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1

Drug-induced DNA repair: X-ray structure of a DNA-ditercalinium complex  

SciTech Connect

Ditercalinium is a synthetic anticancer drug that binds to DNA by bis-intercalation and activates DNA repair processes. In prokaryotes, noncovalent DNA-ditercalinium complexes are incorrectly recognized by the uvrABC repair system as covalent lesions on DNA. In eukaryotes, mitochondrial DNA is degraded by excess and futile DNA repair. Using x-ray crystallography, the authors have determined, to 1.7 {angstrom} resolution, the three-dimensional structure of a complex of ditercalinium bound to the double-stranded DNA fragment (d(CGCG)){sub 2}. The DNA in the complex with ditercalinium is kinked (by 15{degrees}) and severely unsound (by 36{degrees}) with exceptionally wide major and minor grooves. Recognition of the DNA-ditercalinium complex by uvrABC in prokaryotes, and by mitochondrial DNA repair systems in eukaryotes, might be related to drug-induced distortion of the DNA helix.

Gao, Qi; Williams, L.D.; Egli, M.; Rabinovich, D.; Rich, A. (Massachusetts Inst. of Tech., Cambridge (United States)); Chen, Shunle; Quigley, G.J. (Hunter College, New York, NY (United States))

1991-03-15

2

UC Davis researchers discover complexities of DNA repair:  

Cancer.gov

An international team of scientists led by UC Davis researchers has discovered that DNA repair in cancer cells is not a one-way street as previously believed. Their findings show instead that recombination, an important DNA repair process, has a self-correcting mechanism that allows DNA to make a virtual u-turn and start over.

3

Cryo-EM Imaging of DNA-PK DNA Damage Repair Complexes  

SciTech Connect

Exposure to low levels of ionizing radiation causes DNA double-strand breaks (DSBs) that must be repaired for cell survival. Higher eukaryotes respond to DSBs by arresting the cell cycle, presumably to repair the DNA lesions before cell division. In mammalian cells, the nonhomologous end-joining DSB repair pathway is mediated by the 470 kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs) together with the DNA-binding factors Ku70 and Ku80. Mouse knock-out models of these three proteins are all exquisitely sensitive to low doses of ionizing radiation. In the presence of DNA ends, Ku binds to the DNA and then recruits DNA-PKcs. After formation of the complex, the kinase activity associated with DNA-PKcs becomes activated. This kinase activity has been shown to be essential for repairing DNA DSBs in vivo since expression of a kinase-dead form of DNA-PKcs in a mammalian cell line that lacks DNA-PKcs fails to complement the radiosensitive phenotype. The immense size of DNA-PKcs suggests that it may also serve as a docking site for other DNA repair proteins. Since the assembly of the DNA-PK complex onto DNA is a prerequisite for DSB repair, it is critical to obtain structural information on the complex. Cryo-electron microscopy (cryo-EM) and single particle reconstruction methods provide a powerful way to image large macromolecular assemblies at near atomic (10-15 ?) resolution. We have already used cryo-EM methods to examine the structure of the isolated DNA-PKcs protein. This structure reveals numerous cavities throughout the protein that may allow passage of single or double-stranded DNA. Pseudo two-fold symmetry was found for the monomeric protein, suggesting that DNA-PKcs may interact with two DNA ends or two Ku heterodimers simultaneously. Here we propose to study the structure of the cross-linked DNA-PKcs/Ku/DNA complex. Difference imaging with our published DNA-PKcs structure will enable us to elucidate the architecture of the complex. A second objective is to locate the kinase domain of DNA-PKcs by determining the structure of a kinase deletion mutant both as an isolated protein and as part of a DNA-PKcs/Ku/DNA complex. A third objective is to pursue higher resolution studies of DNA-PKcs and the DNA-PKcs/Ku/DNA complex. If the crystal structure determination of DNA-PKcs is completed during the project period, the atomic coordinates of DNA-PKcs will be modeled within the cryo-EM structure of the complex. In order to achieve these goals, a collaborative effort is proposed between Dr. Phoebe Stewart at UCLA, whose laboratory has expertise in cryo-EM reconstruction methods, and Dr. David Chen at the Lawrence Berkeley National Laboratory, who has a long-standing interest in DNA repair. Advantages of the cryo-EM structural method include the fact that the sample is imaged in a frozen-hydrated and unstained state, avoiding artifacts associated with drying and staining in other EM approaches. Also crystals of the sample are not needed for the single particle reconstruction method and only microgram quantities of sample are required. Cryo-EM structural information of macromolecular assemblies is complementary to both atomic structures of individual component molecules, as well as low resolution information obtained from x-ray and neutron scattering. Knowledge of the geometrical arrangement of the complex, and the position of the essential DNA-PKcs kinase domain, should lead to a greater understanding of the molecular events in DNA double-strand break repair following exposure to low doses of radiation.

Phoebe L. Stewart

2005-06-27

4

Structure of the Rad50 DNA double-strand break repair protein in complex with DNA.  

PubMed

The Mre11-Rad50 nuclease-ATPase is an evolutionarily conserved multifunctional DNA double-strand break (DSB) repair factor. Mre11-Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50(NBD) (nucleotide-binding domain) in complex with Mre11(HLH) (helix-loop-helix domain), AMPPNP, and double-stranded DNA. DNA binds between both coiled-coil domains of the Rad50 dimer with main interactions to a strand-loop-helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double-strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP-bound state. PMID:25349191

Rojowska, Anna; Lammens, Katja; Seifert, Florian U; Direnberger, Carolin; Feldmann, Heidi; Hopfner, Karl-Peter

2014-12-01

5

Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair  

SciTech Connect

DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.

Cappadocia, Laurent; Maréchal, Alexandre; Parent, Jean-Sébastien; Lepage, Étienne; Sygusch, Jurgen; Brisson, Normand (Montreal)

2010-09-07

6

Stochastic and reversible assembly of a multiprotein DNA repair complex ensures accurate target site recognition and efficient repair.  

PubMed

To understand how multiprotein complexes assemble and function on chromatin, we combined quantitative analysis of the mammalian nucleotide excision DNA repair (NER) machinery in living cells with computational modeling. We found that individual NER components exchange within tens of seconds between the bound state in repair complexes and the diffusive state in the nucleoplasm, whereas their net accumulation at repair sites evolves over several hours. Based on these in vivo data, we developed a predictive kinetic model for the assembly and function of repair complexes. DNA repair is orchestrated by the interplay of reversible protein-binding events and progressive enzymatic modifications of the chromatin substrate. We demonstrate that faithful recognition of DNA lesions is time consuming, whereas subsequently, repair complexes form rapidly through random and reversible assembly of NER proteins. Our kinetic analysis of the NER system reveals a fundamental conflict between specificity and efficiency of chromatin-associated protein machineries and shows how a trade off is negotiated through reversibility of protein binding. PMID:20439997

Luijsterburg, Martijn S; von Bornstaedt, Gesa; Gourdin, Audrey M; Politi, Antonio Z; Moné, Martijn J; Warmerdam, Daniël O; Goedhart, Joachim; Vermeulen, Wim; van Driel, Roel; Höfer, Thomas

2010-05-01

7

Structure of the FANCI-FANCD2 Complex: Insights into the Fanconi Anemia DNA Repair Pathway  

SciTech Connect

Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the {approx}300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.

Joo, Woo; Xu, Guozhou; Persky, Nicole S.; Smogorzewska, Agata; Rudge, Derek G.; Buzovetsky, Olga; Elledge, Stephen J.; Pavletich, Nikola P. (Harvard-Med); (Cornell); (MSKCC)

2011-08-29

8

Structure of the FANCI-FANCD2 Complex: Insights into the Fanconi Anemia DNA Repair Pathway  

SciTech Connect

Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the {approx}300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.

W Joo; G Xu; n Persky; A Smogorzewska; D Rudge; O Buzovetsky; S Elledge; N Pavletich

2011-12-31

9

The Arabidopsis SWR1 Chromatin-Remodeling Complex Is Important for DNA Repair, Somatic Recombination, and Meiosis[W][OPEN  

PubMed Central

All processes requiring interaction with DNA are attuned to occur within the context of the complex chromatin structure. As it does for programmed transcription and replication, this also holds true for unscheduled events, such as repair of DNA damage. Lesions such as double-strand breaks occur randomly; their repair requires that enzyme complexes access DNA at potentially any genomic site. This is achieved by chromatin remodeling factors that can locally slide, evict, or change nucleosomes. Here, we show that the Swi2/Snf2-related (SWR1 complex), known to deposit histone H2A.Z, is also important for DNA repair in Arabidopsis thaliana. Mutations in genes for Arabidopsis SWR1 complex subunits PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1, ACTIN-RELATED PROTEIN6, and SWR1 COMPLEX6 cause hypersensitivity to various DNA damaging agents. Even without additional genotoxic stress, these mutants show symptoms of DNA damage accumulation. The reduced DNA repair capacity is connected with impaired somatic homologous recombination, in contrast with the hyper-recombinogenic phenotype of yeast SWR1 mutants. This suggests functional diversification between lower and higher eukaryotes. Finally, reduced fertility and irregular gametogenesis in the Arabidopsis SWR1 mutants indicate an additional role for the chromatin-remodeling complex during meiosis. These results provide evidence for the importance of Arabidopsis SWR1 in somatic DNA repair and during meiosis. PMID:23780875

Rosa, Marisa; Von Harder, Mona; Aiese Cigliano, Riccardo; Schlögelhofer, Peter; Mittelsten Scheid, Ortrun

2013-01-01

10

Structural insights into the functions of the FANCM-FAAP24 complex in DNA repair  

PubMed Central

Fanconi anemia (FA) is a genetically heterogeneous disorder associated with deficiencies in the FA complementation group network. FA complementation group M (FANCM) and FA-associated protein 24 kDa (FAAP24) form a stable complex to anchor the FA core complex to chromatin in repairing DNA interstrand crosslinks. Here, we report the first crystal structure of the C-terminal segment of FANCM in complex with FAAP24. The C-terminal segment of FANCM and FAAP24 both consist of a nuclease domain at the N-terminus and a tandem helix-hairpin-helix (HhH)2 domain at the C-terminus. The FANCM-FAAP24 complex exhibits a similar architecture as that of ApXPF. However, the variations of several key residues and the electrostatic property at the active-site region render a catalytically inactive nuclease domain of FANCM, accounting for the lack of nuclease activity. We also show that the first HhH motif of FAAP24 is a potential binding site for DNA, which plays a critical role in targeting FANCM-FAAP24 to chromatin. These results reveal the mechanistic insights into the functions of FANCM-FAAP24 in DNA repair. PMID:24003026

Yang, Hui; Zhang, Tianlong; Tao, Ye; Wang, Fang; Tong, Liang; Ding, Jianping

2013-01-01

11

Optimality in DNA repair  

PubMed Central

DNA within cells is subject to damage from various sources. Organisms have evolved a number of mechanisms to repair DNA damage. The activity of repair enzymes carries its own risk, however, because the repair of two nearby lesions may lead to the breakup of DNA and result in cell death. We propose a mathematical theory of the damage and repair process in the important scenario where lesions are caused in bursts. We use this model to show that there is an optimum level of repair enzymes within cells which optimises the cell's response to damage. This optimal level is explained as the best trade-off between fast repair and a low probability of causing double-stranded breaks. We derive our results analytically and test them using stochastic simulations, and compare our predictions with current biological knowledge. PMID:21945337

Richard, Morgiane; Fryett, Matthew; Miller, Samantha; Booth, Ian; Grebogi, Celso; Moura, Alessandro

2012-01-01

12

Direct interaction between XRCC1 and UNG2 facilitates rapid repair of uracil in DNA by XRCC1 complexes  

PubMed Central

Uracil-DNA glycosylase, UNG2, interacts with PCNA and initiates post-replicative base excision repair (BER) of uracil in DNA. The DNA repair protein XRCC1 also co-localizes and physically interacts with PCNA. However, little is known about whether UNG2 and XRCC1 directly interact and participate in a same complex for repair of uracil in replication foci. Here, we examine localization pattern of these proteins in live and fixed cells and show that UNG2 and XRCC1 are likely in a common complex in replication foci. Using pull-down experiments we demonstrate that UNG2 directly interacts with the nuclear localization signal-region (NLS) of XRCC1. Western blot and functional analysis of immunoprecipitates from whole cell extracts prepared from S-phase enriched cells demonstrate the presence of XRCC1 complexes that contain UNG2 in addition to separate XRCC1 and UNG2 associated complexes with distinct repair features. XRCC1 complexes performed complete repair of uracil with higher efficacy than UNG2 complexes. Based on these results, we propose a model for a functional role of XRCC1 in replication associated BER of uracil. PMID:20466601

Akbari, Mansour; Solvang-Garten, Karin; Hanssen-Bauer, Audun; Lieske, Nora Valeska; Pettersen, Henrik Sahlin; Pettersen, Grete Klippenvåg; Wilson, David M.; Krokan, Hans E.; Otterlei, Marit

2010-01-01

13

The Mre11-Rad50-Xrs2 Complex Is Required for Yeast DNA Postreplication Repair  

PubMed Central

Yeast DNA postreplication repair (PRR) bypasses replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA, namely translesion synthesis (TLS) and error-free PRR, which are regulated via sequential ubiquitination of proliferating cell nuclear antigen (PCNA). We previously demonstrated that error-free PRR utilizes homologous recombination to facilitate template switching. To our surprise, genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which are also required for homologous recombination, are epistatic to TLS mutations. Further genetic analyses indicated that two other nucleases involved in double-strand end resection, Sae2 and Exo1, are also variably required for efficient lesion bypass. The involvement of the above genes in TLS and/or error-free PRR could be distinguished by the mutagenesis assay and their differential effects on PCNA ubiquitination. Consistent with the observation that the MRX complex is required for both branches of PRR, the MRX complex was found to physically interact with Rad18 in vivo. In light of the distinct and overlapping activities of the above nucleases in the resection of double-strand breaks, we propose that the interplay between distinct single-strand nucleases dictate the preference between TLS and error-free PRR for lesion bypass. PMID:25343618

Ball, Lindsay G.; Hanna, Michelle D.; Lambrecht, Amanda D.; Mitchell, Bryan A.; Ziola, Barry; Cobb, Jennifer A.; Xiao, Wei

2014-01-01

14

Identification and Characterization of a Human DNA Double-Strand Break Repair Complex  

SciTech Connect

The authors have used atomic force microscopy (AFM) to characterize the assembly and structure of the macromolecular assemblies involved in DNA repair. They have demonstrated using AFM that the DNA-dependent protein kinase can play a structural role in the repair of DNA double-strand breaks (DSBs) by physically holding DNA ends together. They have extended these studies to include other DNA damage response proteins, these efforts have resulted in important and novel findings regarding the ATM protein. Specifically, the work has demonstrated, for the first time, that the ATM protein binds with specificity to a DNA end. This finding is the first to implicate the ATM protein in the detection of DNA damage by direct physical interaction with DSBs.

Chen, D.J.; Cary, R.B.

1999-07-12

15

DNA Repair and Global Sumoylation Are Regulated by Distinct Ubc9 Noncovalent Complexes ?  

PubMed Central

Global sumoylation, SUMO chain formation, and genome stabilization are all outputs generated by a limited repertoire of enzymes. Mechanisms driving selectivity for each of these processes are largely uncharacterized. Here, through crystallographic analyses we show that the SUMO E2 Ubc9 forms a noncovalent complex with a SUMO-like domain of Rad60 (SLD2). Ubc9:SLD2 and Ubc9:SUMO noncovalent complexes are structurally analogous, suggesting that differential recruitment of Ubc9 by SUMO or Rad60 provides a novel means for such selectivity. Indeed, deconvoluting Ubc9 function by disrupting either the Ubc9:SLD2 or Ubc9:SUMO noncovalent complex reveals distinct roles in facilitating sumoylation. Ubc9:SLD2 acts in the Nse2 SUMO E3 ligase-dependent pathway for DNA repair, whereas Ubc9:SUMO instead promotes global sumoylation and chain formation, via the Pli1 E3 SUMO ligase. Moreover, this Pli1-dependent SUMO chain formation causes the genome instability phenotypes of SUMO-targeted ubiquitin ligase (STUbL) mutants. Overall, we determine that, unexpectedly, Ubc9 noncovalent partner choice dictates the role of sumoylation in distinct cellular pathways. PMID:21444718

Prudden, John; Perry, J. Jefferson P.; Nie, Minghua; Vashisht, Ajay A.; Arvai, Andrew S.; Hitomi, Chiharu; Guenther, Grant; Wohlschlegel, James A.; Tainer, John A.; Boddy, Michael N.

2011-01-01

16

Recombinational DNA Repair in Bacteria  

E-print Network

Recombinational DNA Repair in Bacteria: Postreplication Kevin P Rice,University of Wisconsin Recombinational DNA repair represents the primary function for homologous DNA recombination in bacteria. Most of this repair occurs at replication forks that are stalled at sites of DNA damage. Introduction Deoxyribonucleic

Cox, Michael M.

17

ATM protein is indispensable to repair complex-type DNA double strand breaks induced by high LET heavy ion irradiation.  

NASA Astrophysics Data System (ADS)

ATM (ataxia telangiectasia-mutated) protein responsible for a rare genetic disease with hyperradiosensitivity, is the one of the earliest repair proteins sensing DNA double-strand breaks (DSB). ATM is known to phosphorylate DNA repair proteins such as MRN complex (Mre11, Rad50 and NBS1), 53BP1, Artemis, Brca1, gamma-H2AX, and MDC. We studied the interactions between ATM and DNA-PKcs, a crucial NHEJ repair protein, after cells exposure to high and low LET irradiation. Normal human (HFL III, MRC5VA) and AT homozygote (AT2KY, AT5BIVA, AT3BIVA) cells were irradiated with X-rays and high LET radiation (carbon ions: 290MeV/n initial energy at 70 keV/um, and iron ions: 500MeV/n initial energy at 200KeV/um), and several critical end points were examined. AT cells with high LET irradiation showed a significantly higher radiosensitivity when compared with normal cells. The behavior of DNA DSB repair was monitored by immuno-fluorescence techniques using DNA-PKcs (pThr2609, pSer2056) and ATM (pSer1981) antibodies. In normal cells, the phosphorylation of DNA-PKcs was clearly detected after high LET irradiation, though the peak of phosphorylation was delayed when compared to X-irradiation. In contrast, almost no DNA-PKcs phosphorylation foci were detected in AT cells irradiated with high LET radiation. A similar result was also observed in normal cells treated with 10 uM ATM kinase specific inhibitor (KU55933) one hour before irradiation. These data suggest that the phosphorylation of DNA-PKcs with low LET X-rays is mostly ATM-independent, and the phosphorylation of DNA-PKcs with high LET radiation seems to require ATM probably due to its complex nature of DSB induced. Our study indicates that high LET heavy ion irradiation which we can observe in the space environment would provide a useful tool to study the fundamental mechanism associated with DNA DSB repair.

Sekine, Emiko; Yu, Dong; Fujimori, Akira; Anzai, Kazunori; Okayasu, Ryuichi

18

Chemical Trapping of the Dynamic MutS-MutL Complex Formed in DNA Mismatch Repair in Escherichia coli*  

PubMed Central

The ternary complex comprising MutS, MutL, and DNA is a key intermediate in DNA mismatch repair. We used chemical cross-linking and fluorescence resonance energy transfer (FRET) to study the interaction between MutS and MutL and to shed light onto the structure of this complex. Via chemical cross-linking, we could stabilize this dynamic complex and identify the structural features of key events in DNA mismatch repair. We could show that in the complex between MutS and MutL the mismatch-binding and connector domains of MutS are in proximity to the N-terminal ATPase domain of MutL. The DNA- and nucleotide-dependent complex formation could be monitored by FRET using single cysteine variants labeled in the connector domain of MutS and the transducer domain of MutL, respectively. In addition, we could trap MutS after an ATP-induced conformational change by an intramolecular cross-link between Cys-93 of the mismatch-binding domain and Cys-239 of the connector domain. PMID:21454657

Winkler, Ines; Marx, Andreas D.; Lariviere, Damien; Heinze, Roger J.; Cristovao, Michele; Reumer, Annet; Curth, Ute; Sixma, Titia K.; Friedhoff, Peter

2011-01-01

19

Complex Formation by the Human Rad51B and Rad51C DNA Repair Proteins and Their Activities in Vitro*  

E-print Network

Complex Formation by the Human Rad51B and Rad51C DNA Repair Proteins and Their Activities in Vitro. In addition to Rad51 pro- tein, five paralogs have been identified: Rad51B/ Rad51L1, Rad51C/Rad51L2, Rad51D-(His)6, and Rad51C proteins were individually expressed employ- ing the baculovirus system, and each

Kowalczykowski, Stephen C.

20

DNA end resection is needed for the repair of complex lesions in G1-phase human cells.  

PubMed

ABSTRACT Repair of DNA double strand breaks (DSBs) is influenced by the chemical complexity of the lesion. Clustered lesions (complex DSBs) are generally considered more difficult to repair and responsible for early and late cellular effects after exposure to genotoxic agents. Resection is commonly used by the cells as part of the homologous recombination (HR) pathway in S- and G2-phase. In contrast, DNA resection in G1-phase may lead to an error-prone microhomology-mediated end joining. We induced DNA lesions with a wide range of complexity by irradiation of mammalian cells with X-rays or accelerated ions of different velocity and mass. We found replication protein A (RPA) foci indicating DSB resection both in S/G2- and G1-cells, and the fraction of resection-positive cells correlates with the severity of lesion complexity throughout the cell cycle. Besides RPA, Ataxia telangiectasia and Rad3-related (ATR) was recruited to complex DSBs both in S/G2- and G1-cells. Resection of complex DSBs is driven by meiotic recombination 11 homolog A (MRE11), CTBP-interacting protein (CtIP), and exonuclease 1 (EXO1) but seems not controlled by the Ku heterodimer or by phosphorylation of H2AX. Reduced resection capacity by CtIP depletion increased cell killing and the fraction of unrepaired DSBs after exposure to densely ionizing heavy ions, but not to X-rays. We conclude that in mammalian cells resection is essential for repair of complex DSBs in all phases of the cell-cycle and targeting this process sensitizes mammalian cells to cytotoxic agents inducing clustered breaks, such as in heavy-ion cancer therapy. PMID:25486192

Averbeck, Nicole B; Ringel, Oliver; Herrlitz, Maren; Jakob, Burkhard; Durante, Marco; Taucher-Scholz, Gisela

2014-08-15

21

A novel ruthenium(II)–polypyridyl complex inhibits cell proliferation and induces cell apoptosis by impairing DNA damage repair  

PubMed Central

Ruthenium complexes are widely recognized as one of the most promising DNA damaging chemotherapeutic drugs. The main goal of this study was to explore the anticancer activity and underlying mechanisms of [Ru(phen)2(p-BrPIP)](ClO4)2, a novel chemically synthesized ruthenium (Ru) complex. To this end, we employed MTT assays to determine the anticancer activity of the complex, and performed single-cell gel electrophoresis (SCGE) and Western blotting to evaluate DNA damage. Our results showed that the Ru(II)–poly complex caused severe DNA damage, possibly by downregulating key factors involved in DNA repair pathways, such as proliferating cell nuclear antigen (PCNA) and ring finger protein 8 (RNF8). In addition, this complex induced cell apoptosis by upregulating both p21 and p53. Taken together, our findings demonstrate that the Ru(II)–poly complex exhibits antitumour activity by inducing cell apoptosis, which results from the accumulation of large amounts of unrepaired DNA damage. PMID:24070188

Yang, Qingyuan; Zhang, Zhao; Mei, Wenjie; Sun, Fenyong

2014-01-01

22

Dynamics of DNA Mismatch Repair  

NASA Astrophysics Data System (ADS)

DNA mismatch repair protects the genome from spontaneous mutations by recognizing errors, excising damage, and re-synthesizing DNA in a pathway that is highly conserved. Mismatch recognition is accomplished by the MutS family of proteins which are weak ATPases that bind specifically to damaged DNA, but the specific molecular mechanisms by which these proteins recognize damage and initiate excision are not known. Previous structural investigations have implied that protein-induced conformational changes are central to mismatch recognition. Because damage detection is a highly dynamic process in which conformational changes of the protein-DNA complexes occur on a time scale of a few seconds, it is difficult to obtain meaningful kinetic information with traditional ensemble techniques. In this work, we use single molecule fluorescence resonance energy transfer (smFRET) to study the conformational dynamics of fluorescently labeled DNA substrates in the presence of the mismatch repair protein MutS from E. coli and its human homolog MSH2/MSH6. Our studies allow us to obtain quantitative kinetic information about the rates of binding and dissociation and to determine the conformational states for each protein-DNA complex.

