016 Elsevier GmbH. All rights reserved.

Nanoparticulate carbon black in cigarette smoke induces DNA cleavage and Th17-mediated emphysema

PubMed Central

You, Ran; Lu, Wen; Shan, Ming; Berlin, Jacob M; Samuel, Errol LG; Marcano, Daniela C; Sun, Zhengzong; Sikkema, William KA; Yuan, Xiaoyi; Song, Lizhen; Hendrix, Amanda Y; Tour, James M; Corry, David B; Kheradmand, Farrah

2015-01-01

Chronic inhalation of cigarette smoke is the major cause of sterile inflammation and pulmonary emphysema. The effect of carbon black (CB), a universal constituent of smoke derived from the incomplete combustion of organic material, in smokers and non-smokers is less known. In this study, we show that insoluble nanoparticulate carbon black (nCB) accumulates in human myeloid dendritic cells (mDCs) from emphysematous lung and in CD11c+ lung antigen presenting cells (APC) of mice exposed to smoke. Likewise, nCB intranasal administration induced emphysema in mouse lungs. Delivered by smoking or intranasally, nCB persisted indefinitely in mouse lung, activated lung APCs, and promoted T helper 17 cell differentiation through double-stranded DNA break (DSB) and ASC-mediated inflammasome assembly in phagocytes. Increasing the polarity or size of CB mitigated many adverse effects. Thus, nCB causes sterile inflammation, DSB, and emphysema and explains adverse health outcomes seen in smokers while implicating the dangers of nCB exposure in non-smokers. DOI: http://dx.doi.org/10.7554/eLife.09623.001 PMID:26437452