Coats, Julie; Lin, Yuyen; Rasnik, Ivan

2009-11-01

23

Stalled transcription complexes promote DNA repair at Nia M. Haines, Young-In T. Kim, Abigail J. Smith, and Nigel J. Savery1  

E-print Network

Stalled transcription complexes promote DNA repair at a distance Nia M. Haines, Young-In T. Kim that a lesion must cause RNA polymerase (RNAP) to stall if it is to be a substrate for accelerated repair. We have examined the substrate requirements for TCR using a system in which transcription stalling

Dever, Jennifer A.

24

DNA Repair by Reversal of DNA Damage  

PubMed Central

Endogenous and exogenous factors constantly challenge cellular DNA, generating cytotoxic and/or mutagenic DNA adducts. As a result, organisms have evolved different mechanisms to defend against the deleterious effects of DNA damage. Among these diverse repair pathways, direct DNA-repair systems provide cells with simple yet efficient solutions to reverse covalent DNA adducts. In this review, we focus on recent advances in the field of direct DNA repair, namely, photolyase-, alkyltransferase-, and dioxygenase-mediated repair processes. We present specific examples to describe new findings of known enzymes and appealing discoveries of new proteins. At the end of this article, we also briefly discuss the influence of direct DNA repair on other fields of biology and its implication on the discovery of new biology. PMID:23284047

Yi, Chengqi; He, Chuan

2013-01-01

25

DNA repair in Mycoplasma gallisepticum  

PubMed Central

Background DNA repair is essential for the maintenance of genome stability in all living beings. Genome size as well as the repertoire and abundance of DNA repair components may vary among prokaryotic species. The bacteria of the Mollicutes class feature a small genome size, absence of a cell wall, and a parasitic lifestyle. A small number of genes make Mollicutes a good model for a “minimal cell” concept. Results In this work we studied the DNA repair system of Mycoplasma gallisepticum on genomic, transcriptional, and proteomic levels. We detected 18 out of 22 members of the DNA repair system on a protein level. We found that abundance of the respective mRNAs is less than one per cell. We studied transcriptional response of DNA repair genes of M. gallisepticum at stress conditions including heat, osmotic, peroxide stresses, tetracycline and ciprofloxacin treatment, stationary phase and heat stress in stationary phase. Conclusions Based on comparative genomic study, we determined that the DNA repair system M. gallisepticum includes a sufficient set of proteins to provide a cell with functional nucleotide and base excision repair and mismatch repair. We identified SOS-response in M. gallisepticum on ciprofloxacin, which is a known SOS-inducer, tetracycline and heat stress in the absence of established regulators. Heat stress was found to be the strongest SOS-inducer. We found that upon transition to stationary phase of culture growth transcription of DNA repair genes decreases dramatically. Heat stress does not induce SOS-response in a stationary phase. PMID:24148612

2013-01-01

26

PTEN in DNA damage repair  

PubMed Central

The ability of DNA repair in a cell is vital to its genomic integrity and thus to the normal functioning of an organism. Phosphatase and tensin homolog (PTEN) is a well-established tumor suppressor gene that induces apoptosis and controls cell growth by inhibiting the PI3K/AKT pathway. In various human cancers, PTEN is frequently found to be mutated, deleted, or epigenetically silenced. Recent new findings have demonstrated that PTEN also plays a critical role in DNA damage repair and DNA damage response. This review summarizes the recent progress in the function of PTEN in DNA damage repair, especially in double strand break repair and nucleotide excision repair. In addition, we will discuss the role of PTEN in DNA damage response through its interaction with the Chk1 and p53 pathways. We will focus on the newly discovered mechanisms and the potential implications in cancer prevention and therapeutic intervention. PMID:22266095

Ming, Mei; He, Yu-Ying

2012-01-01

27

DNA repair and synthetic lethality  

PubMed Central

Tumors often have DNA repair defects, suggesting additional inhibition of other DNA repair pathways in tumors may lead to synthetic lethality. Accumulating data demonstrate that DNA repair-defective tumors, in particular homologous recombination (HR), are highly sensitive to DNA-damaging agents. Thus, HR-defective tumors exhibit potential vulnerability to the synthetic lethality approach, which may lead to new therapeutic strategies. It is well known that poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP) inhibitors show the synthetically lethal effect in tumors defective in BRCA1 or BRCA2 genes encoded proteins that are required for efficient HR. In this review, we summarize the strategies of targeting DNA repair pathways and other DNA metabolic functions to cause synthetic lethality in HR-defective tumor cells. PMID:22010575

Guo, Gong-she; Zhang, Feng-mei; Gao, Rui-jie; Delsite, Robert; Feng, Zhi-hui; Powell, Simon N

2011-01-01

28

DNA repair in cultured keratinocytes  

SciTech Connect

Most of our understanding of DNA repair mechanisms in human cells has come from the study of these processes in cultured fibroblasts. The unique properties of keratinocytes and their pattern of terminal differentiation led us to a comparative examination of their DNA repair properties. The relative repair capabilities of the basal cells and the differentiated epidermal keratinocytes as well as possible correlations of DNA repair capacity with respect to age of the donor have been examined. In addition, since portions of human skin are chronically exposed to sunlight, the repair response to ultraviolet (UV) irradiation (254 nm) when the cells are conditioned by chronic low-level UV irradiation has been assessed. The comparative studies of DNA repair in keratinocytes from infant and aged donors have revealed no significant age-related differences for repair of UV-induced damage to DNA. Sublethal UV conditioning of cells from infant skin had no appreciable effect on either the repair or normal replication response to higher, challenge doses of UVL. However, such conditioning resulted in attenuated repair in keratinocytes from adult skin after UV doses above 25 J/m2. In addition, a surprising enhancement in replication was seen in conditioned cells from adult following challenge UV doses.

Liu, S.C.; Parsons, S.; Hanawalt, P.C.

1983-07-01

29

DNA repair meets the RNA world.  

PubMed

The ability to repair damaged DNA and to maintain genome stability is the utmost importance for the survival of any species. Hence, it is not surprising to find that DNA repair mechanisms are evolutionarily conserved and are expected to evolve to maintain the existence of species. In the last few years, there has been an exponential increase in the evidence linking RNA processing with DNA repair programs. For instance, the well-studied DNA base excision repair (BER) enzyme apurinic/apyrimidinic endonuclease 1 can cleave RNA molecules, regulate mRNA levels, and associate physically with proteins involved in RNA processing. It is now clear that not only the expression of noncoding RNAs are changed upon DNA damage, they can modulate the expression of genes involved in the genome stability programs. The five reviews in this Forum provide the up-to-date knowledge on DNA repair, with a focus on BER, and a perspective on how the two ancient biochemical pathways are linked. The contributions demonstrate the complexity of such interactions, but also pointed out the opportunities for new therapeutic interventions. Future in vivo studies on the link between DNA repair processes and RNA metabolism should contribute to our basic understanding of physiology, disease, and treatment strategies. PMID:24252191

Lee, Chow H

2014-02-01

30

DNA Repair Deficiency in Neurodegeneration  

PubMed Central

Deficiency in repair of nuclear and mitochondrial DNA damage has been linked to several neurodegenerative disorders. Many recent experimental results indicate that the post-mitotic neurons are particularly prone to accumulation of unrepaired DNA lesions potentially leading to progressive neurodegeneration. Nucleotide excision repair is the cellular pathway responsible for removing helix-distorting DNA damage and deficiency in such repair is found in a number of diseases with neurodegenerative phenotypes, including Xeroderma Pigmentosum and Cockayne syndrome. The main pathway for repairing oxidative base lesions is base excision repair, and such repair is crucial for neurons given their high rates of oxygen metabolism. Mismatch repair corrects base mispairs generated during replication and evidence indicates that oxidative DNA damage can cause this pathway to expand trinucleotide repeats, thereby causing Huntington’s disease. Single-strand breaks are common DNA lesions and are associated with the neurodegenerative diseases, ataxia-oculomotor apraxia-1 and spinocerebellar ataxia with axonal neuropathy-1. DNA double-strand breaks are toxic lesions and two main pathways exist for their repair: homologous recombination and non-homologous end-joining. Ataxia telangiectasia and related disorders with defects in these pathways illustrate that such defects can lead to early childhood neurodegeneration. Aging is a risk factor for neurodegeneration and accumulation of oxidative mitochondrial DNA damage may be linked with the age-associated neurodegenerative disorders Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Mutation in the WRN protein leads to the premature aging disease Werner syndrome, a disorder that features neurodegeneration. In this article we review the evidence linking deficiencies in the DNA repair pathways with neurodegeneration. PMID:21550379

Jeppesen, Dennis Kjølhede; Bohr, Vilhelm A.; Stevnsner, Tinna

2011-01-01

31

DNA demethylation by DNA repair  

E-print Network

Active DNA demethylation underlies key facets of reproduction in flowering plants and mammals and serves a general genome housekeeping function in plants. A family of 5-methylcytosine DNA glycosylases catalyzes plant ...

Gehring, Mary

32

Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage  

SciTech Connect

The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA{sup -/-}, FANCC{sup -/-}, FANCA{sup -/-}C{sup -/-}, FANCD2{sup -/-} and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical {gamma}H2AX-staining assay. Although the sensitivity of FANCA{sup -/-}, FANCC{sup -/-} and FANCA{sup -/-}C{sup -/-} cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2{sup -/-} cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, {gamma}H2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex. -- Research highlights: {yields} We examined to clarify the repair pathways of formaldehyde-induced DNA damage. Formaldehyde induces DNA double strand breaks (DSBs). {yields} DSBs are repaired through the Fanconi anemia (FA) repair pathway. {yields} This pathway is independent of the FA nuclear core complex. {yields} We also found that homologous recombination repair was induced by formaldehyde.

Noda, Taichi [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan) [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Takahashi, Akihisa [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)] [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Kondo, Natsuko [Particle Radiation Oncology Research Center, Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan)] [Particle Radiation Oncology Research Center, Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Mori, Eiichiro [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)] [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Okamoto, Noritomo [Department of Otorhinolaryngology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)] [Department of Otorhinolaryngology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Nakagawa, Yosuke [Department of Oral and Maxillofacial Surgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)] [Department of Oral and Maxillofacial Surgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Ohnishi, Ken [Department of Biology, Ibaraki Prefectual University of Health Sciences, 4669-2 Ami, Ami-mati, Inasiki-gun, Ibaraki 300-0394 (Japan)] [Department of Biology, Ibaraki Prefectual University of Health Sciences, 4669-2 Ami, Ami-mati, Inasiki-gun, Ibaraki 300-0394 (Japan); Zdzienicka, Malgorzata Z. [Department of Molecular Cell Genetics, Collegium Medicum in Bydgoszcz, Nicolaus-Copernicus-University in Torun, ul. Sklodowskiej-Curie 9, 85-094 Bydgoszcz (Poland)] [Department of Molecular Cell Genetics, Collegium Medicum in Bydgoszcz, Nicolaus-Copernicus-University in Torun, ul. Sklodowskiej-Curie 9, 85-094 Bydgoszcz (Poland); Thompson, Larry H. [Biosciences and Biotechnology Division, L452, Lawrence Livermore National Laboratory, P.O. Box 808, Livermore, CA 94551-0808 (United States)] [Biosciences and Biotechnology Division, L452, Lawrence Livermore National Laboratory, P.O. Box 808, Livermore, CA 94551-0808 (United States); Helleday, Thomas [Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ (United Kingdom) [Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ (United Kingdom); Department of Genetics, Microbiology and Toxicology Stockholm University, SE-106 91 Stockholm (Sweden); Asada, Hideo [Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)] [Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); and others

2011-01-07

33

Smad7 enhances ATM activity by facilitating the interaction between ATM and Mre11-Rad50-Nbs1 complex in DNA double-strand break repair.  

PubMed

Genomic instability is one of the representative causes in genetic disorder, where the proper cellular response to DNA damage is essential in maintaining genomic stability. ATM and the Mre11-Rad50-Nbs1 (MRN) complex play critical roles in the cellular response to DNA damage such as DNA double-strand break (DSB). In this study, we report that Smad7 is indispensible in DNA damage response as a novel component of MRN complex. Smad7 enhances cell survival against DNA damage by accelerating ATM dependent DNA repair signaling. In Smad7-deficient mouse embryonic fibroblast cells, the loss of Smad7 decreases ATM activation and inhibits recruitment of ATM to the sites of DSBs. Smad7 interacts with Nbs1, a member of MRN complex, and enhances the interaction between ATM and Nbs1 upon DNA damage response, leading to phosphorylation of downstream substrates. Ectopic expression of Smad7 in the skin of mice enhances the phosphorylation of ATM upon X-irradiation. We found that effect of Smad7 on enhancing DNA repair is independent of its inhibitory activity of TGF-? signaling. Taken together, our results highlight a critical function of Smad7 in DSB response and establish the novel mechanism in which Smad7 facilitates the recruitment of ATM to the MRN complex through direct interaction with Nbs1. PMID:25063542

Park, Sujin; Kang, Jin Muk; Kim, Staci Jakyong; Kim, Hyojung; Hong, Suntaek; Lee, Young Jae; Kim, Seong-Jin

2015-02-01

34

Inhibitors of DNA Repair and Response to Ionising Radiation  

Microsoft Academic Search

\\u000a Ionising radiation, and most chemotherapeutic agents currently used to treat cancer, target DNA to cause cytotoxicity. The\\u000a cellular response to DNA damage is a complex set of intra-cellular processes involving multiple DNA repair pathways, leading\\u000a either to cell death or to survival if the lesions are repaired or bypassed. Thus, the multiple and redundant pathways involved\\u000a in the repair of

Barbara Vischioni; Nils H. Nicolay; Ricky A. Sharma; Thomas Helleday

35

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

PubMed Central

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

Schipler, Agnes; Iliakis, George

2013-01-01

36

DNA repair by the MRN complex: break it to make it.  

PubMed

Genomic instability in ataxia telangiectasia-like disorder and Nijmegen breakage syndrome is due to disruption of the Mre11-Rad50-Nbs1 complex. Buis et al. (2008) and Williams et al. (2008) now reveal the importance of the nuclease activity of Mre11 for mammalian genome maintenance and present a molecular view of its active site. PMID:18854148

Kanaar, Roland; Wyman, Claire

2008-10-01

37

The Cockayne syndrome B protein, involved in transcription-coupled DNA repair, resides in an RNA polymerase II-containing complex.  

PubMed Central

Transcription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair- and transcription-competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10-15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell-free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency. PMID:9312053

van Gool, A J; Citterio, E; Rademakers, S; van Os, R; Vermeulen, W; Constantinou, A; Egly, J M; Bootsma, D; Hoeijmakers, J H

1997-01-01

38

Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro  

NASA Technical Reports Server (NTRS)

The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.

Lio, Yi-Ching; Mazin, Alexander V.; Kowalczykowski, Stephen C.; Chen, David J.

2003-01-01

39

Small molecule versus DNA repair nanomachine  

PubMed Central

The MRN protein megacomplex mediates repair of double-stranded DNA breaks (DSBs) by tethering together broken ends of chromosomes and signaling a cascade of events required for DNA repair. The first small-molecule inhibitor that disrupts MRN function provides a valuable new tool for functional studies of DSB repair in cells. PMID:18202674

Stivers, James T

2009-01-01

40

DNA Repair Defects and Chromosomal Aberrations  

NASA Technical Reports Server (NTRS)

Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of the DNA repair-defective cell lines were smaller than those of normal cells, with the DNA-PK-deficient cells having RBEs near unity. To further investigate the sensitivity differences that were observed in ATM and NBS deficient cells, chromosomal aberrations were analyzed in normal lung fibroblast cells treated with KU-55933 (a specific ATM kinase inhibitor) or Mirin (an Mre11- Rad50-Nbs1 complex inhibitor involved in activation of ATM). We also performed siRNA knockdown of these proteins. Preliminary data indicate that chromosome exchanges increase in cells treated with the specific ATM inhibitor. Possible cytogenetic signatures of acute and low dose-rate gamma irradiation in ATM or nibrin deficient and suppressed cells will be discussed.

Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

2009-01-01

41

Base Excision Repair and Lesion-Dependent Subpathways for Repair of Oxidative DNA Damage  

PubMed Central

Abstract Nuclear and mitochondrial genomes are under continuous assault by a combination of environmentally and endogenously derived reactive oxygen species, inducing the formation and accumulation of mutagenic, toxic, and/or genome-destabilizing DNA lesions. Failure to resolve these lesions through one or more DNA-repair processes is associated with genome instability, mitochondrial dysfunction, neurodegeneration, inflammation, aging, and cancer, emphasizing the importance of characterizing the pathways and proteins involved in the repair of oxidative DNA damage. This review focuses on the repair of oxidative damage–induced lesions in nuclear and mitochondrial DNA mediated by the base excision repair (BER) pathway in mammalian cells. We discuss the multiple BER subpathways that are initiated by one of 11 different DNA glycosylases of three subtypes: (a) bifunctional with an associated ?-lyase activity; (b) monofunctional; and (c) bifunctional with an associated ?,?-lyase activity. These three subtypes of DNA glycosylases all initiate BER but yield different chemical intermediates and hence different BER complexes to complete repair. Additionally, we briefly summarize alternate repair events mediated by BER proteins and the role of BER in the repair of mitochondrial DNA damage induced by ROS. Finally, we discuss the relation of BER and oxidative DNA damage in the onset of human disease. Antioxid. Redox Signal. 14, 2491–2507. PMID:20649466

Svilar, David; Goellner, Eva M.; Almeida, Karen H.

2011-01-01

42

Effect of acrylamide on hepatocellular DNA repair  

SciTech Connect

Acrylamide has recently been reported to induce tumors in laboratory animals. The effect of acrylamide on unscheduled DNA synthesis using the hepatocyte primary culture (HPC)/DNA repair test was examined. Isolated hepatocytes were exposed to acrylamide and (3H)thymidine ( (3H)TdR) for 18 hr. Incorporation of (3H)TdR into DNA was determined by autoradiography. No DNA repair was observed at acrylamide concentrations up to 10(-2) M. These findings were confirmed using density gradients. Acrylamide concentrations exceeding 10(-2) M were cytotoxic to hepatocytes. Because both autoradiography and density gradients measure DNA repair as an endpoint, the ability of acrylamide to inhibit these repair processes was also determined. Acrylamide had no effect on the repair of UV-damaged DNA. These results show that acrylamide is not genotoxic in isolated hepatocytes.

Miller, M.J.; McQueen, C.A.

1986-01-01

43

Chromatin structure and DNA damage repair  

PubMed Central

The integrity of the genome is continuously challenged by both endogenous and exogenous DNA damaging agents. These damaging agents can induce a wide variety of lesions in the DNA, such as double strand breaks, single strand breaks, oxidative lesions and pyrimidine dimers. The cell has evolved intricate DNA damage response mechanisms to counteract the genotoxic effects of these lesions. The two main features of the DNA damage response mechanisms are cell-cycle checkpoint activation and, at the heart of the response, DNA repair. For both damage signalling and repair, chromatin remodelling is most likely a prerequisite. Here, we discuss current knowledge on chromatin remodelling with respect to the cellular response to DNA damage, with emphasis on the response to lesions resolved by nucleotide excision repair. We will discuss the role of histone modifications as well as their displacement or exchange in nucleotide excision repair and make a comparison with their requirement in transcription and double strand break repair. PMID:19014481

Dinant, Christoffel; Houtsmuller, Adriaan B; Vermeulen, Wim

2008-01-01

44

Conditional DNA repair mutants enable highly precise genome engineering  

PubMed Central

Oligonucleotide-mediated multiplex genome engineering is an important tool for bacterial genome editing. The efficient application of this technique requires the inactivation of the endogenous methyl-directed mismatch repair system that in turn leads to a drastically elevated genomic mutation rate and the consequent accumulation of undesired off-target mutations. Here, we present a novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli. Our method relies on the transient suppression of DNA repair during mismatch carrying oligonucleotide integration. Using temperature-sensitive control of methyl-directed mismatch repair protein activity during multiplex genome engineering, we reduced the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement. PMID:24500200

Nyerges, Ákos; Csörg?, Bálint; Nagy, István; Latinovics, Dóra; Szamecz, Béla; Pósfai, György; Pál, Csaba

2014-01-01

45

Recognition and repair of chemically heterogeneous structures at DNA ends.  

PubMed

Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not "clean." Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase ? (POL?). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini. Environ. Mol. Mutagen. 56:1-21, 2015. © 2014 Wiley Periodicals, Inc. PMID:25111769

Andres, Sara N; Schellenberg, Matthew J; Wallace, Bret D; Tumbale, Percy; Williams, R Scott

2015-01-01

46

Robustness of DNA Repair through Collective Rate Control  

PubMed Central

DNA repair and other chromatin-associated processes are carried out by enzymatic macromolecular complexes that assemble at specific sites on the chromatin fiber. How the rate of these molecular machineries is regulated by their constituent parts is poorly understood. Here we quantify nucleotide-excision DNA repair in mammalian cells and find that, despite the pathways' molecular complexity, repair effectively obeys slow first-order kinetics. Theoretical analysis and data-based modeling indicate that these kinetics are not due to a singular rate-limiting step. Rather, first-order kinetics emerge from the interplay of rapidly and reversibly assembling repair proteins, stochastically distributing DNA lesion repair over a broad time period. Based on this mechanism, the model predicts that the repair proteins collectively control the repair rate. Exploiting natural cell-to-cell variability, we corroborate this prediction for the lesion-recognition factor XPC and the downstream factor XPA. Our findings provide a rationale for the emergence of slow time scales in chromatin-associated processes from fast molecular steps and suggest that collective rate control might be a widespread mode of robust regulation in DNA repair and transcription. PMID:24499930

Manders, Erik; von Bornstaedt, Gesa; van Driel, Roel; Höfer, Thomas

2014-01-01

47

The virion of Cafeteria roenbergensis virus (CroV) contains a complex suite of proteins for transcription and DNA repair.  

PubMed

Cafeteria roenbergensis virus (CroV) is a giant virus of the Mimiviridae family that infects the marine phagotrophic flagellate C. roenbergensis. CroV possesses a DNA genome of ~730 kilobase pairs that is predicted to encode 544 proteins. We analyzed the protein composition of purified CroV particles by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified 141 virion-associated CroV proteins and 60 host proteins. Data are available via ProteomeXchange with identifier PXD000993. Predicted functions could be assigned to 36% of the virion proteins, which include structural proteins as well as enzymes for transcription, DNA repair, redox reactions and protein modification. Homologs of 36 CroV virion proteins have previously been found in the virion of Acanthamoeba polyphaga mimivirus. The overlapping virion proteome of CroV and Mimivirus reveals a set of conserved virion protein functions that were presumably present in the last common ancestor of the Mimiviridae. PMID:24973308

Fischer, Matthias G; Kelly, Isabelle; Foster, Leonard J; Suttle, Curtis A

2014-10-01

48

Molecular mechanisms of DNA repair inhibition by caffeine  

SciTech Connect

Caffeine potentiates the mutagenic and lethal effects of genotoxic agents. It is thought that this is due, at least in some organisms, to inhibition of DNA repair. However, direct evidence for inhibition of repair enzymes has been lacking. Using purified Escherichia coli DNA photolyase and (A)BC excinuclease, we show that the drug inhibits photoreactivation and nucleotide excision repair by two different mechanisms. Caffeine inhibits photoreactivation by interfering with the specific binding of photolyase to damaged DNA, and it inhibits nucleotide excision repair by promoting nonspecific binding of the damage-recognition subunit, UvrA, of (A)BC excinuclease. A number of other intercalators, including acriflavin and ethidium bromide, appear to inhibit the excinuclease by a similar mechanism--that is, by trapping the UvrA subunit in nonproductive complexes on undamaged DNA.

Selby, C.P.; Sancar, A. (Univ. of North Carolina School of Medicine, Chapel Hill (USA))

1990-05-01

49

Does DNA repair occur during somatic hypermutation?  

PubMed Central

Activation-induced deaminase (AID) initiates a flood of DNA damage in the immunoglobulin loci, leading to abasic sites, single-strand breaks and mismatches. It is compelling that some proteins in the canonical base excision and mismatch repair pathways have been hijacked to increase mutagenesis during somatic hypermutation. Thus, the AID-induced mutagenic pathways involve a mix of DNA repair proteins and low fidelity DNA polymerases to create antibody diversity. In this review, we analyze the roles of base excision repair, mismatch repair, and mutagenesis during somatic hypermutation of rearranged variable genes. The emerging view is that faithful base excision repair occurs simultaneously with mutagenesis, whereas faithful mismatch repair is mostly absent. PMID:22728014

Saribasak, Huseyin; Gearhart, Patricia J.

2012-01-01

50

Structure of the catalytic region of DNA ligase IV in complex with an Artemis fragment sheds light on double-strand break repair.  

PubMed

Nonhomologous end joining (NHEJ) is central to the repair of double-stranded DNA breaks throughout the cell cycle and plays roles in the development of the immune system. Although three-dimensional structures of most components of NHEJ have been defined, those of the catalytic region of DNA ligase IV (LigIV), a specialized DNA ligase known to work in NHEJ, and of Artemis have remained unresolved. Here, we report the crystal structure at 2.4 Å resolution of the catalytic region of LigIV (residues 1-609) in complex with an Artemis peptide. We describe interactions of the DNA-binding domain of LigIV with the continuous epitope of Artemis, which, together, form a three-helix bundle. A kink in the first helix of LigIV introduced by a conserved VPF motif gives rise to a hydrophobic pocket, which accommodates a conserved tryptophan from Artemis. We provide structural insights into features of LigIV among human DNA ligases. PMID:23523427

Ochi, Takashi; Gu, Xiaolong; Blundell, Tom L

2013-04-01

51

Structure of the Catalytic Region of DNA Ligase IV in Complex with an Artemis Fragment Sheds Light on Double-Strand Break Repair  

PubMed Central

Summary Nonhomologous end joining (NHEJ) is central to the repair of double-stranded DNA breaks throughout the cell cycle and plays roles in the development of the immune system. Although three-dimensional structures of most components of NHEJ have been defined, those of the catalytic region of DNA ligase IV (LigIV), a specialized DNA ligase known to work in NHEJ, and of Artemis have remained unresolved. Here, we report the crystal structure at 2.4 Å resolution of the catalytic region of LigIV (residues 1–609) in complex with an Artemis peptide. We describe interactions of the DNA-binding domain of LigIV with the continuous epitope of Artemis, which, together, form a three-helix bundle. A kink in the first helix of LigIV introduced by a conserved VPF motif gives rise to a hydrophobic pocket, which accommodates a conserved tryptophan from Artemis. We provide structural insights into features of LigIV among human DNA ligases. PMID:23523427

Ochi, Takashi; Gu, Xiaolong; Blundell, Tom L.

2013-01-01

52

Mechanisms of assembly of the enzyme-ssDNA complexes required for recombination-dependent DNA synthesis and repair in bacteriophage T4  

SciTech Connect

During late stages of bacteriophage T4 infection in E. coli, the initiation of phage DNA replication is dependent on the homologous recombination activity of the T4 uvsX protein. In vitro, uvsX protein initiates DNA synthesis on a duplex template by inserting the 3{prime} end of a homologous ssDNA molecule into the duplex. The resulting D-loop structure serves as a primer-template junction for the assembly of the T4 replication fork. Two key steps in this initiation process are (A) the assembly of uvsX-ssDNA complexes necessary for recombination activity and for the priming of lead-strand DNA synthesis, and (B) the assembly of the T4 primosome (gp41 helicase/gp61 primase complex) onto the single-stranded template for lagging-strand synthesis. Our laboratory is focusing on the mechanisms of these two different but related enzyme-ssDNA assembly processes. In this extended abstract, we describe recent efforts in our laboratory to elucidate the mechanism by which the gp41 helicase enzyme is assembled onto gp32-covered ssDNA, a process requiring the activity of a special helicase assembly factor, the T4 gp59 protein.

Morrical, S.; Hempstead, K.; Morrical, M. [Univ. of Vermont College of Medicine, Burlington, VT (United States)

1994-12-31

53

Importance of ligand structure in DNA/protein binding, mutagenicity, excision repair and nutritional aspects of chromium(III) complexes.  

PubMed

Chromium is extensively used in leather, chrome plating and refining industries. On one hand the occupational exposure to chromium leads to cancer, whereas on the contrary certain Cr(III) compounds have been proposed as nutritional supplements for Type II diabetes and as muscle building agents. Despite the positive outlook of chromium as a bio-essential element, there is increasing concern over the therapeutic application of Cr(III) based supplements, its bioavailability and toxicity profile. In this perspective, we discuss the role of ligand structure in mediating the interaction of chromium(III) complexes with DNA/protein, their mutagenic outcomes, adduct reparability and as nutritional supplements. PMID:23247426

Vaidyanathan, V G; Asthana, Yamini; Nair, Balachandran Unni

2013-02-21

54

Role of DNA repair protein ERCC1 in skin cancer   

E-print Network

Nucleotide excision repair (NER) is one of the major repair systems for removal of DNA lesions. The NER pathway has evolved mainly to repair UV-induced DNA damage and is also active against a broad range of endogenously ...

Song, Liang

2009-01-01

55

Nuclear position dictates DNA repair pathway choice  

PubMed Central

Faithful DNA repair is essential to avoid chromosomal rearrangements and promote genome integrity. Nuclear organization has emerged as a key parameter in the formation of chromosomal translocations, yet little is known as to whether DNA repair can efficiently occur throughout the nucleus and whether it is affected by the location of the lesion. Here, we induce DNA double-strand breaks (DSBs) at different nuclear compartments and follow their fate. We demonstrate that DSBs induced at the nuclear membrane (but not at nuclear pores or nuclear interior) fail to rapidly activate the DNA damage response (DDR) and repair by homologous recombination (HR). Real-time and superresolution imaging reveal that DNA DSBs within lamina-associated domains do not migrate to more permissive environments for HR, like the nuclear pores or the nuclear interior, but instead are repaired in situ by alternative end-joining. Our results are consistent with a model in which nuclear position dictates the choice of DNA repair pathway, thus revealing a new level of regulation in DSB repair controlled by spatial organization of DNA within the nucleus. PMID:25366693

Lemaître, Charlène; Grabarz, Anastazja; Tsouroula, Katerina; Andronov, Leonid; Furst, Audrey; Pankotai, Tibor; Heyer, Vincent; Rogier, Mélanie; Attwood, Kathleen M.; Kessler, Pascal; Dellaire, Graham; Klaholz, Bruno; Reina-San-Martin, Bernardo; Soutoglou, Evi

2014-01-01

56

DNA repair phenotype and dietary antioxidant supplementation  

Microsoft Academic Search

weeks. The mean baseline levels of DNA repair incisions were 65·2 (95 % CI 60·4, 70·0) and 86·1 (95 % CI 76·2, 99·9) among the male smokers and well-nourished subjects, respectively. The male smokers also had high baseline levels of oxidised guanines in MNBC. After supplementation, only the male smokers supplemented with slow-release vitamin C tablets had increased DNA repair

Serena Guarnieri; Steffen Loft; Patrizia Riso; Marisa Porrini; Lotte Risom; Henrik E. Poulsen; Lars O. Dragsted; Peter Møller

2008-01-01

57

Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways  

PubMed Central

During immunoglobulin (Ig) diversification, activation-induced deaminase (AID) initiates somatic hypermutation and class switch recombination by catalysing the conversion of cytosine to uracil. The synergy between AID and DNA repair pathways is fundamental for the introduction of mutations, however the molecular and biochemical mechanisms underlying this process are not fully elucidated. We describe a novel method to efficiently decipher the composition and activity of DNA repair pathways that are activated by AID-induced lesions. The in vitro resolution (IVR) assay combines AID based deamination and DNA repair activities from a cellular milieu in a single assay, thus avoiding synthetically created DNA-lesions or genetic-based readouts. Recombinant GAL4-AID fusion protein is targeted to a plasmid containing GAL4 binding sites, allowing for controlled cytosine deamination within a substrate plasmid. Subsequently, the Xenopus laevis egg extract provides a source of DNA repair proteins and functional repair pathways. Our results demonstrated that DNA repair pathways which are in vitro activated by AID-induced lesions are reminiscent of those found during AID-induced in vivo Ig diversification. The comparative ease of manipulation of this in vitro systems provides a new approach to dissect the complex DNA repair pathways acting on defined physiologically lesions, can be adapted to use with other DNA damaging proteins (e.g. APOBECs), and provide a means to develop and characterise pharmacological agents to inhibit these potentially oncogenic processes. PMID:24349193

Franchini, Don-Marc; Incorvaia, Elisabetta; Rangam, Gopinath; Coker, Heather A.; Petersen-Mahrt, Svend K.

2013-01-01

58

Repair of DNA Double-Strand Breaks  

NASA Astrophysics Data System (ADS)

The genetic information of cells continuously undergoes damage induced by intracellular processes including energy metabolism, DNA replication and transcription, and by environmental factors such as mutagenic chemicals and UV and ionizing radiation. This causes numerous DNA lesions, including double strand breaks (DSBs). Since cells cannot escape this damage or normally function with a damaged genome, several DNA repair mechanisms have evolved. Although most "single-stranded" DNA lesions are rapidly removed from DNA without permanent damage, DSBs completely break the DNA molecule, presenting a real challenge for repair mechanisms, with the highest risk among DNA lesions of incorrect repair. Hence, DSBs can have serious consequences for human health. Therefore, in this chapter, we will refer only to this type of DNA damage. In addition to the biochemical aspects of DSB repair, which have been extensively studied over a long period of time, the spatio-temporal organization of DSB induction and repair, the importance of which was recognized only recently, will be considered in terms of current knowledge and remaining questions.

Falk, Martin; Lukasova, Emilie; Kozubek, Stanislav

59

DNA repair in human bronchial epithelial cells  

SciTech Connect

The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz(a)anthracene; benzo(a)pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents.

Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

1982-01-01

60

Part 1: Mechanisms of DNA Repair  

NSDL National Science Digital Library

The most deleterious form of DNA damage is a double-strand break (DSB), which can arise from errors in DNA replication, from the failure of topoisomerases to complete their cycles of DNA cutting and rejoining, from mechanical stress and from the action of endonucleases that cleave DNA. Here we review how DSBs can be repaired either by nonhomologous end-joining mechanisms or by several homologous recombination pathways including single-strand annealing, gene conversion and break-induced replication.

Jim Haber (Brandeis University;Department of Biology and Rosenstiel Basic Medical Sciences Research Center)

2009-12-01

61

Repair of alkylated DNA: Recent advances  

Microsoft Academic Search

Cytotoxic and mutagenic methylated bases in DNA can be generated by endogenous and environmental alkylating agents. Such damaged bases are removed by three distinct strategies. The abundant toxic lesion 3-methyladenine (3-alkyladenine) is excised by a specific DNA glycosylase that initiates a base excision-repair process. The toxic lesions 1-methyladenine and 3-methylcytosine are corrected by oxidative DNA demethylation catalyzed by DNA dioxygenases.

Barbara Sedgwick; Paul A. Bates; Johanna Paik; Susan C. Jacobs; Tomas Lindahl

2007-01-01

62

Eukaryotic damaged DNA-binding proteins: DNA repair proteins or transcription factors?  

SciTech Connect

Recognition and removal of structural defects in the genome, caused by diverse physical and chemical agents, are among the most important cell functions. Proteins that recognize and bind to modified DNA, and thereby initiate damage-induced recovery processes, have been identified in prokaryotic and eukaryotic cells. Damaged DNA-binding (DDB) proteins from prokaryotes are either DNA repair enzymes or noncatalytic subunits of larger DNA repair complexes that participate in excision repair, or in recombinational repair and SOS-mutagenesis. Although the methods employed may not have allowed detection of all eukaryotic DDB proteins and identification of their functions, it appears that during evolution cells have developed a wide array of DDB proteins that can discriminate among the diversity of DNA conformations found in the eukaryotic nucleus, as well as a gene-sharing feature found in DDB proteins that also act as transcription factors.

Protic, M. [National Institutes of Health, Bethesda, MD (United States)

1994-12-31

63

Chromatin Remodeling, DNA Damage Repair and Aging  

PubMed Central

Cells are constantly exposed to a variety of environmental and endogenous conditions causing DNA damage, which is detected and repaired by conserved DNA repair pathways to maintain genomic integrity. Chromatin remodeling is critical in this process, as the organization of eukaryotic DNA into compact chromatin presents a natural barrier to all DNA-related events. Studies on human premature aging syndromes together with normal aging have suggested that accumulated damages might lead to exhaustion of resources that are required for physiological functions and thus accelerate aging. In this manuscript, combining the present understandings and latest findings, we focus mainly on discussing the role of chromatin remodeling in the repair of DNA double-strand breaks (DSBs) and regulation of aging. PMID:23633913

Liu, Baohua; Yip, Raymond KH; Zhou, Zhongjun

2012-01-01

64

DNA INTERSTRAND CROSSLINK REPAIR IN MAMMALIAN CELLS: STEP BY STEP  

PubMed Central

Interstrand DNA crosslinks (ICLs) are formed by natural products of metabolism and by chemotherapeutic reagents. Work in E. coli identified a two cycle repair scheme involving incisions on one strand on either side of the ICL (unhooking) producing a gapped intermediate with the incised oligonucleotide attached to the intact strand. The gap is filled by recombinational repair or lesion bypass synthesis. The remaining monoadduct is then removed by Nucleotide Excision Repair (NER). Despite considerable effort, our understanding of each step in mammalian cells is still quite limited. In part this reflects the variety of crosslinking compounds, each with distinct structural features, used by different investigators. Also, multiple repair pathways are involved, variably operative during the cell cycle. G1 phase repair requires functions from NER, although the mechanism of recognition has not been determined. Repair can be initiated by encounters with the transcriptional apparatus, or a replication fork. In the case of the latter, the reconstruction of a replication fork, stalled or broken by collision with an ICL, adds to the complexity of the repair process. The enzymology of unhooking, the identity of the lesion bypass polymerases required to fill the first repair gap, and the functions involved in the second repair cycle are all subjects of active inquiry. Here we will review current understanding of each step in ICL repair in mammalian cells. PMID:20039786

Muniandy, Parameswary; Liu, Jia; Majumdar, Alokes; Liu, Su-ting; Seidman, Michael M.

2009-01-01

65

The awakening of DNA repair at Yale.  

PubMed

As a graduate student with Professor Richard Setlow at Yale in the late 1950s, I studied the effects of ultraviolet and visible light on the syntheses of DNA, RNA, and protein in bacteria. I reflect upon my research in the Yale Biophysics Department, my subsequent postdoctoral experiences, and the eventual analyses in the laboratories of Setlow, Paul Howard-Flanders, and myself that constituted the discovery of the ubiquitous pathway of DNA excision repair in the early 1960s. I then offer a brief perspective on a few more recent developments in the burgeoning DNA repair field and their relationships to human disease. PMID:24348216

Hanawalt, Philip C

2013-12-01

66

The Awakening of DNA Repair at Yale  

PubMed Central

As a graduate student with Professor Richard Setlow at Yale in the late 1950s, I studied the effects of ultraviolet and visible light on the syntheses of DNA, RNA, and protein in bacteria. I reflect upon my research in the Yale Biophysics Department, my subsequent postdoctoral experiences, and the eventual analyses in the laboratories of Setlow, Paul Howard-Flanders, and myself that constituted the discovery of the ubiquitous pathway of DNA excision repair in the early 1960s. I then offer a brief perspective on a few more recent developments in the burgeoning DNA repair field and their relationships to human disease. PMID:24348216

Hanawalt, Philip C.

2013-01-01

67

The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites*  

PubMed Central

XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase III?-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair. PMID:22992732

Della-Maria, Julie; Hegde, Muralidhar L.; McNeill, Daniel R.; Matsumoto, Yoshihiro; Tsai, Miaw-Sheue; Ellenberger, Tom; Wilson, David M.; Mitra, Sankar; Tomkinson, Alan E.

2012-01-01

68

The ups and downs of DNA repair biomarkers for PARP inhibitor therapies  

PubMed Central

PARP inhibitors are emerging as a valuable new drug class in the treatment of cancer. Recent discoveries make a compelling case for the complexity of DNA repair biomarker evaluation and underscore the need to examine at multiple biomarkers in a relational manner. This review updates the current trends in DNA repair biomarker strategies in use for the PARP inhibitors and describes the impact of many DNA repair biomarkers on PARP inhibitor benefit in the cancer clinic. PMID:21968427

Wang, XiaoZhe; Weaver, David T

2011-01-01

69

DNA repair synthesis and ligation affect the processing of excised oligonucleotides generated by human nucleotide excision repair.  

PubMed

Ultraviolet (UV) photoproducts are removed from genomic DNA by dual incisions in humans in the form of 24- to 32-nucleotide-long oligomers (canonical 30-mers) by the nucleotide excision repair system. How the small, excised, damage-containing DNA oligonucleotides (sedDNAs) are processed in cells following the dual incision event is not known. Here, we demonstrate that sedDNAs are localized to the nucleus in two biochemically distinct forms, which include chromatin-associated, transcription factor II H-bound complexes and more readily solubilized, RPA-bound complexes. Because the nuclear mobility and repair functions of transcription factor II H and RPA are influenced by post-incision gap-filling events, we examined how DNA repair synthesis and DNA ligation affect sedDNA processing. We found that although these gap filling activities are not essential for the dual incision/sedDNA generation event per se, the inhibition of DNA repair synthesis and ligation is associated with a decrease in UV photoproduct removal rate and an accumulation of RPA-sedDNA complexes in the cell. These findings indicate that sedDNA processing and association with repair proteins following the dual incisions may be tightly coordinated with gap filling during nucleotide excision repair in vivo. PMID:25107903

Kemp, Michael G; Gaddameedhi, Shobhan; Choi, Jun-Hyuk; Hu, Jinchuan; Sancar, Aziz

2014-09-19

70

DNA Repair and Personalized Breast Cancer Therapy  

PubMed Central

Personalized cancer therapy is likely to be one of the next big advances in our search for a cure for cancer. To be able to treat people in an individualized manner, researchers need to know a great deal about their genetic constitution and the DNA repair status of their tumors. Specific knowledge is required regarding the polymorphisms individuals carry and how these polymorphisms influence responses to therapy. Researchers are actively engaged in biomarker discovery and validation for this purpose. In addition, the design of clinical trials must be reassessed to include new information on biomarkers and drug responses. In this review, we focus on personalized breast cancer therapy. The hypothesis we focus upon in this review is that there is connection between the DNA repair profile of individuals, their breast tumor subtypes, and their responses to cancer therapy. We first briefly review cellular DNA repair pathways that are likely to be impacted by breast cancer therapies. Next, we review the phenotypes of breast tumor subtypes with an emphasis on how a DNA repair deficiency might result in tumorigenesis itself and lead to the chemotherapeutic responses that are observed. Specific examples of breast tumor subtypes and their responses to cancer therapy are given, and we discuss possible DNA repair mechanisms that underlie the responses of tumors to various chemotherapeutic agents. Much is known about breast cancer subtypes and the way each of these subtypes responds to chemotherapy. In addition, we discuss novel design of clinical trials that incorporates rapidly emerging information on biomarkers. PMID:20872853

Li, Shu-Xia; Sjolund, Ashley; Harris, Lyndsay; Sweasy, Joann B.

2010-01-01

71

DNA repair responses in human skin cells  

SciTech Connect

Sunlight and some environmental chemical agents produce lesions in the DNA of human skin cells that if unrepaired may interfere with normal functioning of these cells. The most serious outcome of such interactions may be malignancy. It is therefore important to develop an understanding of mechanisms by which the lesions may be repaired or tolerated without deleterious consequences. Our models for the molecular processing of damaged DNA have been derived largely from the study of bacterial systems. Some similarities but significant differences are revealed when human cell responses are tested against these models. It is also of importance to learn DNA repair responses of epidermal keratinocytes for comparison with the more extensive studies that have been carried out with dermal fibroblasts. Our experimental results thus far indicate similarities for the excision-repair of ultraviolet-induced pyrimidine dimers in human keratinocytes and fibroblasts. Both the monoadducts and the interstrand crosslinks produced in DNA by photoactivated 8-methoxypsoralen (PUVA) can be repaired in normal human fibroblasts but not in those from xeroderma pigmentosum patients. The monoadducts, like pyrimidine dimers, are probably the more mutagenic/carcinogenic lesions while the crosslinks are less easily repaired and probably result in more effective blocking of DNA function. It is suggested that a split-dose protocol that maximizes the production of crosslinks while minimizing the yield of monoadducts may be more effective and potentially less carcinogenic than the single ultraviolet exposure regimen in PUVA therapy for psoriasis.

Hanawalt, P.C.; Liu, S.C.; Parsons, C.S.

1981-07-01

72

DNA damage and repair after high LET radiation  

NASA Astrophysics Data System (ADS)

Predictions from biophysical models of interactions of radiation tracks with cellular DNA indicate that clustered DNA damage sites, defined as two or more lesions formed within one or two helical turns of the DNA by passage of a single radiation track, are formed in mammalian cells. These complex DNA damage sites are regarded as a signature of ionizing radiation exposure particularly as the likelihood of clustered damage sites arising endogenously is low. For instance, it was predicted from biophysical modelling that 30-40% of low LET-induced double strand breaks (DSB), a form of clustered damage, are complex with the yield increasing to >90% for high LET radiation, consistent with the reduced reparability of DSB with increasing ionization density of the radiation. The question arises whether the increased biological effects such as mutagenesis, carcinogenesis and lethality is in part related to DNA damage complexity and/or spatial distribution of the damage sites, which may lead to small DNA fragments. With particle radiation it is also important to consider not only delta-rays which may cause clustered damaged sites and may be highly mutagenic but the non-random spatial distribution of DSB which may lead to deletions. In this overview I will concentrate on the molecular aspects of the variation of the complexity of DNA damage on radiation quality and the challenges this complexity presents the DNA damage repair pathways. I will draw on data from micro-irradiations which indicate that the repair of DSBs by non-homologous end joining is highly regulated with pathway choice and kinetics of repair dependent on the chemical complexity of the DSB. In summary the aim is to emphasis the link between the spatial distribution of energy deposition events related to the track, the molecular products formed and the consequence of damage complexity contributing to biological effects and to present some of the outstanding molecular challenges with particle radiation.

O'Neill, Peter; Cucinotta, Francis; Anderson, Jennifer

73

Recombination and DNA Repair in Helicobacter pylori  

PubMed Central

All organisms have pathways that repair the genome, ensuring their survival and that of their progeny. But these pathways also serve to diversify the genome, causing changes on the level of nucleotide, whole gene, and genome structure. Sequencing of bacteria has revealed wide allelic diversity and differences in gene content within the same species, highlighting the importance of understanding pathways of recombination and DNA repair. The human stomach pathogen Helicobacter pylori is an excellent model system for studying these pathways. H. pylori harbors major recombination and repair pathways and is naturally competent, facilitating its ability to diversify its genome. Elucidation of DNA recombination, repair, and diversification programs in this pathogen will reveal connections between these pathways and their importance to infection. PMID:21682641

Dorer, Marion S.; Sessler, Tate H.; Salama, Nina R.

2013-01-01

74

The RASSF1A Tumor Suppressor Regulates XPA-Mediated DNA Repair.  

PubMed

RASSF1A may be the most frequently inactivated tumor suppressor identified in human cancer so far. It is a proapoptotic Ras effector and plays an important role in the apoptotic DNA damage response (DDR). We now show that in addition to DDR regulation, RASSF1A also plays a key role in the DNA repair process itself. We show that RASSF1A forms a DNA damage-regulated complex with the key DNA repair protein xeroderma pigmentosum A (XPA). XPA requires RASSF1A to exert full repair activity, and RASSF1A-deficient cells exhibit an impaired ability to repair DNA. Moreover, a cancer-associated RASSF1A single-nucleotide polymorphism (SNP) variant exhibits differential XPA binding and inhibits DNA repair. The interaction of XPA with other components of the repair complex, such as replication protein A (RPA), is controlled in part by a dynamic acetylation/deacetylation cycle. We found that RASSF1A and its SNP variant differentially regulate XPA protein acetylation, and the SNP variant hyperstabilizes the XPA-RPA70 complex. Thus, we identify two novel functions for RASSF1A in the control of DNA repair and protein acetylation. As RASSF1A modulates both apoptotic DDR and DNA repair, it may play an important and unanticipated role in coordinating the balance between repair and death after DNA damage. PMID:25368379

Donninger, Howard; Clark, Jennifer; Rinaldo, Francesca; Nelson, Nicholas; Barnoud, Thibaut; Schmidt, M Lee; Hobbing, Katharine R; Vos, Michele D; Sils, Brian; Clark, Geoffrey J

2015-01-01

75

40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...  

Code of Federal Regulations, 2014 CFR

...repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests...repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests...growth inhibition of repair deficient bacteria in a set of repair proficient and...

2014-07-01

76

40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...  

Code of Federal Regulations, 2012 CFR

...repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests...repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests...growth inhibition of repair deficient bacteria in a set of repair proficient and...

2012-07-01

77

40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...  

Code of Federal Regulations, 2013 CFR

...repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests...repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests...growth inhibition of repair deficient bacteria in a set of repair proficient and...

2013-07-01

78

40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...  

Code of Federal Regulations, 2011 CFR

...repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests...repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests...growth inhibition of repair deficient bacteria in a set of repair proficient and...

2011-07-01

79

40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...  

Code of Federal Regulations, 2010 CFR

...repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests...repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests...growth inhibition of repair deficient bacteria in a set of repair proficient and...

2010-07-01

80

Sumoylation of the Rad1 nuclease promotes DNA repair and regulates its DNA association  

PubMed Central

The Saccharomyces cerevisiae Rad1-Rad10 complex is a conserved, structure-specific endonuclease important for repairing multiple types of DNA lesions. Upon recruitment to lesion sites, Rad1-Rad10 removes damaged sequences, enabling subsequent gap filling and ligation. Acting at mid-steps of repair, the association and dissociation of Rad1-Rad10 with DNA can influence repair efficiency. We show that genotoxin-enhanced Rad1 sumoylation occurs after the nuclease is recruited to lesion sites. A single lysine outside Rad1's nuclease and Rad10-binding domains is sumoylated in vivo and in vitro. Mutation of this site to arginine abolishes Rad1 sumoylation and impairs Rad1-mediated repair at high doses of DNA damage, but sustains the repair of a single double-stranded break. The timing of Rad1 sumoylation and the phenotype bias toward high lesion loads point to a post-incision role for sumoylation, possibly affecting Rad1 dissociation from DNA. Indeed, biochemical examination shows that sumoylation of Rad1 decreases the complex's affinity for DNA without affecting other protein properties. These findings suggest a model whereby sumoylation of Rad1 promotes its disengagement from DNA after nuclease cleavage, allowing it to efficiently attend to large numbers of DNA lesions. PMID:24753409

Sarangi, Prabha; Bartosova, Zdenka; Altmannova, Veronika; Holland, Cory; Chavdarova, Melita; Lee, Sang Eun; Krejci, Lumir; Zhao, Xiaolan

2014-01-01

81

A Quantitative Assay Reveals Ligand Specificity of the DNA scaffold repair protein XRCC1 and Efficient Disassembly of Complexes of XRCC1 and the Poly (ADP-ribose) polymerase 1 by PAR glycohydrolase.  

PubMed

The posttranslational modification of proteins with poly(ADP-ribose) (PAR) regulates protein-protein interactions in DNA repair, gene expression, chromatin structure, and cell fate determination. The PAR polymerase PARP1 binds to damaged chromatin and synthesizes PAR chains to signal DNA damage and recruit the DNA repair scaffold, XRCC1. Pharmacological blockade of PARP1 enzymatic activity impairs XRCC1-dependent repair of DNA damage and selectively kills cancer cells lacking other DNA repair functions. As such, PARP inhibitors are promising new therapies for repair-deficient tumors such as BRCA mutated breast cancers. Although the XRCC1-PARP1 complex is relevant to the proposed therapeutic mechanism of PARP inhibitors, the physical makeup and dynamics of this complex are not well characterized at the molecular level. Here we describe a fluorescence-based, real-time assay that quantitatively monitors interactions between PARylated PARP1 and XRCC1. Using this assay, we show that the PAR posttranslational modification by itself is a high affinity ligand for XRCC1, requiring a minimum chain length of 7 ADP-ribose units in the oligo(ADP-ribose) ligand for a stable interaction with XRCC1. This discrete binding interface enables the PAR glycohydrolase (PARG) to completely disassemble the PARP1-XRCC1 complex without assistance from a mono(ADP-ribose) glycohydrolase. Our quantitative, real-time assay of PAR-dependent protein-protein interactions and PAR turnover by PARG is an excellent tool for high-throughput screening to identify pharmacological modulators of PAR metabolism that may be useful therapeutic alternatives to PARP inhibitors. PMID:25477519

Kim, In-Kwon; Stegeman, Roderick A; Brosey, Chris A; Ellenberger, Tom

2014-12-01

82

Repair of Oxidative DNA Damage and Cancer: Recent Progress in DNA Base Excision Repair  

PubMed Central

Abstract Significance: Reactive oxygen species (ROS) are generated by exogenous and environmental genotoxins, but also arise from mitochondria as byproducts of respiration in the body. ROS generate DNA damage of which pathological consequence, including cancer is well established. Research efforts are intense to understand the mechanism of DNA base excision repair, the primary mechanism to protect cells from genotoxicity caused by ROS. Recent Advances: In addition to the notion that oxidative DNA damage causes transformation of cells, recent studies have revealed how the mitochondrial deficiencies and ROS generation alter cell growth during the cancer transformation. Critical Issues: The emphasis of this review is to highlight the importance of the cellular response to oxidative DNA damage during carcinogenesis. Oxidative DNA damage, including 7,8-dihydro-8-oxoguanine, play an important role during the cellular transformation. It is also becoming apparent that the unusual activity and subcellular distribution of apurinic/apyrimidinic endonuclease 1, an essential DNA repair factor/redox sensor, affect cancer malignancy by increasing cellular resistance to oxidative stress and by positively influencing cell proliferation. Future Directions: Technological advancement in cancer cell biology and genetics has enabled us to monitor the detailed DNA repair activities in the microenvironment. Precise understanding of the intracellular activities of DNA repair proteins for oxidative DNA damage should provide help in understanding how mitochondria, ROS, DNA damage, and repair influence cancer transformation. Antioxid. Redox Signal. 20, 708–726. PMID:23901781

Scott, Timothy L.; Rangaswamy, Suganya; Wicker, Christina A.

2014-01-01

83

Databases and Bioinformatics Tools for the Study of DNA Repair  

PubMed Central

DNA is continuously exposed to many different damaging agents such as environmental chemicals, UV light, ionizing radiation, and reactive cellular metabolites. DNA lesions can result in different phenotypical consequences ranging from a number of diseases, including cancer, to cellular malfunction, cell death, or aging. To counteract the deleterious effects of DNA damage, cells have developed various repair systems, including biochemical pathways responsible for the removal of single-strand lesions such as base excision repair (BER) and nucleotide excision repair (NER) or specialized polymerases temporarily taking over lesion-arrested DNA polymerases during the S phase in translesion synthesis (TLS). There are also other mechanisms of DNA repair such as homologous recombination repair (HRR), nonhomologous end-joining repair (NHEJ), or DNA damage response system (DDR). This paper reviews bioinformatics resources specialized in disseminating information about DNA repair pathways, proteins involved in repair mechanisms, damaging agents, and DNA lesions. PMID:22091405

Milanowska, Kaja; Rother, Kristian; Bujnicki, Janusz M.

2011-01-01

84

Electrically monitoring DNA repair by photolyase  

NASA Astrophysics Data System (ADS)

Cyclobutane pyrimidine dimers are the major DNA photoproducts produced upon exposure to UV radiation. If left unrepaired, these lesions can lead to replication errors, mutation, and cell death. Photolyase is a light-activated flavoenzyme that binds to pyrimidine dimers in DNA and repairs them in a reaction triggered by electron transfer from the photoexcited flavin cofactor to the dimer. Using gold electrodes modified with DNA duplexes containing a cyclobutane thymine dimer (T<>T), here we probe the electrochemistry of the flavin cofactor in Escherichia coli photolyase. Cyclic and square-wave voltammograms of photolyase deposited on these electrodes show a redox signal at 40 mV versus normal hydrogen electrode, consistent with electron transfer to and from the flavin in the DNA-bound protein. This signal is dramatically attenuated on surfaces where the ?-stacking of the DNA bases is perturbed by the presence of an abasic site below the T<>T, an indication that the redox pathway is DNA-mediated. DNA repair can, moreover, be monitored electrically. Exposure of photolyase on T<>T-damaged DNA films to near-UV/blue light leads to changes in the flavin signal consistent with repair, as confirmed by parallel HPLC experiments. These results demonstrate the exquisite sensitivity of DNA electrochemistry to perturbations in base pair stacking and the applicability of this chemistry to probe reactions of proteins with DNA. Author contributions: M.C.D. and J.K.B. designed research; M.C.D. performed research; A.S. contributed new reagents/analytic tools; M.C.D. analyzed data; and M.C.D. and J.K.B. wrote the paper.This paper was submitted directly (Track II) to the PNAS office.Abbreviations: T<>T, thymine dimer; CT, charge transport.

DeRosa, Maria C.; Sancar, Aziz; Barton, Jacqueline K.

2005-08-01

85

Single cell visualization of DNA repair in vivo  

Microsoft Academic Search

The creation of a DNA double-strand-break constitutes the most dangerous type of DNA damage. Inefficient response to DNA damage may lead to hypersensitivity to cellular stressors, susceptibility to genomic defects and resistance to apoptosis, which can lead to cancer. Current research on DNA repair has enabled numerous breakthroughs in our understanding of the DNA repair mechanisms at the population level.

Azadeh Samadani; Amy Rowat; Jennifer Makridakis; James Haber

2008-01-01

86

Fragile DNA repair mechanism reduces ageing in multicellular model.  

PubMed

DNA damages, as well as mutations, increase with age. It is believed that these result from increased genotoxic stress and decreased capacity for DNA repair. The two causes are not independent, DNA damage can, for example, through mutations, compromise the capacity for DNA repair, which in turn increases the amount of unrepaired DNA damage. Despite this vicious circle, we ask, can cells maintain a high DNA repair capacity for some time or is repair capacity bound to continuously decline with age? We here present a simple mathematical model for ageing in multicellular systems where cells subjected to DNA damage can undergo full repair, go apoptotic, or accumulate mutations thus reducing DNA repair capacity. Our model predicts that at the tissue level repair rate does not continuously decline with age, but instead has a characteristic extended period of high and non-declining DNA repair capacity, followed by a rapid decline. Furthermore, the time of high functionality increases, and consequently slows down the ageing process, if the DNA repair mechanism itself is vulnerable to DNA damages. Although counterintuitive at first glance, a fragile repair mechanism allows for a faster removal of compromised cells, thus freeing the space for healthy peers. This finding might be a first step toward understanding why a mutation in single DNA repair protein (e.g. Wrn or Blm) is not buffered by other repair proteins and therefore, leads to severe ageing disorders. PMID:22567122

Bendtsen, Kristian Moss; Juul, Jeppe; Trusina, Ala

2012-01-01

87

Introduction Telomere biology and DNA repair: Enemies with benefits  

E-print Network

Introduction Telomere biology and DNA repair: Enemies with benefits This special issue features in-depth reviews of telomere biology and DNA repair. Understanding how telomeres function requires insights into the nature and regulation of the cellular pathways that detect and repair DNA lesions. As telomeres block

de Lange, Titia

88

Induction of the SOS DNA repair response in Escherichia coli by nitric oxide donating agents: dinitrosyl iron complexes with thiol-containing ligands and S-nitrosothiols.  

PubMed

The ability of nitric oxide (NO) donor compounds to induce the SOS DNA repair response in Escherichia coli is reported. Dinitrosyl iron complexes with glutathione and cysteine (DNIC) are the most potent SOS-inducers. S-Nitrosothiols (RSNO) mediate a similar response at 10-100 microM, but the response decreases sharply at concentrations above 0.5 mM. Pretreatment of the cells with the chelating agent o-phenanthroline (OP) prevents induction of the SOS response by all agents used. On the other hand, the toxicity of S-nitrosothiols is higher than that of DNIC. The EPR study shows the appearance of an EPR DNIC-type signal after incubation of the cells with S-nitrosoglutathione because of mutual transformation between RSNO and DNIC in the presence of accessible iron inside the cells. Pretreatment of the cells with OP leads to a decrease in this signal. Analysis of NO donor effects reveals a dual role of the iron ions in reactivity and toxicity of the compounds studied, i.e. (i) stabilization of the cytotoxic RSNO and (ii) generation of the SOS signal. PMID:10431802

Lobysheva, I I; Stupakova, M V; Mikoyan, V D; Vasilieva, S V; Vanin, A F

1999-07-01

89

Importance of DNA repair in tumor suppression  

NASA Astrophysics Data System (ADS)

The transition from a normal to cancerous cell requires a number of highly specific mutations that affect cell cycle regulation, apoptosis, differentiation, and many other cell functions. One hallmark of cancerous genomes is genomic instability, with mutation rates far greater than those of normal cells. In microsatellite instability (MIN tumors), these are often caused by damage to mismatch repair genes, allowing further mutation of the genome and tumor progression. These mutation rates may lie near the error catastrophe found in the quasispecies model of adaptive RNA genomes, suggesting that further increasing mutation rates will destroy cancerous genomes. However, recent results have demonstrated that DNA genomes exhibit an error threshold at mutation rates far lower than their conservative counterparts. Furthermore, while the maximum viable mutation rate in conservative systems increases indefinitely with increasing master sequence fitness, the semiconservative threshold plateaus at a relatively low value. This implies a paradox, wherein inaccessible mutation rates are found in viable tumor cells. In this paper, we address this paradox, demonstrating an isomorphism between the conservatively replicating (RNA) quasispecies model and the semiconservative (DNA) model with post-methylation DNA repair mechanisms impaired. Thus, as DNA repair becomes inactivated, the maximum viable mutation rate increases smoothly to that of a conservatively replicating system on a transformed landscape, with an upper bound that is dependent on replication rates. On a specific single fitness peak landscape, the repair-free semiconservative system is shown to mimic a conservative system exactly. We postulate that inactivation of post-methylation repair mechanisms is fundamental to the progression of a tumor cell and hence these mechanisms act as a method for the prevention and destruction of cancerous genomes.

Brumer, Yisroel; Shakhnovich, Eugene I.

2004-12-01

90

Small Molecules, Inhibitors of DNA-PK, Targeting DNA Repair, and Beyond  

PubMed Central

Many current chemotherapies function by damaging genomic DNA in rapidly dividing cells ultimately leading to cell death. This therapeutic approach differentially targets cancer cells that generally display rapid cell division compared to normal tissue cells. However, although these treatments are initially effective in arresting tumor growth and reducing tumor burden, resistance and disease progression eventually occur. A major mechanism underlying this resistance is increased levels of cellular DNA repair. Most cells have complex mechanisms in place to repair DNA damage that occurs due to environmental exposures or normal metabolic processes. These systems, initially overwhelmed when faced with chemotherapy induced DNA damage, become more efficient under constant selective pressure and as a result chemotherapies become less effective. Thus, inhibiting DNA repair pathways using target specific small molecule inhibitors may overcome cellular resistance to DNA damaging chemotherapies. Non-homologous end joining a major mechanism for the repair of double-strand breaks (DSB) in DNA is regulated in part by the serine/threonine kinase, DNA dependent protein kinase (DNA-PK). The DNA-PK holoenzyme acts as a scaffold protein tethering broken DNA ends and recruiting other repair molecules. It also has enzymatic activity that may be involved in DNA damage signaling. Because of its’ central role in repair of DSBs, DNA-PK has been the focus of a number of small molecule studies. In these studies specific DNA-PK inhibitors have shown efficacy in synergizing chemotherapies in vitro. However, compounds currently known to specifically inhibit DNA-PK are limited by poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives. Future improvement in DNA-PK inhibition will likely be achieved by designing new molecules based on the recently reported crystallographic structure of DNA-PK. Computer based drug design will not only assist in identifying novel functional moieties to replace the metabolically labile morpholino group but will also facilitate the design of molecules to target the DNA-PKcs/Ku80 interface or one of the autophosphorylation sites. PMID:23386830

Davidson, David; Amrein, Lilian; Panasci, Lawrence; Aloyz, Raquel

2012-01-01

91

After sun reversal of DNA damage: enhancing skin repair  

Microsoft Academic Search

UV-induced DNA damage has been directly linked to skin cancer, and DNA repair is an important protection against this neoplasm. This is illustrated by the genetic disease xeroderma pigmentosum wherein a serious defect in DNA repair of cyclobutane pyrimidine dimers dramatically increases the rate of skin cancer. In other instances in which skin cancer rates are elevated, deficits in DNA

Daniel B. Yarosh; Matthew T. Canning; Danielle Teicher; David A. Brown

2005-01-01

92

ISWI chromatin remodeling complexes in the DNA damage response.  

PubMed

Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA. PMID:25486562

Aydin, Ozge Z; Vermeulen, Wim; Lans, Hannes

2014-10-01

93

Identification of novel DNA repair proteins via primary sequence, secondary structure, and homology  

PubMed Central

Background DNA repair is the general term for the collection of critical mechanisms which repair many forms of DNA damage such as methylation or ionizing radiation. DNA repair has mainly been studied in experimental and clinical situations, and relatively few information-based approaches to new extracting DNA repair knowledge exist. As a first step, automatic detection of DNA repair proteins in genomes via informatics techniques is desirable; however, there are many forms of DNA repair and it is not a straightforward process to identify and classify repair proteins with a single optimal method. We perform a study of the ability of homology and machine learning-based methods to identify and classify DNA repair proteins, as well as scan vertebrate genomes for the presence of novel repair proteins. Combinations of primary sequence polypeptide frequency, secondary structure, and homology information are used as feature information for input to a Support Vector Machine (SVM). Results We identify that SVM techniques are capable of identifying portions of DNA repair protein datasets without admitting false positives; at low levels of false positive tolerance, homology can also identify and classify proteins with good performance. Secondary structure information provides improved performance compared to using primary structure alone. Furthermore, we observe that machine learning methods incorporating homology information perform best when data is filtered by some clustering technique. Analysis by applying these methodologies to the scanning of multiple vertebrate genomes confirms a positive correlation between the size of a genome and the number of DNA repair protein transcripts it is likely to contain, and simultaneously suggests that all organisms have a non-zero minimum number of repair genes. In addition, the scan result clusters several organisms' repair abilities in an evolutionarily consistent fashion. Analysis also identifies several functionally unconfirmed proteins that are highly likely to be involved in the repair process. A new web service, INTREPED, has been made available for the immediate search and annotation of DNA repair proteins in newly sequenced genomes. Conclusion Despite complexity due to a multitude of repair pathways, combinations of sequence, structure, and homology with Support Vector Machines offer good methods in addition to existing homology searches for DNA repair protein identification and functional annotation. Most importantly, this study has uncovered relationships between the size of a genome and a genome's available repair repetoire, and offers a number of new predictions as well as a prediction service, both which reduce the search time and cost for novel repair genes and proteins. PMID:19154573

Brown, JB; Akutsu, Tatsuya

2009-01-01

94

The hMre11\\/hRad50 Protein Complex and Nijmegen Breakage Syndrome: Linkage of Double-Strand Break Repair to the Cellular DNA Damage Response  

Microsoft Academic Search

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by increased cancer incidence, cell cycle checkpoint defects, and ionizing radiation sensitivity. We have isolated the gene encoding p95, a member of the hMre11\\/hRad50 double-strand break repair complex. The p95 gene mapped to 8q21.3, the region that contains the NBS locus, and p95 was absent from NBS cells established from

James P. Carney; Richard S. Maser; Heidi Olivares; Elizabeth M. Davis; Michelle Le Beau; John R. Yates; Lara Hays; William F. Morgan; John H. J. Petrini

1998-01-01

95

Print the story Watching DNA Repair in Real Time  

E-print Network

Print the story Watching DNA Repair in Real Time Direct observations of DNA are giving new insights into how genetic material is copied and repaired. "We can monitor the process directly, and that gives us of an enzyme called RecA attach themselves along a DNA strand, stretching it out and forming a filament

Kowalczykowski, Stephen C.

96

Comet assay to measure DNA repair: approach and applications  

PubMed Central

Cellular repair enzymes remove virtually all DNA damage before it is fixed; repair therefore plays a crucial role in preventing cancer. Repair studied at the level of transcription correlates poorly with enzyme activity, and so assays of phenotype are needed. In a biochemical approach, substrate nucleoids containing specific DNA lesions are incubated with cell extract; repair enzymes in the extract induce breaks at damage sites; and the breaks are measured with the comet assay. The nature of the substrate lesions defines the repair pathway to be studied. This in vitro DNA repair assay has been modified for use in animal tissues, specifically to study the effects of aging and nutritional intervention on repair. Recently, the assay was applied to different strains of Drosophila melanogaster proficient and deficient in DNA repair. Most applications of the repair assay have been in human biomonitoring. Individual DNA repair activity may be a marker of cancer susceptibility; alternatively, high repair activity may result from induction of repair enzymes by exposure to DNA-damaging agents. Studies to date have examined effects of environment, nutrition, lifestyle, and occupation, in addition to clinical investigations. PMID:25202323

Azqueta, Amaya; Slyskova, Jana; Langie, Sabine A. S.; O’Neill Gaivão, Isabel; Collins, Andrew

2014-01-01

97

DNA Computing Complexity Analysis Using DNA/DNA Hybridization Kinetics  

E-print Network

DNA Computing Complexity Analysis Using DNA/DNA Hybridization Kinetics Soo­Yong Shin 1 , Eun Jeong the complexity of DNA computing. The complexity of any computational algorithm is typically measured in terms of time and space. In DNA computing, the time complexity can be measured by the total reaction time

98

DNA repair and radiation sensitivity in mammalian cells  

SciTech Connect

Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

Chen, D.J.C.; Stackhouse, M. [Los Alamos National Lab., NM (United States); Chen, D.S. [Rochester Univ., NY (United States). Dept. of Radiation Oncology

1993-02-01

99

DNA repair and radiation sensitivity in mammalian cells  

SciTech Connect

Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

Chen, D.J.C.; Stackhouse, M. (Los Alamos National Lab., NM (United States)); Chen, D.S. (Rochester Univ., NY (United States). Dept. of Radiation Oncology)

1993-01-01

100

Androgen receptor signaling regulates DNA repair in prostate cancers  

PubMed Central

We demonstrate that the androgen receptor (AR) regulates a transcriptional program of DNA repair genes that promotes prostate cancer radioresistance, providing a potential mechanism by which androgen deprivation therapy (ADT) synergizes with ionizing radiation (IR). Using a model of castration-resistant prostate cancer, we show that second-generation antiandrogen therapy results in downregulation of DNA repair genes. Next, we demonstrate that primary prostate cancers display a significant spectrum of AR transcriptional output which correlates with expression of a set of DNA repair genes. Employing RNA-seq and ChIP-seq, we define which of these DNA repair genes are both induced by androgen and represent direct AR targets. We establish that prostate cancer cells treated with IR plus androgen demonstrate enhanced DNA repair and decreased DNA damage and furthermore that antiandrogen treatment causes increased DNA damage and decreased clonogenic survival. Finally, we demonstrate that antiandrogen treatment results in decreased classical non-homologous end joining. PMID:24027196

Polkinghorn, William R.; Parker, Joel S.; Lee, Man X.; Kass, Elizabeth M.; Spratt, Daniel E.; Iaquinta, Phillip J.; Arora, Vivek K.; Yen, Wei-Feng; Cai, Ling; Zheng, Deyou; Carver, Brett S.; Chen, Yu; Watson, Philip A.; Shah, Neel P.; Fujisawa, Sho; Goglia, Alexander G.; Gopalan, Anuradha; Hieronymus, Haley; Wongvipat, John; Scardino, Peter T.; Zelefsky, Michael J.; Jasin, Maria; Chaudhuri, Jayanta; Powell, Simon N.; Sawyers, Charles L.

2013-01-01

101

DNA-PK: a dynamic enzyme in a versatile DSB repair pathway  

PubMed Central

DNA double stranded breaks (DSBs) are the most cytoxic DNA lesion as the inability to properly repair them can lead to genomic instability and tumorigenesis. The prominent DSB repair pathway in humans is non-homologous end-joining (NHEJ). In the simplest sense, NHEJ mediates the direct re-ligation of the broken DNA molecule. However, NHEJ is a complex and versatile process that can repair DSBs with a variety of damages and ends via the utilization of a significant number of proteins. In this review we will describe the important factors and mechanisms modulating NHEJ with emphasis given to the versatility of this repair process and the DNA-PK complex. PMID:24680878

Davis, Anthony J.; Chen, Benjamin P.C.; Chen, David J.

2014-01-01

102

Structure of the DNA repair helicase XPD.  

PubMed

The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalyzing DNA duplex opening localized to the transcription start site or site of DNA damage, respectively. XPD has a 5' to 3' polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigmentosum, trichothiodystrophy, and Cockayne's syndrome, characterized by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 A crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans. PMID:18510925

Liu, Huanting; Rudolf, Jana; Johnson, Kenneth A; McMahon, Stephen A; Oke, Muse; Carter, Lester; McRobbie, Anne-Marie; Brown, Sara E; Naismith, James H; White, Malcolm F

2008-05-30

103

DNA repair. Mechanism of DNA interstrand cross-link processing by repair nuclease FAN1.  

PubMed

DNA interstrand cross-links (ICLs) are highly toxic lesions associated with cancer and degenerative diseases. ICLs can be repaired by the Fanconi anemia (FA) pathway and through FA-independent processes involving the FAN1 nuclease. In this work, FAN1-DNA crystal structures and biochemical data reveal that human FAN1 cleaves DNA successively at every third nucleotide. In vitro, this exonuclease mechanism allows FAN1 to excise an ICL from one strand through flanking incisions. DNA access requires a 5'-terminal phosphate anchor at a nick or a 1- or 2-nucleotide flap and is augmented by a 3' flap, suggesting that FAN1 action is coupled to DNA synthesis or recombination. FAN1's mechanism of ICL excision is well suited for processing other localized DNA adducts as well. PMID:25430771

Wang, Renjing; Persky, Nicole S; Yoo, Barney; Ouerfelli, Ouathek; Smogorzewska, Agata; Elledge, Stephen J; Pavletich, Nikola P

2014-11-28

104

DNA Repair and Genome Maintenance in Bacillus subtilis  

PubMed Central

Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis. PMID:22933559

Lenhart, Justin S.; Schroeder, Jeremy W.; Walsh, Brian W.

2012-01-01

105

DNA Computing Complexity Analysis Using DNA/DNA Hybridization Kinetics  

E-print Network

DNA Computing Complexity Analysis Using DNA/DNA Hybridization Kinetics Soo-Yong Shin1 , Eun Jeong of DNA computing. The complexity of any computational algorithm is typically measured in terms of time and space. In DNA computing, the time complexity can be measured by the total reaction time

106

DNA repair after X-irradiation: lessons from plants.  

PubMed

The effects of low-dose radiation causing DNA damage continue to be subjects of interest. Problems with existing approaches to low-dose DNA damage are that single-strand breaks (the predominant radiation-induced lesion) are very rapidly repaired and that results using current methods for measuring DNA damage can be difficult to interpret. As a novel approach, we conducted studies using plants (rye grass and the model plant Arabidopsis) exposed to X-rays and used the alkaline comet assay to measure DNA damage and repair after exposures. Consistent with previous studies, we detected so-called 'rapid' and 'slow' phases of DNA repair after acute exposures of 5 and 15 Gy. After exposures corresponding to 2 Gy and lower, 'rapid' repair was so fast that it was difficult to detect. We also found that the so-called 'slow' phase in both plants actually consisted of two components; an initial period of negligible repair lasting 80-120 min followed by a period of rapid repair lasting <30 min. Using Arabidopsis mutants homozygous for both ATM and BRCA1, we found that both of these genes are required for DNA repair during the 3-h period of our experiments, indicating that the 'slow' phase involves a homologous repair (HR) of double-strand breaks and clustered single-strand breaks. The lag of repair in the 'slow' phase presumably involves induction of expression of genes involved in HR repair such as BRCA1 and RAD51. PMID:25527727

Einset, John; Collins, Andrew R

2015-01-01

107

DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment  

PubMed Central

Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting. PMID:25099886

Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A.

2014-01-01

108

Formation and Repair of Tobacco Carcinogen-Derived Bulky DNA Adducts  

PubMed Central

DNA adducts play a central role in chemical carcinogenesis. The analysis of formation and repair of smoking-related DNA adducts remains particularly challenging as both smokers and nonsmokers exposed to smoke are repetitively under attack from complex mixtures of carcinogens such as polycyclic aromatic hydrocarbons and N-nitrosamines. The bulky DNA adducts, which usually have complex structure, are particularly important because of their biological relevance. Several known cellular DNA repair pathways have been known to operate in human cells on specific types of bulky DNA adducts, for example, nucleotide excision repair, base excision repair, and direct reversal involving O6-alkylguanine DNA alkyltransferase or AlkB homologs. Understanding the mechanisms of adduct formation and repair processes is critical for the assessment of cancer risk resulting from exposure to cigarette smoke, and ultimately for developing strategies of cancer prevention. This paper highlights the recent progress made in the areas concerning formation and repair of bulky DNA adducts in the context of tobacco carcinogen-associated genotoxic and carcinogenic effects. PMID:21234336

Hang, Bo

2010-01-01

109

DNA repair systems as targets of cadmium toxicity  

SciTech Connect

Cadmium (Cd) is a heavy metal and a potent carcinogen implicated in tumor development through occupational and environmental exposure. Recent evidence suggests that proteins participating in the DNA repair systems, especially in excision and mismatch repair, are sensitive targets of Cd toxicity. Cd by interfering and inhibiting these DNA repair processes might contribute to increased risk for tumor formation in humans. In the present review, the information available on the interference of Cd with DNA repair systems and their inhibition is summarized. These actions could possibly explain the indirect contribution of Cd to mutagenic effects and/or carcinogenicity.

Giaginis, Constantinos [Department of Forensic Medicine and Toxicology, University of Athens, Medical School, 75 M. Asias str., Goudi, GR11527 Athens (Greece); Gatzidou, Elisavet [Department of Forensic Medicine and Toxicology, University of Athens, Medical School, 75 M. Asias str., Goudi, GR11527 Athens (Greece); Theocharis, Stamatios [Department of Forensic Medicine and Toxicology, University of Athens, Medical School, 75 M. Asias str., Goudi, GR11527 Athens (Greece)]. E-mail: theocharis@ath.forthnet.gr

2006-06-15

110

Mismatch Repair Mutants in Yeast Are Not Defective in Transcription-Coupled DNA Repair of Uv-Induced DNA Damage  

PubMed Central

Transcription-coupled repair, the targeted repair of the transcribed strands of active genes, is defective in bacteria, yeast, and human cells carrying mutations in mfd, RAD26 and ERCC6, respectively. Other factors probably are also uniquely involved in transcription-repair coupling. Recently, a defect was described in transcription-coupled repair for Escherichia coli mismatch repair mutants and human tumor cell lines with mutations in mismatch repair genes. We examined removal of UV-induced DNA damage in yeast strains mutated in mismatch repair genes in an effort to confirm a defect in transcription-coupled repair in this system. In addition, we determined the contribution of the mismatch repair gene MSH2 to transcription-coupled repair in the absence of global genomic repair using rad7? mutants. We also determined whether the Rad26-independent transcription-coupled repair observed in rad26? and rad7? rad26? mutants depends on MSH2 by examining repair deficiencies of rad26? msh2? and rad7? rad26? msh2? mutants. We found no defects in transcription-coupled repair caused by mutations in the mismatch repair genes MSH2, MLH1, PMS1, and MSH3. Yeast appears to differ from bacteria and human cells in the capacity for transcription-coupled repair in a mismatch repair mutant background. PMID:8807287

Sweder, K. S.; Verhage, R. A.; Crowley, D. J.; Crouse, G. F.; Brouwer, J.; Hanawalt, P. C.

1996-01-01

111

DNA repair in microgravity: studies on bacteria and mammalian cells in the experiments REPAIR and KINETICS  

Microsoft Academic Search

The impact of microgravity on cellular repair processes was tested in the space experiments REPAIR and KINETICS, which were performed during the IML-2 mission in the Biorack of ESA: (a) survival of spores of Bacillus subtilis HA101 after UV-irradiation (up to 340 J m?2) in the experiment REPAIR; (b) in the experiment KINETICS the kinetics of DNA repair in three

G. Horneck; P. Rettberg; C. Baumstark-Khan; H. Rink; S. Kozubek; M. Schäfer; C. Schmitz

1996-01-01

112

Brca1 Controls Homology-Directed DNA Repair  

Microsoft Academic Search

Germline mutations in BRCA1 confer a high risk of breast and ovarian tumors. The role of BRCA1 in tumor suppression is not yet understood, but both transcription and repair functions have been ascribed. Evidence that BRCA1 is involved in DNA repair stems from its association with RAD51, a homolog of the yeast protein involved in the repair of DNA double-strand

Mary Ellen Moynahan; Joanne W Chiu; Beverly H Koller; Maria Jasin

1999-01-01

113

An Overview of DNA Repair in Amyotrophic Lateral Sclerosis  

PubMed Central

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is an adult onset neurodegenerative disorder characterised by the degeneration of cortical and spinal cord motor neurons, resulting in progressive muscular weakness and death. Increasing evidence supports mitochondrial dysfunction and oxidative DNA damage in ALS motor neurons. Several DNA repair enzymes are activated following DNA damage to restore genome integrity, and impairments in DNA repair capabilities could contribute to motor neuron degeneration. After a brief description of the evidence of DNA damage in ALS, this paper focuses on the available data on DNA repair activity in ALS neuronal tissue and disease animal models. Moreover, biochemical and genetic data on DNA repair in ALS are discussed in light of similar findings in other neurodegenerative diseases. PMID:22125427

Coppedè, Fabio

2011-01-01

114

Alkyltransferase-like proteins: Molecular switches between DNA repair pathways  

PubMed Central

Alkyltransferase-like proteins (ATLs) play a role in the protection of cells from the biological effects of DNA alkylation damage. Although ATLs share functional motifs with the DNA repair protein and cancer chemotherapy target O6-alkylguanine-DNA alkyltransferase, they lack the reactive cysteine residue required for alkyltransferase activity, so its mechanism for cell protection was previously unknown. Here, we review recent advances in unravelling the enigmatic cellular protection provided by ATLs against the deleterious effects of DNA alkylation damage. We discuss exciting new evidence that ATLs aid in the repair of DNA O6-alkylguanine lesions through a novel repair cross-talk between DNA-alkylation base damage responses and the DNA nucleotide excision repair pathway. PMID:20502938

Tubbs, Julie L.; Tainer, John A.

2011-01-01

115

DNA Polymerase ? Catalytic Domains Are Dispensable for DNA Replication, DNA Repair, and Cell Viability  

Microsoft Academic Search

DNA polymerase ? (Pol ?) is believed to play an essential catalytic role during eukaryotic DNA replication and is thought to participate in recombination and DNA repair. That Pol ? is essential for progression through S phase and for viability in budding and fission yeasts is a central element of support for that view. We show that the amino-terminal portion

Tapio Kesti; Karin Flick; Sirkka Keränen; Juhani E Syväoja; Curt Wittenberg

1999-01-01

116

Role of LrpC from Bacillus subtilis in DNA transactions during DNA repair and recombination  

PubMed Central

Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein, which binds both single-stranded (ss) and double-stranded (ds) DNA and facilitates the formation of higher order protein–DNA complexes in vitro. LrpC binds at different sites within the same DNA molecule promoting intramolecular ligation. When bound to separate molecules, it promotes intermolecular ligation, and joint molecule formation between a circular ssDNA and a homologous ssDNA-tailed linear dsDNA. LrpC binding showed a higher affinity for 4-way (Holliday) junctions in their open conformation, when compared with curved dsDNA. Consistent with these biochemical activities, an lrpC null mutant strain rendered cells sensitive to DNA damaging agents such as methyl methanesulfonate and 4-nitroquinoline-1-oxide, and showed a segregation defect. These findings collectively suggest that LrpC may be involved in DNA transactions during DNA repair and recombination. PMID:16407330

López-Torrejón, Gema; Martínez-Jiménez, María I.; Ayora, Silvia

2006-01-01

117

Supramolecular Complexes of DNA  

NASA Astrophysics Data System (ADS)

Deoxyribose nucleic acid or DNA is a linear polymer in the form of a double strand, synthesised by sequential polymerisation of a large number of units chosen from among the nucleic bases called purines (adenosine A and guanosine G) and pyrimidines (cytosine C and thymidine T). DNA contains all the genetic information required for life. It exists in the form of a limited number (a few dozen) of very big molecules, called chromosomes. This genetic information is first of all transcribed. In this process, a restricted fragment of the DNA called a gene is copied in the form of ribonucleic acid, or RNA. This RNA is itself a polymer, but with a single strand in which the sequence of nucleic acids is schematically analogous to the sequence on one of the two strands of the transcribed DNA. Finally, this RNA is translated into a protein, yet another linear polymer. The proteins make up the main part of the active constituents ensuring the survival of the cell. Any loss of information, either by mutation or by deletion of the DNA, will cause an imbalance in the cell's metabolism that may in turn lead to incurable pathologies. Several strategies have been developed to reduce the consequences of such genetic deficiencies or, more generally, to act, by amplifying or suppressing them, on the mechanisms leading from the reading of the genetic information to the production of proteins: Strategies aiming to introduce synthetic DNA or RNA, which selectively block the expression of certain genes, are now being studied by an increasing number of research scientists and pharmacologists. They use antisense oligodeoxyribonucleotides or interfering oligoribonucleotides and they already have clinical applications. This kind of therapy is often called gene pharmacology. Other, more ambitious strategies aim to repair in situ mutated or incomplete DNA within the chromosomes themselves, by introducing short sequences of DNA or RNA which recognise and take the place of mutations. This is the underlying principle of genetic correction. Yet other strategies aim to reintroduce the deficient DNA fragments into the cells in the form of genes. Indeed, in certain diseases, the only solution is to bring genetic information back into the cells by transferring exogeneous DNA into the cell nucleus. This approach goes by the name of gene therapy.

Zuber, G.; Scherman, D.

118

Glyceraldehyde-3-phosphate dehydrogenase is required for efficient repair of cytotoxic DNA lesions in Escherichia coli.  

PubMed

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with diverse biological functions in human cells. In bacteria, moonlighting GAPDH functions have only been described for the secreted protein in pathogens or probiotics. At the intracellular level, we previously reported the interaction of Escherichia coli GAPDH with phosphoglycolate phosphatase, a protein involved in the metabolism of the DNA repair product 2-phosphoglycolate, thus suggesting a putative role of GAPDH in DNA repair processes. Here, we provide evidence that GAPDH is required for the efficient repair of DNA lesions in E. coli. We show that GAPDH-deficient cells are more sensitive to bleomycin or methyl methanesulfonate. In cells challenged with these genotoxic agents, GAPDH deficiency results in reduced cell viability and filamentous growth. In addition, the gapA knockout mutant accumulates a higher number of spontaneous abasic sites and displays higher spontaneous mutation frequencies than the parental strain. Pull-down experiments in different genetic backgrounds show interaction between GAPDH and enzymes of the base excision repair pathway, namely the AP-endonuclease Endo IV and uracil DNA glycosylase. This finding suggests that GAPDH is a component of a protein complex dedicated to the maintenance of genomic DNA integrity. Our results also show interaction of GAPDH with the single-stranded DNA binding protein. This interaction may recruit GAPDH to the repair sites and implicates GAPDH in DNA repair pathways activated by profuse DNA damage, such as homologous recombination or the SOS response. PMID:25603270

Ferreira, Elaine; Giménez, Rosa; Cañas, María Alexandra; Aguilera, Laura; Aguilar, Juan; Badia, Josefa; Baldomà, Laura

2015-03-01

119

Targeting abnormal DNA double strand break repair in cancer  

PubMed Central

A major challenge in cancer treatment is the development of therapies that target cancer cells with little or no toxicity to normal tissues and cells. Alterations in DNA double strand break (DSB) repair in cancer cells include both elevated and reduced levels of key repair proteins and changes in the relative contributions of the various DSB repair pathways. These differences can result in increased sensitivity to DSB-inducing agents and increased genomic instability. The development of agents that selectively inhibit the DSB repair pathways that cancer cells are more dependent upon will facilitate the design of therapeutic strategies that exploit the differences in DSB repair between normal and cancer cells. Here, we discuss the pathways of DSB repair, alterations in DSB repair in cancer, inhibitors of DSB repair and future directions for cancer therapies that target DSB repair. PMID:20697770

Rassool, Feyruz V.

2010-01-01

120

Repair of endogenous DNA base lesions modulate lifespan in mice.  

PubMed

The accumulation of DNA damage is thought to contribute to the physiological decay associated with the aging process. Here, we report the results of a large-scale study examining longevity in various mouse models defective in the repair of DNA alkylation damage, or defective in the DNA damage response. We find that the repair of spontaneous DNA damage by alkyladenine DNA glycosylase (Aag/Mpg)-initiated base excision repair and O(6)-methylguanine DNA methyltransferase (Mgmt)-mediated direct reversal contributes to maximum life span in the laboratory mouse. We also uncovered important genetic interactions between Aag, which excises a wide variety of damaged DNA bases, and the DNA damage sensor and signaling protein, Atm. We show that Atm plays a role in mediating survival in the face of both spontaneous and induced DNA damage, and that Aag deficiency not only promotes overall survival, but also alters the tumor spectrum in Atm(-/-) mice. Further, the reversal of spontaneous alkylation damage by Mgmt interacts with the DNA mismatch repair pathway to modulate survival and tumor spectrum. Since these aging studies were performed without treatment with DNA damaging agents, our results indicate that the DNA damage that is generated endogenously accumulates with age, and that DNA alkylation repair proteins play a role in influencing longevity. PMID:24994062

Meira, Lisiane B; Calvo, Jennifer A; Shah, Dharini; Klapacz, Joanna; Moroski-Erkul, Catherine A; Bronson, Roderick T; Samson, Leona D

2014-09-01

121

Structural Insights Into DNA Repair by RNase T—An Exonuclease Processing 3? End of Structured DNA in Repair Pathways  

PubMed Central

DNA repair mechanisms are essential for preservation of genome integrity. However, it is not clear how DNA are selected and processed at broken ends by exonucleases during repair pathways. Here we show that the DnaQ-like exonuclease RNase T is critical for Escherichia coli resistance to various DNA-damaging agents and UV radiation. RNase T specifically trims the 3? end of structured DNA, including bulge, bubble, and Y-structured DNA, and it can work with Endonuclease V to restore the deaminated base in an inosine-containing heteroduplex DNA. Crystal structure analyses further reveal how RNase T recognizes the bulge DNA by inserting a phenylalanine into the bulge, and as a result the 3? end of blunt-end bulge DNA can be digested by RNase T. In contrast, the homodimeric RNase T interacts with the Y-structured DNA by a different binding mode via a single protomer so that the 3? overhang of the Y-structured DNA can be trimmed closely to the duplex region. Our data suggest that RNase T likely processes bulge and bubble DNA in the Endonuclease V–dependent DNA repair, whereas it processes Y-structured DNA in UV-induced and various other DNA repair pathways. This study thus provides mechanistic insights for RNase T and thousands of DnaQ-like exonucleases in DNA 3?-end processing. PMID:24594808

Hsiao, Yu-Yuan; Fang, Woei-Horng; Lee, Chia-Chia; Chen, Yi-Ping; Yuan, Hanna S.

2014-01-01

122

Spatiotemporal dynamics of DNA repair proteins following laser microbeam induced DNA damage – When is a DSB not a DSB??  

PubMed Central

The formation of DNA lesions poses a constant threat to cellular stability. Repair of endogenously and exogenously produced lesions has therefore been extensively studied, although the spatiotemporal dynamics of the repair processes has yet to be fully understood. One of the most recent advances to study the kinetics of DNA repair has been the development of laser microbeams to induce and visualize recruitment and loss of repair proteins to base damage in live mammalian cells. However, a number of studies have produced contradictory results that are likely caused by the different laser systems used reflecting in part the wavelength dependence of the damage induced. Additionally, the repair kinetics of laser microbeam induced DNA lesions have generally lacked consideration of the structural and chemical complexity of the DNA damage sites, which are known to greatly influence their reparability. In this review, we highlight the key considerations when embarking on laser microbeam experiments and interpreting the real time data from laser microbeam irradiations. We compare the repair kinetics from live cell imaging with biochemical and direct quantitative cellular measurements for DNA repair. PMID:23688615

Reynolds, Pamela; Botchway, Stanley W.; Parker, Anthony W.; O’Neill, Peter

2013-01-01

123

DNA repair in cancer: emerging targets for personalized therapy  

PubMed Central

Genomic deoxyribonucleic acid (DNA) is under constant threat from endogenous and exogenous DNA damaging agents. Mammalian cells have evolved highly conserved DNA repair machinery to process DNA damage and maintain genomic integrity. Impaired DNA repair is a major driver for carcinogenesis and could promote aggressive cancer biology. Interestingly, in established tumors, DNA repair activity is required to counteract oxidative DNA damage that is prevalent in the tumor microenvironment. Emerging clinical data provide compelling evidence that overexpression of DNA repair factors may have prognostic and predictive significance in patients. More recently, DNA repair inhibition has emerged as a promising target for anticancer therapy. Synthetic lethality exploits intergene relationships where the loss of function of either of two related genes is nonlethal, but loss of both causes cell death. Exploiting this approach by targeting DNA repair has emerged as a promising strategy for personalized cancer therapy. In the current review, we focus on recent advances with a particular focus on synthetic lethality targeting in cancer. PMID:24600246

Abbotts, Rachel; Thompson, Nicola; Madhusudan, Srinivasan

2014-01-01

124

Mitochondrial DNA repair and association with aging - an update  

PubMed Central

Mitochondrial DNA is constantly exposed to oxidative injury. Due to its location close to the main site of reactive oxygen species, the inner mitochondrial membrane, mtDNA is more susceptible than nuclear DNA to oxidative damage. The accumulation of DNA damage is thought to play a critical role in the aging process and to be particularly deleterious in post-mitotic cells. Thus, DNA repair is an important mechanism for maintenance of genomic integrity. Despite the importance of mitochondria in the aging process, it was thought for many years that mitochondria lacked an enzymatic DNA repair system comparable to that in the nuclear compartment. However, it is now well established that DNA repair actively takes place in mitochondria. Oxidative DNA damage processing, base excision repair mechanisms were the first to be described in these organelles, and consequently the best understood. However, new proteins and novel DNA repair pathways, thought to be exclusively present in the nucleus, have recently been described also to be present in mitochondria. Here we review the main mitochondrial DNA repair pathways and their association with the aging process. PMID:20096766

Gredilla, Ricardo; Bohr, Vilhelm A.; Stevnsner, Tinna

2010-01-01

125

DNA repair in cancer: emerging targets for personalized therapy.  

PubMed

Genomic deoxyribonucleic acid (DNA) is under constant threat from endogenous and exogenous DNA damaging agents. Mammalian cells have evolved highly conserved DNA repair machinery to process DNA damage and maintain genomic integrity. Impaired DNA repair is a major driver for carcinogenesis and could promote aggressive cancer biology. Interestingly, in established tumors, DNA repair activity is required to counteract oxidative DNA damage that is prevalent in the tumor microenvironment. Emerging clinical data provide compelling evidence that overexpression of DNA repair factors may have prognostic and predictive significance in patients. More recently, DNA repair inhibition has emerged as a promising target for anticancer therapy. Synthetic lethality exploits intergene relationships where the loss of function of either of two related genes is nonlethal, but loss of both causes cell death. Exploiting this approach by targeting DNA repair has emerged as a promising strategy for personalized cancer therapy. In the current review, we focus on recent advances with a particular focus on synthetic lethality targeting in cancer. PMID:24600246

Abbotts, Rachel; Thompson, Nicola; Madhusudan, Srinivasan

2014-01-01

126

Probing the Structure and Function of the Escherichia coli DNA Alkylation Repair AlkB Protein through Chemical Cross-Linking  

E-print Network

that relies on protein/DNA interactions, enables us to trap and isolate homogeneous AlkB/DNA complexesProbing the Structure and Function of the Escherichia coli DNA Alkylation Repair AlkB Protein@uchicago.edu The Escherichia coli AlkB protein has been known to play an important role in alkylated DNA damage repair;1

He, Chuan

127

Repair of DNA breaks after irradiation in misonidazole or oxygen  

SciTech Connect

Biphasic kinetics (half-times 7' and 250') adequately describe the aerobic repair of DNA breaks after irradiation of mammalian cells. The proportions repaired by the two components are affected by conditions prior to, during and after irradiation. DNA of cells irradiated in hypoxia have approximately twice as much of the damage which is repaired by the slow component as cells irradiated in air. If, instead of oxygen, misonidazole is present during hypoxic radiation, the repair resembles the repair of oxic damage more closely than repair of hypoxic damage. The biphasic nature of the repair curves is interpreted to be due to two classes of initial damage, proportions of which can be altered by sensitizers such as O/sub 2/ and misonidazole.

Skov, K.A.

1984-08-01

128

DNA repair: Dynamic defenders against cancer and aging  

SciTech Connect

You probably weren't thinking about your body's cellular DNA repair systems the last time you sat on the beach in the bright sunshine. Fortunately, however, while you were subjecting your DNA to the harmful effects of ultraviolet light, your cells were busy repairing the damage. The idea that our genetic material could be damaged by the sun was not appreciated in the early days of molecular biology. When Watson and Crick discovered the structure of DNA in 1953 [1], it was assumed that DNA is fundamentally stable since it carries the blueprint of life. However, over 50 years of research have revealed that our DNA is under constant assault by sunlight, oxygen, radiation, various chemicals, and even our own cellular processes. Cleverly, evolution has provided our cells with a diverse set of tools to repair the damage that Mother Nature causes. DNA repair processes restore the normal nucleotide sequence and DNA structure of the genome after damage [2]. These responses are highly varied and exquisitely regulated. DNA repair mechanisms are traditionally characterized by the type of damage repaired. A large variety of chemical modifications can alter normal DNA bases and either lead to mutations or block transcription if not repaired, and three distinct pathways exist to remove base damage. Base excision repair (BER) corrects DNA base alterations that do not distort the overall structure of the DNA helix such as bases damaged by oxidation resulting from normal cellular metabolism. While BER removes single damaged bases, nucleotide excision repair (NER) removes short segments of nucleotides (called oligonucleotides) containing damaged bases. NER responds to any alteration that distorts the DNA helix and is the mechanism responsible for repairing bulky base damage caused by carcinogenic chemicals such as benzo [a]pyrene (found in cigarette smoke and automobile exhaust) as well as covalent linkages between adjacent pyrimidine bases resulting from the ultraviolet (UV) component of sunlight. NER can be divided into two classes based on where the repair occurs. NER occurring in DNA that is not undergoing transcription (i.e., most of the genome) is called global genome repair (GGR or GGNER), while NER taking place in the transcribed strand of active genes is called transcription-coupled repair (TCR or TC-NER). We will explore NER in more detail below. Mismatch repair (MMR) is another type of excision repair that specifically removes mispaired bases resulting from replication errors. DNA damage can also result in breaks in the DNA backbone, in one or both strands. Single-strand breaks (SSBs) are efficiently repaired by a mechanism that shares common features with the later steps in BER. Double-strand breaks (DSBs) are especially devastating since by definition there is no intact complementary strand to serve as a template for repair, and even one unrepaired DSB can be lethal [3]. In cells that have replicated their DNA prior to cell division, the missing information can be supplied by the duplicate copy, or sister chromatid, and DSBs in these cells are faithfully repaired by homologous recombination involving the exchange of strands of DNA between the two copies. However, most cells in the body are non-dividing, and in these cells the major mechanism for repairing DSBs is by non-homologous end joining (NHEJ), which as the name implies involves joining two broken DNA ends together without a requirement for homologous sequence and which therefore has a high potential for loss of genetic information.

Fuss, Jill O.; Cooper, Priscilla K.

2006-04-01

129

Distinct structural alterations in PCNA block DNA mismatch repair  

PubMed Central

During DNA replication, mismatches and small loops in the DNA resulting from insertions or deletions are repaired by the mismatch repair (MMR) machinery. Proliferating cell nuclear antigen (PCNA) plays an important role in both mismatch-recognition and resynthesis stages of MMR. Previously, two mutant forms of PCNA were identified that cause defects in MMR with little, if any, other defects. The C22Y mutant PCNA protein completely blocks MutS?-dependent MMR, and the C81R mutant PCNA protein partially blocks both MutS?-dependent and MutS?-dependent MMR. In order to understand the structural and mechanistic basis by which these two amino acid substitutions in PCNA proteins block MMR, we solved the X-ray crystal structures of both mutant proteins and carried out further biochemical studies. We found that these amino acid substitutions lead to subtle, distinct structural changes in PCNA. The C22Y substitution alters the positions of the ?-helices lining the central hole of the PCNA ring, whereas the C81R substitution creates a distortion in an extended loop near the PCNA subunit interface. We conclude that the structural integrity of the ?-helices lining the central hole and this loop are both necessary to form productive complexes with MutS ? and mismatch-containing DNA. PMID:23869605

Dieckman, Lynne M.; Boehm, Elizabeth M.; Hingorani, Manju M.; Washington, M. Todd

2013-01-01

130

Repair of DNA-polypeptide crosslinks by human excision nuclease  

NASA Astrophysics Data System (ADS)

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome. damage recognition | nucleotide excision repair

Reardon, Joyce T.; Sancar, Aziz

2006-03-01

131

Situation-dependent repair of DNA damage in yeast  

SciTech Connect

The concept of channelling of lesions in DNA into defined repair systems has been used to explain many aspects of induced and spontaneous mutation. The channelling hypothesis states that lesions excluded from one repair process will be taken up by another repair process. This is a simplification. The three known modes of repair of damage induced by radiation are not equivalent modes of repair; they are, instead, different solutions to the problem of replacement of damaged molecules with new molecules which have the same informational content as those that were damaged. The mode of repair that is used is the result of the response to the situation in which the damage takes place. Thus, when the most likely mode of repair does not take place, then the situation changes with respect to the repair of the lesion; the lesion may enter the replication fork and be reparable by another route.

von Borstel, R.C.; Hastings, P.J.

1985-01-01

132

DNA repair endonuclease ERCC1-XPF as a novel therapeutic target to overcome chemoresistance in cancer therapy.  

PubMed

The ERCC1-XPF complex is a structure-specific endonuclease essential for the repair of DNA damage by the nucleotide excision repair pathway. It is also involved in other key cellular processes, including DNA interstrand crosslink (ICL) repair and DNA double-strand break (DSB) repair. New evidence has recently emerged, increasing our understanding of its requirement in these additional roles. In this review, we focus on the protein-protein and protein-DNA interactions made by the ERCC1 and XPF proteins and discuss how these coordinate ERCC1-XPF in its various roles. In a number of different cancers, high expression of ERCC1 has been linked to a poor response to platinum-based chemotherapy. We discuss prospects for the development of DNA repair inhibitors that target the activity, stability or protein interactions of the ERCC1-XPF complex as a novel therapeutic strategy to overcome chemoresistance. PMID:22941649

McNeil, Ewan M; Melton, David W

2012-11-01

133

The Rad9 protein enhances survival and promotes DNA repair following exposure to ionizing radiation  

SciTech Connect

Following DNA damage cells initiate cell cycle checkpoints to allow time to repair sustained lesions. Rad9, Rad1, and Hus1 proteins form a toroidal complex, termed the 9-1-1 complex, that is involved in checkpoint signaling. 9-1-1 shares high structural similarity to the DNA replication protein proliferating cell nuclear antigen (PCNA) and 9-1-1 has been shown in vitro to stimulate steps of the repair process known as long patch base excision repair. Using a system that allows conditional repression of the Rad9 protein in human cell culture, we show that Rad9, and by extension, the 9-1-1 complex, enhances cell survival, is required for efficient exit from G2-phase arrest, and stimulates the repair of damaged DNA following ionizing radiation. These data provide in vivo evidence that the human 9-1-1 complex participates in DNA repair in addition to its previously described role in DNA damage sensing.

Brandt, Patrick D. [Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642 (United States); Helt, Christopher E. [Department of Radiation Oncology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642 (United States); Keng, Peter C. [Department of Radiation Oncology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642 (United States); Bambara, Robert A. [Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642 (United States)]. E-mail: robert_bambara@urmc.rochester.edu

2006-08-18

134

Transcription bypass of DNA lesions enhances cell survival but attenuates transcription coupled DNA repair.  

PubMed

Transcription-coupled DNA repair (TCR) is a subpathway of nucleotide excision repair (NER) dedicated to rapid removal of DNA lesions in the transcribed strand of actively transcribed genes. The precise nature of the TCR signal and how the repair machinery gains access to lesions imbedded in stalled RNA polymerase II (RNAP II) complexes in eukaryotic cells are still enigmatic. RNAP II has an intrinsic capacity for transcription bypass of DNA lesions by incorporation or misincorporation of nucleotides across the lesions. It has been suggested that transcription bypass of lesions, which exposes the lesions, may be required for TCR. Here, we show that E1103G mutation of Rpb1, the largest subunit of RNAP II, which promotes transcription bypass of UV-induced cyclobutane pyrimidine dimers (CPDs), increases survival of UV irradiated yeast cells but attenuates TCR. The increased cell survival is independent of any NER subpathways. In contrast, G730D mutation of Rpb1, which impairs transcription bypass of CPDs, enhances TCR. Our results suggest that transcription bypass of lesions attenuates TCR but enhances cell tolerance to DNA lesions. Efficient stalling of RNAP II is essential for efficient TCR. PMID:25389266

Li, Wentao; Selvam, Kathiresan; Ko, Tengyu; Li, Shisheng

2014-12-01

135

Transcription bypass of DNA lesions enhances cell survival but attenuates transcription coupled DNA repair  

PubMed Central

Transcription-coupled DNA repair (TCR) is a subpathway of nucleotide excision repair (NER) dedicated to rapid removal of DNA lesions in the transcribed strand of actively transcribed genes. The precise nature of the TCR signal and how the repair machinery gains access to lesions imbedded in stalled RNA polymerase II (RNAP II) complexes in eukaryotic cells are still enigmatic. RNAP II has an intrinsic capacity for transcription bypass of DNA lesions by incorporation or misincorporation of nucleotides across the lesions. It has been suggested that transcription bypass of lesions, which exposes the lesions, may be required for TCR. Here, we show that E1103G mutation of Rpb1, the largest subunit of RNAP II, which promotes transcription bypass of UV-induced cyclobutane pyrimidine dimers (CPDs), increases survival of UV irradiated yeast cells but attenuates TCR. The increased cell survival is independent of any NER subpathways. In contrast, G730D mutation of Rpb1, which impairs transcription bypass of CPDs, enhances TCR. Our results suggest that transcription bypass of lesions attenuates TCR but enhances cell tolerance to DNA lesions. Efficient stalling of RNAP II is essential for efficient TCR. PMID:25389266

Li, Wentao; Selvam, Kathiresan; Ko, Tengyu; Li, Shisheng

2014-01-01

136

Fanconi Anemia: A Signal Transduction and DNA Repair Pathway  

PubMed Central

Fanconi anemia (FA) is a fascinating, rare genetic disorder marked by congenital defects, bone marrow failure, and cancer susceptibility. Research in recent years has led to the elucidation of FA as a DNA repair disorder and involved multiple pathways as well as having wide applicability to common cancers, including breast, ovarian, and head and neck. This review will describe the clinical aspects of FA as well as the current state of its molecular pathophysiology. In particular, work from the Kupfer laboratory will be described that demonstrates how the FA pathway interacts with multiple DNA repair pathways, including the mismatch repair system and signal transduction pathway of the DNA damage response. PMID:24348213

Kupfer, Gary M.

2013-01-01

137

Poly-ADP-ribosylation signaling during DNA damage repair.  

PubMed

Poly-ADP-ribosylation is a post-translational modification generated in high amounts by poly-ADP-ribose polymerases (PARPs) in response to cellular stress, especially genotoxic stimuli. DNA damage-induced PARylation significantly changes local chromatin structure and triggers the accumulation of several DNA damage response (DDR) proteins at the DNA lesions. In this review, we will discuss the regulation of chromatin structure and DNA damage repair machineries by DNA damage-induced poly-ADP-ribosylation. PMID:25553460

Golia, Barbara; Singh, Hari R; Timinszky, Gyula

2015-01-01

138

A review of DNA repair and possible DNA-repair adjuvants and selected natural anti-oxidants.  

PubMed

Few other organs have the environmental exposure-neoplasia relationship that has been observed between epithelial cutaneous malignancy and UVB exposure. A significant DNA type of defective linking of DNA nucleotides involves pyrimidine dimers. Important insight into the molecular processes that affect the response of cells to UVB have been provided by the study of rare inherited diseases characterized by DNA repair defects. Nucleotide excision repair is the best characterized of these and its importance is illustrated by the disease, xeroderma pigmentosum. This heterogenous disorder clinically characterized by malignant tumor development and molecularly by distinct alterations in the nucleotide excision repair apparatus. More recently, other DNA mechanisms have been shown to have some role in skin cancer, such as DNA-mismatch repair and double-stranded DNA breaks. Herein, we discuss the DNA-repair adjuvants a aqueous extract of Urcaria tomentosa (AC-11, Optigenex, Inc.), and T4 endonuclease V that is prepared in a liposome lotion (Dimericine, Applied Genetics Inc. Dermatics). The positive effects on the integrity DNA of other substances (from nature, heat shock proteins and cytokines) including IL-12, Polypodium leucotomos, and ubiquitin are also reviewed. Understanding DNA repair mechanisms is far from complete; further understanding will provide insight into the pathogenesis of cancer and pave the way for efficacious therapeutic agents. PMID:18328204

Emanuel, Patrick; Scheinfeld, Noah

2007-01-01

139

The role of DNA repair in brain related disease pathology  

PubMed Central

Oxidative DNA damage is implicated in brain aging, neurodegeneration and neurological diseases. Damage can be created by normal cellular metabolism, which accumulates with age, or by acute cellular stress conditions which create bursts of oxidative damage. Brain cells have a particularly high basal level of metabolic activity and use distinct oxidative damage repair mechanisms to remove oxidative damage from DNA and dNTP pools. Accumulation of this damage in the background of a functional DNA repair response is associated with normal aging, but defective repair in brain cells can contribute to neurological dysfunction. Emerging research strongly associates three common neurodegenerative conditions, Alzheimer’s, Parkinson’s and stroke, with defects in the ability to repair chronic or acute oxidative damage in neurons. This review explores the current knowledge of the role of oxidative damage repair in preserving brain function and highlights the emerging models and methods being used to advance our knowledge of the pathology of neurodegenerative disease. PMID:23721970

Canugovi, Chandrika; Misiak, Magdalena; Ferarelli, Leslie K.; Croteau, Deborah L.; Bohr, Vilhelm A.

2013-01-01

140

Small RNAs: emerging key players in DNA double-strand break repair.  

PubMed

DNA double-strand break (DSB) is the most deleterious form of DNA damage and poses great threat to genome stability. Eukaryotes have evolved complex mechanisms to repair DSBs through coordinated actions of protein sensors, transducers, and effectors. DSB-induced small RNAs (diRNAs) or Dicer/Drosha-dependent RNAs (DDRNAs) have been recently discovered in plants and vertebrates, adding an unsuspected RNA component into the DSB repair pathway. DiRNAs/DDRNAs control DNA damage response (DDR) activation by affecting DDR foci formation and cell cycle checkpoint enforcement and are required for efficient DSB repair. Here, we summarize the findings of diRNAs/DDRNAs and discuss the possible mechanisms through which they act to facilitate DSB repair. PMID:24026293

Ba, Zhaoqing; Qi, Yijun

2013-10-01

141

Single cell visualization of DNA repair in vivo.  

NASA Astrophysics Data System (ADS)

The creation of a DNA double-strand-break constitutes the most dangerous type of DNA damage. Inefficient response to DNA damage may lead to hypersensitivity to cellular stressors, susceptibility to genomic defects and resistance to apoptosis, which can lead to cancer. Current research on DNA repair has enabled numerous breakthroughs in our understanding of the DNA repair mechanisms at the population level. However, similar understanding at the level of single cells has been lacking mainly because of two reasons: 1) population level measurements do not visualize the repair process and therefore the exact mechanism by which the donor and recipient sequences are brought together is not well understood. 2) they are only sensitive to the mean of a distribution and usually hide the cell-to-cell variability of the repair processes. In my lab we utilize a multidisciplinary approach to address specific aspects of the DNA repair at the single cell level. By tagging several locations on DNA, its dynamic is visualized. furthermore the exact timing of the repair process is measured. In our experiments, individual cells are followed over long periods of time and many cellular generations in a microfluidic device, in which a precise control of the microenvironment of the cells is possible

Samadani, Azadeh; Rowat, Amy; Makridakis, Jennifer; Haber, James

2008-03-01

142

Effect of metals on mutagenesis and DNA repair.  

PubMed Central

Unlike the situation with organic compounds, metals do not show a high correlation between carcinogenicity and mutagenicity. An agent may be mutagenic by causing misreplication of DNA due to alterations of the DNA template, decreased fidelity of DNA polymerase, or inhibition of the proofreading of DNA replication. In addition, bacteria have an inducible, error-prone DNA repair system (SOS repair) whose activity results in mutagenesis. In the best studied example of metal mutagenesis, chromate, there is little evidence for the involvement of the SOS system. Metals may act as comutagens by inhibiting the repair of damage to DNA caused by another agent. This has been demonstrated for arsenite. Comutagens would not be detected by standard screening methods. PMID:7274183

Rossman, T G

1981-01-01

143

Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay  

PubMed Central

Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss, and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionizing radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (?20–50%) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumor suppressor protein ARF, and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase ?, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A–DDB1–STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair capacity. PMID:25076968

Nickson, Catherine M.; Parsons, Jason L.

2014-01-01

144

D-ribose inhibits DNA repair synthesis in human lymphocytes  

SciTech Connect

D-ribose is cytotoxic for quiescent human lymphocytes and severely inhibits their PHA-induced proliferation at concentrations (25-50 mM) at which other simple sugars are ineffective. In order to explain these effects, DNA repair synthesis was evaluated in PHA-stimulated human lymphocytes treated with hydroxyurea and irradiated. D-ribose, in contrast to other reducing sugars, did not induce repair synthesis and therefore did not apparently damage DNA in a direct way, although it markedly inhibited gamma ray-induced repair. Taking into account that lymphocytes must rejoin physiologically-formed DNA strand breaks in order to enter the cell cycle, we suggest that D-ribose exerts its cytotoxic activity by interfering with metabolic pathways critical for the repair of DNA breaks.

Zunica, G.; Marini, M.; Brunelli, M.A.; Chiricolo, M.; Franceschi, C.

1986-07-31

145

MicroRNAs in the DNA Damage/Repair Network and Cancer  

PubMed Central

Cancer is a multistep process characterized by various and different genetic lesions which cause the transformation of normal cells into tumor cells. To preserve the genomic integrity, eukaryotic cells need a complex DNA damage/repair response network of signaling pathways, involving many proteins, able to induce cell cycle arrest, apoptosis, or DNA repair. Chemotherapy and/or radiation therapy are the most commonly used therapeutic approaches to manage cancer and act mainly through the induction of DNA damage. Impairment in the DNA repair proteins, which physiologically protect cells from persistent DNA injury, can affect the efficacy of cancer therapies. Recently, increasing evidence has suggested that microRNAs take actively part in the regulation of the DNA damage/repair network. MicroRNAs are endogenous short noncoding molecules able to regulate gene expression at the post-transcriptional level. Due to their activity, microRNAs play a role in many fundamental physiological and pathological processes. In this review we report and discuss the role of microRNAs in the DNA damage/repair and cancer. PMID:24616890

Tessitore, Alessandra; Cicciarelli, Germana; Del Vecchio, Filippo; Gaggiano, Agata; Verzella, Daniela; Fischietti, Mariafausta; Vecchiotti, Davide; Capece, Daria; Zazzeroni, Francesca; Alesse, Edoardo

2014-01-01

146

[Ionizing radiation-induced DNA damage and its repair in human cells]. Progress report, [April 1, 1993--February 28, 1994  

SciTech Connect

The excision of radiation-induced lesions in DNA by a DNA repair enzyme complex, namely the UvrABC nuclease complex, has been investigated. Irradiated DNA was treated with the enzyme complex. DNA fractions were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results showed that a number pyrimidine- and purine-derived lesions in DNA were excised by the UvrABC nuclease complex and that the enzyme complex does not act on radiation-induced DNA lesions as a glycosylase. This means that it does not excise individual base products, but it excises oligomers containing these lesions. A number of pyrimidine-derived lesions that were no substrates for other DNA repair enzymes investigated in our laboratory were substrates for the UvrABC nuclease complex.

Not Available

1994-07-01

147

Transcript-RNA-templated DNA recombination and repair.  

PubMed

Homologous recombination is a molecular process that has multiple important roles in DNA metabolism, both for DNA repair and genetic variation in all forms of life. Generally, homologous recombination involves the exchange of genetic information between two identical or nearly identical DNA molecules; however, homologous recombination can also occur between RNA molecules, as shown for RNA viruses. Previous research showed that synthetic RNA oligonucleotides can act as templates for DNA double-strand break (DSB) repair in yeast and human cells, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements. Here we report that endogenous transcript RNA mediates homologous recombination with chromosomal DNA in yeast Saccharomyces cerevisiae. We developed a system to detect the events of homologous recombination initiated by transcript RNA following the repair of a chromosomal DSB occurring either in a homologous but remote locus, or in the same transcript-generating locus in reverse-transcription-defective yeast strains. We found that RNA-DNA recombination is blocked by ribonucleases H1 and H2. In the presence of H-type ribonucleases, DSB repair proceeds through a complementary DNA intermediate, whereas in their absence, it proceeds directly through RNA. The proximity of the transcript to its chromosomal DNA partner in the same locus facilitates Rad52-driven homologous recombination during DSB repair. We demonstrate that yeast and human Rad52 proteins efficiently catalyse annealing of RNA to a DSB-like DNA end in vitro. Our results reveal a novel mechanism of homologous recombination and DNA repair in which transcript RNA is used as a template for DSB repair. Thus, considering the abundance of RNA transcripts in cells, RNA may have a marked impact on genomic stability and plasticity. PMID:25186730

Keskin, Havva; Shen, Ying; Huang, Fei; Patel, Mikir; Yang, Taehwan; Ashley, Katie; Mazin, Alexander V; Storici, Francesca

2014-11-20

148

Role of Nicotinamide in DNA Damage, Mutagenesis, and DNA Repair  

PubMed Central

Nicotinamide is a water-soluble amide form of niacin (nicotinic acid or vitamin B3). Both niacin and nicotinamide are widely available in plant and animal foods, and niacin can also be endogenously synthesized in the liver from dietary tryptophan. Nicotinamide is also commercially available in vitamin supplements and in a range of cosmetic, hair, and skin preparations. Nicotinamide is the primary precursor of nicotinamide adenine dinucleotide (NAD+), an essential coenzyme in ATP production and the sole substrate of the nuclear enzyme poly-ADP-ribose polymerase-1 (PARP-1). Numerous in vitro and in vivo studies have clearly shown that PARP-1 and NAD+ status influence cellular responses to genotoxicity which can lead to mutagenesis and cancer formation. This paper will examine the role of nicotinamide in the protection from carcinogenesis, DNA repair, and maintenance of genomic stability. PMID:20725615

Surjana, Devita; Halliday, Gary M.; Damian, Diona L.

2010-01-01

149

Essential role of ?-human 8-oxoguanine DNA glycosylase 1 in mitochondrial oxidative DNA repair.  

PubMed

8-Oxoguanine (8-OG) is the major mutagenic base lesion in DNA caused by reactive oxygen species (ROS) and accumulates in both nuclear and mitochondrial DNA (mtDNA). In humans, 8-OG is primarily removed by human 8-OG DNA glycosylase 1 (hOGG1) through the base excision repair (BER) pathway. There are two major hOGG1 isoforms, designated ?- and ?-hOGG1, generated by alternative splicing, and they have distinct subcellular localization: cell nuclei and mitochondria, respectively. Using yeast two-hybrid screening assays, we found that ?- but not ?-hOGG1 directly interacts with the mitochondrial protein NADH:ubiquinone oxidoreductase 1 beta subcomplex 10 (NDUFB10), an integral factor in Complex 1 on the mitochondrial inner membrane. Using coimmunoprecipitation and immunofluorescence studies, we found that this interaction was greatly increased by hydrogen peroxide-induced oxidative stress, suggesting that ?- but not ?-hOGG1 is localized in the mitochondrial inner membrane. Analyses of nuclear and mtDNA damage showed that the ?- but not ?- hogg1 knockdown (KD) cells were severely defective in mitochondrial BER, indicating an essential requirement of ?-hOGG1 for mtDNA repair. ?-hogg1 KD cells were also found to be mildly deficient in Complex I activity, suggesting that ?-hOGG1 is an accessory factor for the mitochondrial integral function for ATP synthesis. In summary, our findings define ?-hOGG1 as an important factor for mitochondrial BER and as an accessory factor in the mitochondrial Complex I function. PMID:23055259

Su, Yu-Hung; Lee, Yen-Ling; Chen, Sung-Fang; Lee, Yun-Ping; Hsieh, Yi-Hsuan; Tsai, Jui-He; Hsu, Jye-Lin; Tian, Wei-Ting; Huang, Wenya

2013-01-01

150

Condensation of DNA--a putative obstruction for repair process in abasic clustered DNA damage.  

PubMed

Clustered DNA damages are defined as two or more closely located DNA damage lesions that may be present within a few helical turns of the DNA double strand. These damages are potential signatures of ionizing radiation and are often found to be repair resistant. Types of damaged lesions frequently found inside clustered DNA damage sites include oxidized bases, abasic sites, nucleotide dimers, strand breaks or their complex combinations. In this study, we used a bistranded two-lesion abasic cluster DNA damage model to access the repair process of DNA in condensate form. Oligomer DNA duplexes (47 bp) were designed to have two deoxyuridine in the middle of the sequences, three bases apart in opposite strands. The deoxyuridine residues were converted into abasic sites by treatment with UDG enzyme creating an abasic clustered damage site in a precise position in each of the single strand of the DNA duplex. This oligomer duplex having compatible cohesive ends was ligated to pUC19 plasmid, linearized with HindIII restriction endonuclease. The plasmid-oligomer conjugate was transformed into condensates by treating them with spermidine. The efficiency of strand cleavage action of ApeI enzyme on the abasic sites was determined by denaturing PAGE after timed incubation of the oligomer duplex and the oligomer-plasmid conjugate in presence and absence of spermidine. The efficiency of double strand breaks was determined similarly by native PAGE. Quantitative gel analysis revealed that rate of abasic site cleavage is reduced in the DNA condensates as compared to the oligomer DNA duplex or the linear ligated oligomer-plasmid conjugates. Generation of double strand break is significantly reduced also, suggesting that their creation is not proportionate to the number of abasic sites cleaved in the condensate model. All these suggest that the ApeI enzyme have difficulty to access the abasic sites located deep into the condensates leading to repair refractivity of the damages. In addition, we found that presence of a polyamine such as spermidine has no notable effect in the incision activity of ApeI enzyme in linear oligomer DNA duplexes in our experimental concentration. PMID:23582211

Singh, Vandana; Das, Prolay

2013-06-01

151

Targeting the DNA double strand breaks repair for cancer therapy.  

PubMed

Among several types of DNA lesions, the DNA double strand breaks (DSBs) are one of the most deleterious and harmful. Mammalian cells mount a coordinated response to DSBs with the aim of appropriately repair the DNA damage. Indeed, failure of the DNA damage response (DDR) can lead to the development of cancer-prone genetic diseases. The identification and development of drugs targeting proteins involved in the DDR is even more investigated, as it gives the possibility to specifically target cancer cells. Indeed, the administration of DNA repair inhibitors could be combined with chemo- and radiotherapy, thus improving the eradication of tumor cells. Here, we provide an overview about DSBs damage response, focusing on the role of the DSBs repair mechanisms, of chromatin modifications, and of the cancer susceptibility gene BRCA1 which plays a multifunctional role in controlling genome integrity. Moreover, the most investigated DSBs enzyme inhibitors tested as potential therapeutic agents for anti-cancer therapy are reported. PMID:20423312

Gullotta, Francesca; De Marinis, Elisabetta; Ascenzi, Paolo; di Masi, Alessandra

2010-01-01

152

DNA repair mechanisms in dividing and non-dividing cells  

PubMed Central

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye towards how these pathways may regulate the development of neurological disease. PMID:23684800

Iyama, Teruaki; Wilson, David M.

2013-01-01

153

DNA Binding Properties of the Actin-Related Protein Arp8 and Its Role in DNA Repair  

PubMed Central

Actin and actin-related proteins (Arps), which are members of the actin family, are essential components of many of these remodeling complexes. Actin, Arp4, Arp5, and Arp8 are found to be evolutionarily conserved components of the INO80 chromatin remodeling complex, which is involved in transcriptional regulation, DNA replication, and DNA repair. A recent report showed that Arp8 forms a module in the INO80 complex and this module can directly capture a nucleosome. In the present study, we showed that recombinant human Arp8 binds to DNAs, and preferentially binds to single-stranded DNA. Analysis of the binding of adenine nucleotides to Arp8 mutants suggested that the ATP-binding pocket, located in the evolutionarily conserved actin fold, plays a regulatory role in the binding of Arp8 to DNA. To determine the cellular function of Arp8, we derived tetracycline-inducible Arp8 knockout cells from a cultured human cell line. Analysis of results obtained after treating these cells with aphidicolin and camptothecin revealed that Arp8 is involved in DNA repair. Together with the previous observation that Arp8, but not ?-H2AX, is indispensable for recruiting INO80 complex to DSB in human, results of our study suggest an individual role for Arp8 in DNA repair. PMID:25299602

Murakami, Hirokazu; Otawa, Kenji; Tachiwana, Hiroaki; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-ich; Kurumizaka, Hitoshi; Harata, Masahiko

2014-01-01

154

Insights into the DNA repair process by the formamidopyrimidine-DNA glycosylase investigated by molecular dynamics  

PubMed Central

Formamidopyrimidine-DNA glycosylase (Fpg) identifies and removes 8-oxoguanine from DNA. All of the X-ray structures of Fpg complexed to an abasic site containing DNA exhibit a common disordered region present in the C-terminal domain of the enzyme. However, this region is believed to be involved in the damaged base binding site when the initial protein/DNA complex is formed. The dynamic behavior of the disordered polypeptide (named Loop) in relation to the supposed scenario for the DNA repair mechanism was investigated by molecular dynamics on different models, derived from the X-ray structure of Lactococcus lactis Fpg bound to an abasic site analog-containing DNA and of Bacillus stearothermophilus Fpg bound to 8-oxoG. This study shows that the presence of the damaged base influences the dynamics of the whole enzyme and that the Loop location is dependent on the presence and on the conformation of the 8-oxoG in its binding site. In addition, from our results, the conformation of the 8-oxoG seems to be favored in syn in the L. lactis models, in agreement with the available X-ray structure from B. stearothermophilus Fpg and with a possible catalytic role of the flexibility of the Loop region. PMID:15273302

Amara, Patricia; Serre, Laurence; Castaing, Bertrand; Thomas, Aline

2004-01-01

155

DNA repair: a changing geography? (1964-2008).  

PubMed

This article aims to explain the current state of DNA Repair studies' global geography by focusing on the genesis of the community. Bibliometric data is used to localize scientific activities related to DNA Repair at the city level. The keyword "DNA Repair" was introduced first by American scientists. It started to spread after 1964 that is to say, after P. Howard-Flanders (Yale University), P. Hanawalt (Stanford University) and R. Setlow (Oak Ridge Laboratories) found evidence for Excision Repair mechanisms. It was the first stage in the emergence of an autonomous scientific community. In this article, we will try to assess to what extent the geo-history of this scientific field is determinant in understanding its current geography. In order to do so, we will localize the places where the first "DNA Repair" publications were signed fifty years ago and the following spatial diffusion process, which led to the current geography of the field. Then, we will focus on the evolution of the research activity of "early entrants" in relation to the activity of "latecomers". This article is an opportunity to share with DNA Repair scientists some research results of a dynamic field in Science studies: spatial scientometrics. PMID:23669398

Maisonobe, Marion; Giglia-Mari, Giuseppina; Eckert, Denis

2013-07-01

156

Repair of double-strand DNA breaks by the human nonhomologous DNA end joining pathway: the iterative processing model.  

PubMed

Naturally-occurring ionizing radiation and reactive oxygen species (ROS) from oxidative metabolism are factors that have challenged all life forms during the course of evolution. Ionizing radiation (IR) and reactive oxygen species cause a diverse set of double-strand DNA end configurations. Non-homologous DNA end joining (NHEJ) is an optimal DNA repair pathway for dealing with such a diverse set of DNA lesions. NHEJ can carry out nucleolytic, polymerization, and ligation operations on each strand independently. This iterative processing nature of NHEJ is ideal for repair of pathologic and physiologic double-strand breaks because it permits sequential action of the NHEJ enzymes on each DNA end and on each strand. The versatility of the Artemis:DNA-PKcs endonuclease in cleaving 5' and 3' overhangs, hairpins, gaps, flaps, and various loop conformations makes it well-suited for DNA end modifications on oxidized overhangs. In addition, the ability to cleave stem-loop and hairpin structures permits it to open terminal fold-back configurations that may arise at DNA ends after IR damage. The ability of the XRCC4:DNA ligase IV complex to ligate one strand without ligation of the other permits additional end joining flexibility in NHEJ and raises the possibility of optional involvement of repair proteins from other pathways. PMID:16082219

Ma, Yunmei; Lu, Haihui; Schwarz, Klaus; Lieber, Michael R

2005-09-01

157

Loss of Urokinase Receptor Sensitizes Cells to DNA Damage and Delays DNA Repair  

PubMed Central

DNA damage induced by numerous exogenous or endogenous factors may have irreversible consequences on the cell leading to cell cycle arrest, senescence and cell death. The DNA damage response (DDR) is powerful signaling machinery triggered in response to DNA damage, to provide DNA damage recognition, signaling and repair. Most anticancer drugs induce DNA damage, and DNA repair in turn attenuates therapeutic efficiency of those drugs. Approaches delaying DNA repair are often used to increase efficiency of treatment. Recent data show that ubiquitin-proteasome system is essential for signaling and repair of DNA damage. However, mechanisms providing regulation of proteasome intracellular localization, activity, and recruitment to DNA damage sites are elusive. Even less investigated are the roles of extranuclear signaling proteins in these processes. In this study, we report the involvement of the serine protease urokinase-type plasminogen activator receptor (uPAR) in DDR-associated regulation of proteasome. We show that in vascular smooth muscle cells (VSMC) uPAR activates DNA single strand break repair signaling pathway. We provide evidence that uPAR is essential for functional assembly of the 26S proteasome. We further demonstrate that uPAR mediates DNA damage-induced phosphorylation, nuclear import, and recruitment of the regulatory subunit PSMD6 to proteasome. We found that deficiency of uPAR and PSMD6 delays DNA repair and leads to decreased cell survival. These data may offer new therapeutic approaches for diseases such as cancer, cardiovascular and neurodegenerative disorders. PMID:24987841

Narayanaswamy, Pavan B.; Hodjat, Mahshid; Haller, Hermann; Dumler, Inna; Kiyan, Yulia

2014-01-01

158

BRG1 promotes DNA double-strand break repair by facilitating the replacement of RPA with RAD51.  

PubMed

DNA double-strand breaks (DSBs) are a type of lethal DNA damage. To repair DSBs, a tight coordination between the factors modulating chromatin structure and the DNA repair machinery is required. BRG1, the ATPase subunit of the chromatin remodeling complex SWItch/Sucrose NonFermentable (SWI/SNF), is often linked to tumourigenesis and genome instability, and its role in DSB repair remains largely unclear. In the present study, we showed that BRG1 is recruited to DSB sites and enhances DSB repair. Using DR-GFP and EJ5-GFP reporter systems, we demonstrated that BRG1 facilitates homologous recombination (HR) repair rather than nonhomologous end-joining (NHEJ) repair. Moreover, the BRG1/RAD52 complex mediates the replacement of RPA with RAD51 onto single-stranded DNA (ssDNA) to initiate DNA strand invasion. Loss of BRG1 results in the failure of RAD51 loading onto ssDNAs, abnormal HR repair and enhanced DSB-induced lethality. Our present study provides a mechanistic insight into how BRG1, which is known to be a chromatin remodeling modulator, plays a substantial role in the HR repair pathway in mammalian cells. PMID:25395584

Qi, Wenjing; Wang, Ruoxi; Chen, Hongyu; Wang, Xiaolin; Xiao, Ting; Boldogh, Istvan; Ba, Xueqing; Han, Liping; Zeng, Xianlu

2014-11-13

159

Hypermutation in Burkholderia cepacia complex is mediated by DNA mismatch repair inactivation and is highly prevalent in cystic fibrosis chronic respiratory infection.  

PubMed

The Burkholderia cepacia complex (Bcc) represents an important group of pathogens involved in long-term lung infection in cystic fibrosis (CF) patients. A positive selection of hypermutators, linked to antimicrobial resistance development, has been previously reported for Pseudomonas aeruginosa in this chronic infection setting. Hypermutability, however, has not yet been systematically evaluated in Bcc species. A total of 125 well characterized Bcc isolates recovered from 48 CF patients, 10 non-CF patients and 15 environmental samples were analyzed. In order to determine the prevalence of mutators their spontaneous mutation rates to rifampicin resistance were determined. In addition, the genetic basis of the mutator phenotypes was investigated by sequencing the mutS and mutL genes, the main components of the mismatch repair system (MRS). The overall prevalence of hypermutators in the collection analyzed was 13.6%, with highest occurrence (40.7%) among the chronically infected CF patients, belonging mainly to B. cenocepacia, B. multivorans, B. cepacia, and B. contaminans -the most frequently recovered Bcc species from CF patients worldwide. Thirteen (76.5%) of the hypermutators were defective in mutS and/or mutL. Finally, searching for a possible association between antimicrobial resistance and hypermutability, the resistance-profiles to 17 antimicrobial agents was evaluated. High antimicrobial resistance rates were documented for all the Bcc species recovered from CF patients, but, except for ciprofloxacin, a significant association with hypermutation was not detected. In conclusion, in the present study we demonstrate for the first time that, MRS-deficient Bcc species mutators are highly prevalent and positively selected in CF chronic lung infections. Hypermutation therefore, might be playing a key role in increasing bacterial adaptability to the CF-airway environment, facilitating the persistence of chronic lung infections. PMID:25217078

Martina, Pablo; Feliziani, Sofía; Juan, Carlos; Bettiol, Marisa; Gatti, Blanca; Yantorno, Osvaldo; Smania, Andrea M; Oliver, Antonio; Bosch, Alejandra

2014-11-01

160

Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair  

PubMed Central

Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER. PMID:23890570

German, Peter; Szaniszlo, Peter; Hajas, Gyorgy; Radak, Zsolt; Bacsi, Attila; Hazra, Tapas K.; Hegde, Muralidhar L.; Ba, Xueqing; Boldogh, Istvan

2013-01-01

161

DNA repair in murine embryonic stem cells and differentiated cells  

SciTech Connect

Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.

Tichy, Elisia D. [Department of Cell and Cancer Biology, University of Cincinnati, Cincinnati, OH 45267 (United States)], E-mail: tichyed@email.uc.edu; Stambrook, Peter J. [Department of Cell and Cancer Biology, University of Cincinnati, Cincinnati, OH 45267 (United States)

2008-06-10

162

Recombination repair pathway in the maintenance of chromosomal integrity against DNA interstrand crosslinks  

Microsoft Academic Search

DNA interstrand crosslinks (ICL) present a major threat to cell viability and genome integrity. In eukaryotic cells, the ICLs have been suggested to be repaired by a complex process involving Xpf\\/Ercc1-mediated endonucleolytic incision and homologous recombination (HR). However, the entire feature of the ICL tolerating mechanism is still poorly understood. Here we studied chromosome aberrations (CA) and sister chromatid exchanges

M. S. Sasaki; M. Takata; E. Sonoda; A. Tachibana; S. Takeda

2004-01-01

163

DNA repair is limiting for haematopoietic stem cells during ageing  

Microsoft Academic Search

Accumulation of DNA damage leading to adult stem cell exhaustion has been proposed to be a principal mechanism of ageing. Here we address this question by taking advantage of the highly specific role of DNA ligase IV in the repair of DNA double-strand breaks by non-homologous end-joining, and by the discovery of a unique mouse strain with a hypomorphic Lig4Y288C

Anastasia Nijnik; Lisa Woodbine; Caterina Marchetti; Sara Dawson; Teresa Lambe; Cong Liu; Neil P. Rodrigues; Tanya L. Crockford; Erik Cabuy; Alessandro Vindigni; Tariq Enver; John I. Bell; Predrag Slijepcevic; Christopher C. Goodnow; Penelope A. Jeggo; Richard J. Cornall

2007-01-01

164

Alkyltransferase-like proteins: molecular switches between DNA repair pathways  

Microsoft Academic Search

Alkyltransferase-like proteins (ATLs) play a role in the protection of cells from the biological effects of DNA alkylation\\u000a damage. Although ATLs share functional motifs with the DNA repair protein and cancer chemotherapy target O\\u000a 6-alkylguanine-DNA alkyltransferase, they lack the reactive cysteine residue required for alkyltransferase activity, so its\\u000a mechanism for cell protection was previously unknown. Here we review recent advances

Julie L. TubbsJohn; John A. Tainer

2010-01-01

165

Solvating, manipulating, damaging, and repairing DNA in a computer  

NASA Astrophysics Data System (ADS)

This work highlights four different topics in modeling of DNA: (i) the importance of water and ions together with the structure and function of DNA; the hydration structure around the ions appears to be the determining factor in the ion coordination to DNA, as demonstrated in the results of our MD simulations; (ii) how MD simulations can be used to simulate single molecule manipulation experiments as a complement to reveal the structural dynamics of the studied biomolecules; (iii) how damaged DNA can be studied in computer simulations; and (iv) how repair of damaged DNA can be studied theoretically.

Bunta, Juraj; Dahlberg, Martin; Eriksson, Leif; Korolev, Nikolai; Laaksonen, Aatto; Lohikoski, Raimo; Lyubartsev, Alexander; Pinak, Miroslav; Schyman, Patric

166

Direct Observation of Thymine Dimer Repair in DNA by Photolyase  

NASA Astrophysics Data System (ADS)

Departments of Physics, Chemistry, and Biochemistry, Programs of Biophysics, Chemical Physics, and Biochemistry, The Ohio State University, Columbus, 191 West Woodruff Avenue, OH 43210. Photolyase uses light energy to split ultraviolet-induced cyclobutane pyrimidine dimers in damaged DNA, but its molecular mechanism has never been directly revealed. We report here the direct mapping of catalytic processes through femtosecond synchronization of the enzymatic dynamics with the repair function. We observed direct electron transfer from the excited flavin cofactor to the dimer in 170 ps and back electron transfer from the repaired thymines in 560 ps. Both reactions are strongly modulated by active-site solvation to achieve maximum repair efficiency. These results show that the photocycle of DNA repair by photolyase is through a radical mechanism and completed on subnanosecond time scale at the dynamic active site with no net electron change in redox states of the flavin cofactor.

Zhong, Dongping

2006-03-01

167

Thermodynamics of the DNA Damage Repair Steps of Human 8-Oxoguanine DNA Glycosylase  

PubMed Central

Human 8-oxoguanine DNA glycosylase (hOGG1) is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG). In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van’t Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves. PMID:24911585

Kuznetsov, Nikita A.; Kuznetsova, Alexandra A.; Vorobjev, Yuri N.; Krasnoperov, Lev N.; Fedorova, Olga S.

2014-01-01

168

DNA Ligase III is critical for mtDNA integrity but not Xrcc1-mediated nuclear DNA repair  

PubMed Central

DNA replication and repair in mammalian cells involves three distinct DNA ligases; ligase I (Lig1), ligase III (Lig3) and ligase IV (Lig4)1. Lig3 is considered a key ligase during base excision repair because its stability depends upon its nuclear binding partner Xrcc1, a critical factor for this DNA repair pathway2,3. Lig3 is also present in the mitochondria where its role in mitochondrial DNA (mtDNA) maintenance is independent of Xrcc14. However, the biological role of Lig3 is unclear as inactivation of murine Lig3 results in early embryonic lethality5. Here we report that Lig3 is essential for mtDNA integrity but dispensable for nuclear DNA repair. Inactivation of Lig3 in the mouse nervous system resulted in mtDNA loss leading to profound mitochondrial dysfunction, disruption of cellular homeostasis and incapacitating ataxia. Similarly, inactivation of Lig3 in cardiac muscle resulted in mitochondrial dysfunction and defective heart pump function leading to heart failure. However, Lig3 inactivation did not result in nuclear DNA repair deficiency, indicating essential DNA repair functions of Xrcc1 can occur in the absence of Lig3. Instead, we found that Lig1 was critical for DNA repair, but in a cooperative manner with Lig3. Additionally, Lig3 deficiency did not recapitulate the hallmark features of neural Xrcc1 inactivation such as DNA damage-induced cerebellar interneuron loss6, further underscoring functional separation of these DNA repair factors. Therefore, our data reveal that the critical biological role of Lig3 is to maintain mtDNA integrity and not Xrcc1-dependent DNA repair. PMID:21390131

Gao, Yankun; Katyal, Sachin; Lee, Youngsoo; Zhao, Jingfeng; Rehg, Jerold E.; Russell, Helen R.; McKinnon, Peter J.

2011-01-01

169

Monitoring populations for DNA repair deficiency and for cancer susceptibility.  

PubMed Central

The induction of a mutator phenotype has been hypothesized to cause the accumulation of multiple mutations in the development of cancer. Recent evidence suggests that the mutator phenotype is associated with DNA repair deficiencies. We have been using a challenge assay to study exposed populations to test our hypothesis that exposure to environmental toxicants induce DNA repair deficiency in somatic cells. In this assay, lymphocytes were irradiated in vitro to challenge cells to repair the radiation-induction DNA strand breaks. An increase of chromosome aberrations in the challenged cells from toxicant-exposed populations compared to nonexposed populations is used to indicate abnormal DNA repair response. From studies of cigarette smokers, butadiene-exposed workers, and uranium-exposed residents, the assay showed that these exposed populations had mutagen-induced abnormal DNA repair response. The phenomenon was also demonstrated using experimental animals. Mice were exposed in vivo to two different doses of N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) and their lymphocytes were challenged with one dose of a radiomimetic chemical, bleomycin, in vitro. These challenged lymphocytes showed an MNNG dose-dependent increase of abnormal DNA repair response. In a population that was potentially exposed to teratogens--mothers having children with neural tube defects--lymphocytes from these mothers did not have the abnormal response in our assay. In studies with patients, we reported that lymphocytes from Down's syndrome patients have the abnormal DNA repair response. Lymphocytes from skin cancer-prone patients (epidermodysplasia verruciformis) have normal response to gamma-ray challenge but abnormal response to UV-light challenge. These patient studies also indicate that the challenge assay is useful in documenting the radiosensitivity of Down's syndrome and the UV sensitivity in EV patients. In most cases, the challenge assay is more sensitive in detecting biological effects than the standard chromosome aberration assay. Our series of studies indicates that the challenge assay can be used to document biological effects from exposure to mutagens and that the effect is an abnormal DNA repair response. This abnormality can increase the risk for development of cancer. The repair deficiency is currently being validated using a plasmid transfection (host-reactivation) assay. The need to integrate chromosome aberration and the challenge assays with other relevant assays for better documentation of biological effects and for more precise prediction of health risk will be presented. Our experience in using genetic polymorphism and host-reactivation assays will be discussed. PMID:8781386

Au, W W; Wilkinson, G S; Tyring, S K; Legator, M S; el Zein, R; Hallberg, L; Heo, M Y

1996-01-01

170

Early Steps in the DNA Base Excision/Single-Strand Interruption Repair Pathway in Mammalian Cells  

PubMed Central

Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4–5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3? OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3? phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified that may affect activity and complex formation. The focus of this review are the early steps in mammalian BER for oxidized damage. PMID:18166975

Hegde, Muralidhar L.; Hazra, Tapas K.; Mitra, Sankar

2009-01-01

171

Modeling DNA Repair: Approaching In Vivo Techniques in the Hyperthermophile Sulfolobus Solfataricus  

SciTech Connect

Archaea are found in some of the most extreme environments on earth and represent a third domain of life distinct from Eukarya and Eubacteria. The hyperthermophilic archaeon Sulfolobus solfataricus, isolated from acidic hot springs (80oC, pH 3) in Yellowstone National Park, has emerged as a potential model system for studying human DNA repair processes. Archaea are more closely related to Eukarya than to Eubacteria, suggesting that archaeal DNA repair machinery may model the complex human system much more closely than that of other prokaryotes. DNA repair requires coordinated protein-protein interactions that are frequently transient. Protein complexes that are transient at extreme temperatures where archaea thrive may be more stable at room temperature, allowing for the characterization of otherwise short-lived complexes. However, characterization of these systems in archaea has been limited by the absence of a stable in vivo transformation and expression system. The work presented here is a pilot study in gene cloning and recombinant protein expression in S. solfataricus. Three genes associated with DNA repair were selected for expression: MRE11, PCNA1, and a putative CSB homologue. Though preparation of these recombinant genes followed standard methods, preparation of a suitable vector proved more challenging. The shuttle vector pSSV64, derived from the SSV1 virus and the E. coli vector pBSSK+, was most successfully isolated from the DH5? E. coli strain. Currently, alternative vectors are being designed for more efficient genetic manipulations in S. solfataricus.

Blanton, J.; Fuss, J.; Yannone, S.M.; Tainer, J.A.; Cooper, P.K.

2005-01-01

172

Charge-transport-mediated recruitment of DNA repair enzymes  

NASA Astrophysics Data System (ADS)

Damaged or mismatched bases in DNA can be repaired by base excision repair enzymes (BER) that replace the defective base. Although the detailed molecular structures of many BER enzymes are known, how they colocalize to lesions remains unclear. One hypothesis involves charge transport (CT) along DNA [Yavin et al., Proc. Natl. Acad. Sci. U.S.A. 102, 3546 (2005)]. In this CT mechanism, electrons are released by recently adsorbed BER enzymes and travel along the DNA. The electrons can scatter (by heterogeneities along the DNA) back to the enzyme, destabilizing and knocking it off the DNA, or they can be absorbed by nearby lesions and guanine radicals. We develop a stochastic model to describe the electron dynamics and compute probabilities of electron capture by guanine radicals and repair enzymes. We also calculate first passage times of electron return and ensemble average these results over guanine radical distributions. Our statistical results provide the rules that enable us to perform implicit-electron Monte Carlo simulations of repair enzyme binding and redistribution near lesions. When lesions are electron absorbing, we show that the CT mechanism suppresses wasteful buildup of enzymes along intact portions of the DNA, maximizing enzyme concentration near lesions.

Fok, Pak-Wing; Guo, Chin-Lin; Chou, Tom

2008-12-01

173

Charge transport-mediated recruitment of DNA repair enzymes  

E-print Network

Damaged or mismatched bases in DNA can be repaired by Base Excision Repair (BER) enzymes that replace the defective base. Although the detailed molecular structures of many BER enzymes are known, how they colocalize to lesions remains unclear. One hypothesis involves charge transport (CT) along DNA [Yavin, {\\it et al.}, PNAS, {\\bf 102}, 3546, (2005)]. In this CT mechanism, electrons are released by recently adsorbed BER enzymes and travel along the DNA. The electrons can scatter (by heterogeneities along the DNA) back to the enzyme, destabilizing and knocking it off the DNA, or, they can be absorbed by nearby lesions and guanine radicals. We develop a stochastic model to describe the electron dynamics, and compute probabilities of electron capture by guanine radicals and repair enzymes. We also calculate first passage times of electron return, and ensemble-average these results over guanine radical distributions. Our statistical results provide the rules that enable us to perform implicit-electron Monte-Carlo simulations of repair enzyme binding and redistribution near lesions. When lesions are electron absorbing, we show that the CT mechanism suppresses wasteful buildup of enzymes along intact portions of the DNA, maximizing enzyme concentration near lesions.

Pak-Wing Fok; Chin-Lin Guo; Tom Chou

2008-11-18

174

DNA damage repair and genetic polymorphisms: Assessment of individual sensitivity and repair capacity  

SciTech Connect

Purpose: To study the repair capacity after X-ray irradiation in human peripheral blood cells of healthy subjects, in relation to their genotypes. Methods and Materials: The peripheral blood of 50 healthy subjects was irradiated in vitro with 2 Gy of X rays and the induced DNA damage was measured by Comet assay immediately after irradiation. DNA repair was detected by analyzing the cells at defined time intervals after the exposure. Furthermore, all subjects were genotyped for XRCC1, OGG1, and XPC genes. Results: After X-ray irradiation, persons bearing XRCC1 homozygous variant (codon 399) genotype exhibited significantly lower Tail DNA values than those bearing wild-type and heterozygous genotypes. These results are also confirmed at 30 and 60 min after irradiation. Furthermore, XPC heterozygous subjects (variant codon 939) showed lower residual DNA damage 60 min after irradiation compared with wild-type and homozygous genotypes. Conclusion: The results of the present study show that polymorphisms in DNA repair genes could influence individual DNA repair capacity.

Cornetta, Tommaso [Department of Biology, Universita degli Studi 'Roma TRE', Rome (Italy); Fondazione Don Carlo Gnocchi, Rome (Italy); Festa, Fabiola [Department of Biology, Universita degli Studi 'Roma TRE', Rome (Italy); Testa, Antonella [Section of Toxicology and Biomedical Sciences, ENEA CR Casaccia, Rome (Italy); Cozzi, Renata Prof. [Department of Biology, Universita degli Studi 'Roma TRE', Rome (Italy)]. E-mail: cozzi@uniroma3.it

2006-10-01

175

The DNA repair component Metnase regulates Chk1 stability  

PubMed Central

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylation of downstream effectors. Metnase (also termed SETMAR) is a SET histone methylase and transposase nuclease protein that promotes both DNA double strand break (DSB) repair and re-start of stalled replication forks. We previously found that Chk1 phosphorylation of Metnase on S495 enhanced its DNA DSB repair activity but decreased its ability to re-start stalled replication forks. Here we show that phosphorylated Metnase feeds back to increase the half-life of Chk1. Chk1 half-life is regulated by DDB1 targeting it to Cul4A for ubiquitination and destruction. Metnase decreases Chk1 interaction with DDB1, and decreases Chk1 ubiquitination. These data define a novel pathway for Chk1 regulation, whereby a target of Chk1, Metnase, feeds back to amplify Chk1 stability, and therefore enhance replication fork arrest. PMID:25024738

2014-01-01

176

Functional Aspects of PARP1 in DNA Repair and Transcription  

PubMed Central

Poly (ADP-ribose) polymerase 1 (PARP1) is an ADP-ribosylating enzyme essential for initiating various forms of DNA repair. Inhibiting its enzyme activity with small molecules thus achieves synthetic lethality by preventing unwanted DNA repair in the treatment of cancers. Through enzyme-dependent chromatin remodeling and enzyme-independent motif recognition, PARP1 also plays important roles in regulating gene expression. Besides presenting current findings on how each process is individually controlled by PARP1, we shall discuss how transcription and DNA repair are so intricately linked that disturbance by PARP1 enzymatic inhibition, enzyme hyperactivation in diseases, and viral replication can favor one function while suppressing the other. PMID:24970148

Ko, Hui Ling; Ren, Ee Chee

2012-01-01

177

Evidence for DNA-PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle.  

PubMed Central

Mice homozygous for the scid (severe combined immune deficiency) mutation are defective in the repair of DNA double-strand breaks (DSBs) and are consequently very X-ray sensitive and defective in the lymphoid V(D)J recombination process. Recently, a strong candidate for the scid gene has been identified as the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex. Here, we show that the activity of the DNA-PK complex is regulated in a cell cycle-dependent manner, with peaks of activity found at the G1/early S phase and again at the G2 phase in wild-type cells. Interestingly, only the deficit of the G1/early S phase DNA-PK activity correlated with an increased hypersensitivity to X-irradiation and a DNA DSB repair deficit in synchronized scid pre-B cells. Finally, we demonstrate that the DNA-PK activity found at the G2 phase may be required for exit from a DNA damage-induced G2 checkpoint arrest. These observations suggest the presence of two pathways (DNA-PK-dependent and -independent) of illegitimate mammalian DNA DSB repair and two distinct roles (DNA DSB repair and G2 checkpoint traversal) for DNA-PK in the cellular response to ionizing radiation. PMID:9032269

Lee, S E; Mitchell, R A; Cheng, A; Hendrickson, E A

1997-01-01

178

Repair of mismatched basepairs in mammalian DNA  

SciTech Connect

We have concentrated on three specific areas of our research plan. Our greatest emphasis is on the role of single strand nicks in influencing template strand selection in mismatch repair. We have found, that the ability of a nick in one strand to influence which strand is repaired is not a simple function of distance from the mismatched site but rather that an hot spot where a nick is more likely to have an influence can exist. The second line was production of single-genotype heteroduplexes in order to examine independently the repair of T/G and A/C mispairs within the same sequence context as in our mixed mispair preparations. We have shown preparations of supercoiled heteroduplex can be prepared that were exclusively T/G or exclusively A/C at the mispair site. The third effort has been to understand the difference in repair bias of different cell lines or different transfection conditions as it may relate to different repair systems in the cell. We have identified some of the sources of variation, including cell cycle position. We hope to continue this work to more precisely identify the phase of the cell cycle.

Taylor, J.H.; Hare, J.T.

1991-08-01

179

Exploiting the homologous recombination DNA repair network for targeted cancer therapy  

PubMed Central

Genomic instability is a characteristic of cancer cells. In order to maintain genomic integrity, cells have evolved a complex DNA repair system to detect, signal and repair a diversity of DNA lesions. Homologous recombination (HR)-mediated DNA repair represents an error-free repair mechanism to maintain genomic integrity and ensure high-fidelity transmission of genetic information. Deficiencies in HR repair are of tremendous importance in the etiology of human cancers and at the same time offer great opportunities for designing targeted therapeutic strategies. The increase in the number of proteins identified as being involved in HR repair has dramatically shifted our concept of the proteins involved in this process: traditionally viewed as existing in a linear and simple pathway, today they are viewed as existing in a dynamic and interconnected network. Moreover, exploration of the targets within this network that can be modulated by small molecule drugs has led to the discovery of many effective kinase inhibitors, such as ATM, ATR, DNA-PK, CHK1, and CHK2 inhibitors. In preclinical studies, these inhibitors have been shown to sensitize cancer cells to chemotherapy and radiation therapy. The most exciting discovery in the field of HR repair is the identification of the synthetic lethality relationship between poly (ADP-ribose) polymerase (PARP) inhibitors and HR deficiency. The promises of clinical applications of PARP inhibitors and the concept of synthetic lethality also bring challenges into focus. Future research directions in the area of HR repair include determining how to identify the patients most likely to benefit from PARP inhibitors and developing strategies to overcome resistance to PARP inhibitors. PMID:21603316

Peng, Guang; Lin, Shiaw-Yih

2011-01-01

180

Mtor-Fanconi Anemia DNA Damage Repair Pathway in Cancer  

PubMed Central

mTOR is a serine/threonine kinase and plays a critical role in mammalian cell growth, survival, and metabolism. mTOR is present in two cellular complexes: mTORC1 and mTORC2. Dysregulation of the mTOR pathway has been related to tumorigenesis, poor prognosis and/or chemotherapy resistance in a variety of malignancies. Inhibition of mTORC1 by Rapamycin and its analogs has been explored to treat a number of tumors. However, the effectiveness of patient response is limited and not all patients respond. Second generation of mTOR inhibitors have recently been developed to target mTOR kinase activity and to suppress both mTORC1 and mTORC2. Dual mTORC1/mTORC2 inhibitors generally are more efficacious in preclinical studies and clinical trials. We and others have recently found that dual mTORC1/mTORC2 inhibitors sensitize T-cell acute lymphocytic leukemia and rhabdomyosarcoma cells to DNA damaging agents by suppression of expression of FANCD2 of the Fanconi anemia pathway, an important DNA repair mechanism that is associated with drug resistance of multiple types of cancer. This review will highlight mTOR and the Fanconi anemia pathway in cancer, with a particular attention to our newly discovered connection between mTOR and the Fanconi anemia pathway.

Guo, Fukun

2014-01-01

181

Human AP endonuclease suppresses DNA mismatch repair activity leading to microsatellite instability  

PubMed Central

The multifunctional mammalian apurinic/apyrimidinic (AP) endonuclease (APE) participates in the repair of AP sites in the cellular DNA as well as participating in the redox regulation of the transcription factor function. The function of APE is considered as the rate-limiting step in DNA base excision repair. Paradoxically, an unbalanced increase in APE protein leads to genetic instability. Therefore, we investigated the mechanisms of genetic instability that are induced by APE. Here, we report that the overexpression of APE protein disrupts the repair of DNA mismatches, which results in microsatellite instability (MSI). We found that expression of APE protein led to the suppression of the repair of DNA mismatches in the normal human fibroblast cells. Western blot analysis revealed that hMSH6 protein was markedly reduced in the APE-expressing cells. Moreover, the addition of purified Mut? (MSH2 and MSH6 complex) to the extracts from the APE-expressing cells led to the restoration of mismatch repair (MMR) activity. By performing MMR activity assay and MSI analysis, we found that the co-expression of hMSH6 and APE exhibited the microsatellite stability, whereas the expression of APE alone generated the MSI-high phenotype. The APE-mediated decrease in MMR activity described here demonstrates the presence of a new and highly effective APE-mediated mechanism for MSI. PMID:16147991

Chang, In-Youb; Kim, Soo-Hyun; Cho, Hyun-Ju; Lee, Do Young; Kim, Mi-Hwa; Chung, Myung-Hee; You, Ho Jin

2005-01-01

182

Metal Complexes for DNA-Mediated Charge Transport  

PubMed Central

In all organisms, oxidation threatens the integrity of the genome. DNA-mediated charge transport (CT) may play an important role in the generation and repair of this oxidative damage. In studies involving long-range CT from intercalating Ru and Rh complexes to 5?-GG-3? sites, we have examined the efficiency of CT as a function of distance, temperature, and the electronic coupling of metal oxidants bound to the base stack. Most striking is the shallow distance dependence and the sensitivity of DNA CT to how the metal complexes are stacked in the helix. Experiments with cyclopropylamine-modified bases have revealed that charge occupation occurs at all sites along the bridge. Using Ir complexes, we have seen that the process of DNA-mediated reduction is very similar to that of DNA-mediated oxidation. Studies involving metalloproteins have, furthermore, shown that their redox activity is DNA-dependent and can be DNA-mediated. Long range DNA-mediated CT can facilitate the oxidation of DNA-bound base excision repair proteins to initiate a redox-active search for DNA lesions. DNA CT can also activate the transcription factor SoxR, triggering a cellular response to oxidative stress. Indeed, these studies show that within the cell, redox-active proteins may utilize the same chemistry as that of synthetic metal complexes in vitro, and these proteins may harness DNA-mediated CT to reduce damage to the genome and regulate cellular processes. PMID:21643528

Barton, Jacqueline K.; Olmon, Eric D.; Sontz, Pamela A.

2010-01-01

183

PARP1–TDP1 coupling for the repair of topoisomerase I–induced DNA damage  

PubMed Central

Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3?-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1–PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1. PMID:24493735

Das, Benu Brata; Huang, Shar-yin N.; Murai, Junko; Rehman, Ishita; Amé, Jean-Christophe; Sengupta, Souvik; Das, Subhendu K.; Majumdar, Papiya; Zhang, Hongliang; Biard, Denis; Majumder, Hemanta K.; Schreiber, Valérie; Pommier, Yves

2014-01-01

184

Kinetic analysis of DNA double-strand break repair pathways in Arabidopsis.  

PubMed

Double-strand breaks in genomic DNA (DSB) are potentially lethal lesions which separate parts of chromosome arms from their centromeres. Repair of DSB by recombination can generate mutations and further chromosomal rearrangements, making the regulation of recombination and the choice of recombination pathways of the highest importance. Although knowledge of recombination mechanisms has considerably advanced, the complex interrelationships and regulation of pathways are far from being fully understood. We analyse the different pathways of DSB repair acting in G2/M phase nuclei of irradiated plants, through quantitation of the kinetics of appearance and loss of ?-H2AX foci in Arabidopsis mutants. These analyses show the roles for the four major recombination pathways in post-S-phase DSB repair and that non-homologous recombination pathways constitute the major response. The data suggest a hierarchical organisation of DSB repair in these cells: C-NHEJ acts prior to B-NHEJ which can also inhibit MMEJ. Surprisingly the quadruple ku80 xrcc1 xrcc2 xpf mutant can repair DSB, although with severely altered kinetics. This repair leads to massive genetic instability with more than 50% of mitoses showing anaphase bridges following irradiation. This study thus clarifies the relationships between the different pathways of DSB repair in the living plant and points to the existence of novel DSB repair processes. PMID:21530420

Charbonnel, Cyril; Allain, Elisabeth; Gallego, Maria Eugenia; White, Charles I

2011-06-10

185

Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair  

PubMed Central

There are two main pathways in eukaryotic cells for the repair of DNA double-strand breaks: homologous recombination and nonhomologous end joining. Because eukaryotic genomes are packaged in chromatin, these pathways are likely to require the modulation of chromatin structure. One way to achieve this is by the acetylation of lysine residues on the N-terminal tails of histones. Here we demonstrate that Sin3p and Rpd3p, components of one of the predominant histone deacetylase complexes of Saccharomyces cerevisiae, are required for efficient nonhomologous end joining. We also show that lysine 16 of histone H4 becomes deacetylated in the proximity of a chromosomal DNA double-strand break in a Sin3p-dependent manner. Taken together, these results define a role for the Sin3p/Rpd3p complex in the modulation of DNA repair. PMID:14711989

Jazayeri, Ali; McAinsh, Andrew D.; Jackson, Stephen P.

2004-01-01

186

DNA damage and repair in Stylonychia lemnae (Ciliata, Protozoa).  

PubMed

Irradiation with X rays, UV irradiation after incorporation of bromodeoxyuridine (BU) into the DNA, and cis-platinum (cis-Pt) treatment each cause the loss of micronuclei of Stylonychia lemnae while the macronuclei are not severely affected. The abilities of both nuclei to repair DNA were investigated. Unscheduled DNA synthesis could not be demonstrated after X-ray irradiation, but it was found after treatment with BU/UV and cis-Pt in macro- and micronuclei. The extent of the repair process in the micro- and macronuclei was alike, as indicated by grain counts of [6-3H]thymidine-treated cells. One reason for the different sensitivity of both nuclei to DNA-damaging treatment may be the different number of gene copies in the macro- and micronuclei. PMID:3135386

Ammermann, D

1988-05-01

187

DNA Synthesis Errors Associated with Double-Strand-Break Repair  

PubMed Central

Repair of a site-specific double-strand DNA break (DSB) resulted in increased reversion frequency for a nearby allele. Site-specific DSBs were introduced into the genome of Saccharomyces cerevisiae by the endonuclease encoded by the HO gene. Expression of the HO gene from a galactose-inducible promoter allowed efficient DNA cleavage at a single site in large populations of cells. To determine whether the DNA synthesis associated with repair of DSBs has a higher error rate than that associated with genome duplication, HO-induced DSBs were generated 0.3 kb from revertible alleles of trp1. The reversion rate of the trp1 alleles was ~100-fold higher among cells that had experienced an HO cut than among uninduced cells. The reverted allele was found predominantly on the chromosome that experienced the DNA cleavage. PMID:7672595

Strathern, J. N.; Shafer, B. K.; McGill, C. B.

1995-01-01

188

DNA Repair at Telomeres: Keeping the Ends Intact  

PubMed Central

The molecular era of telomere biology began with the discovery that telomeres usually consist of G-rich simple repeats and end with 3? single-stranded tails. Enormous progress has been made in identifying the mechanisms that maintain and replenish telomeric DNA and the proteins that protect them from degradation, fusions, and checkpoint activation. Although telomeres in different organisms (or even in the same organism under different conditions) are maintained by different mechanisms, the disparate processes have the common goals of repairing defects caused by semiconservative replication through G-rich DNA, countering the shortening caused by incomplete replication, and postreplication regeneration of G tails. In addition, standard DNA repair mechanisms must be suppressed or modified at telomeres to prevent their being recognized and processed as DNA double-strand breaks. Here, we discuss the players and processes that maintain and regenerate telomere structure. PMID:23732473

Webb, Christopher J.; Wu, Yun; Zakian, Virginia A.

2013-01-01

189

DNA repair in reduced genome: the Mycoplasma model.  

PubMed

The occurrence of bacteria with a reduced genome, such as that found in Mycoplasmas, raises the question as to which genes should be enough to guarantee the genomic stability indispensable for the maintenance of life. The aim of this work was to compare nine Mycoplasma genomes in regard to DNA repair genes. An in silico analysis was done using six Mycoplasma species, whose genomes are accessible at GenBank, and M. synoviae, and two strains of M. hyopneumoniae, whose genomes were recently sequenced by The Brazilian National Genome Project Consortium and Southern Genome Investigation Program (Brazil) respectively. Considering this reduced genome model, our comparative analysis suggests that the DNA integrity necessary for life can be primarily maintained by nucleotide excision repair (NER), which is the only complete repair pathway. Furthermore, some enzymes involved with base excision repair (BER) and recombination are also present and can complement the NER activity. The absence of RecR and RecO-like ORFs was observed only in M. genitalium and M. pneumoniae, which can be involved with the conservation of gene order observed between these two species. We also obtained phylogenetic evidence for the recent acquisition of the ogt gene in M. pulmonis and M. penetrans by a lateral transference event. In general, the presence or nonexistence of repair genes is shared by all species analyzed, suggesting that the loss of the majority of repair genes was an ancestral event, which occurred before the divergence of the Mycoplasma species. PMID:16153783

Carvalho, Fabíola Marques; Fonseca, Marbella Maria; Batistuzzo De Medeiros, Sílvia; Scortecci, Kátia Castanho; Blaha, Carlos Alfredo Galindo; Agnez-Lima, Lucymara Fassarella

2005-11-01

190

DNA damage-activated ABL-MyoD signaling contributes to DNA repair in skeletal myoblasts  

PubMed Central

Previous works have established a unique function of MyoD in the control of muscle gene expression during DNA damage response in myoblasts. Phosphorylation by DNA damage-activated ABL tyrosine kinase transiently inhibits MyoD-dependent activation of transcription in response to genotoxic stress. We show here that ABL-MyoD signaling is also an essential component of the DNA repair machinery in myoblasts exposed to genotoxic stress. DNA damage promoted the recruitment of MyoD to phosphorylated Nbs1 (pNbs1)-containing repair foci, and this effect was abrogated by either ABL knockdown or the ABL kinase inhibitor imatinib. Upon DNA damage, MyoD and pNbs1 were detected on the chromatin to MyoD target genes without activating transcription. DNA damage-mediated tyrosine phosphorylation was required for MyoD recruitment to target genes, as the ABL phosphorylation-resistant MyoD mutant (MyoD Y30F) failed to bind the chromatin following DNA damage, while retaining the ability to activate transcription in response to differentiation signals. Moreover, MyoD Y30F exhibited an impaired ability to promote repair in a heterologous system, as compared with MyoD wild type (WT). Consistently, MyoD-null satellite cells (SCs) displayed impaired DNA repair that was rescued by reintroduction of MyoD WT but not by MyoD Y30F. In addition, inhibition of ABL kinase prevented MyoD WT-mediated rescue of DNA repair in MyoD-null SCs. These results identify an unprecedented contribution of MyoD to DNA repair and suggest that ABL-MyoD signaling coordinates DNA repair and transcription in myoblasts. PMID:24056763

Simonatto, M; Marullo, F; Chiacchiera, F; Musaró, A; Wang, J Y J; Latella, L; Puri, P L

2013-01-01

191

Triplex technology in studies of DNA damage, DNA repair, and mutagenesis  

PubMed Central

Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. PMID:21501652

Mukherjee, Anirban; Vasquez, Karen M.

2012-01-01

192

Base excision repair: NMR backbone assignments of Escherichia coli formamidopyrimidine-DNA glycosylase  

SciTech Connect

Oxidative damage is emerging as one of the most important mechanisms responsible for mutagenesis, carcinogenesis, aging, and various diseases (Farr and Kogma, 1991). One of the potential targets for oxidation is cellular DNA. While exposure to exogenous agents, such as ionizing radiation and chemicals, contributes to damaging DNA, the most important oxidative agents are endogenous, such as the reactive free radicals produced during normal oxidative metabolism (Adelman et., 1988). To mitigate the potentially deleterious effects of oxidative DNA damage virtually all aerobic organisms have developed complex repair mechanisms (Petit and Sancar, 1999). One repair mechanism, base excision repair (BER), appears to be responsible for replacing most oxidative DNA damage (David and Williams, 1998). Formamidopyrimidine-DNA glycosylase (Fpg), a 269-residue metalloprotein with a molecular weight of 30.2 kDa, is a key BER enzyme in prokaryotes (Boiteaux et al., 1987). Substrates recognized and released by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoG), 2,6 diamino-4-hydroxy-5-formamido pyrimidine (Fapy-G), the adenine equivalents 8-oxoA and Fapy-A, 5-hydroxycytosine, 5-hydroxyuracil, B ureidoisobutiric acid, and a-R-hydroxy-B-ureidoisobutiric acid (Freidberg et al., 1995). In vitro Fpg bind double-stranded DNA and performs three catalytic activities: (i) DNA glycosylase, (ii) AP lyase, and (iii) deoxyribophosphodiesterase.

Buchko, Garry W.; Wallace, Susan S.; Kennedy, Michael A.

2002-03-01

193

At the intersection of non-coding transcription, DNA repair, chromatin structure, and cellular senescence  

PubMed Central

It is well accepted that non-coding RNAs play a critical role in regulating gene expression. Recent paradigm-setting studies are now revealing that non-coding RNAs, other than microRNAs, also play intriguing roles in the maintenance of chromatin structure, in the DNA damage response, and in adult human stem cell aging. In this review, we will discuss the complex inter-dependent relationships among non-coding RNA transcription, maintenance of genomic stability, chromatin structure, and adult stem cell senescence. DNA damage-induced non-coding RNAs transcribed in the vicinity of the DNA break regulate recruitment of the DNA damage machinery and DNA repair efficiency. We will discuss the correlation between non-coding RNAs and DNA damage repair efficiency and the potential role of changing chromatin structures around double-strand break sites. On the other hand, induction of non-coding RNA transcription from the repetitive Alu elements occurs during human stem cell aging and hinders efficient DNA repair causing entry into senescence. We will discuss how this fine balance between transcription and genomic instability may be regulated by the dramatic changes to chromatin structure that accompany cellular senescence. PMID:23967007

Ohsawa, Ryosuke; Seol, Ja-Hwan; Tyler, Jessica K.

2013-01-01

194

Damage and repair of ancient DNA  

Microsoft Academic Search

Under certain conditions small amounts of DNA can survive for long periods of time and can be used as polymerase chain reaction (PCR) substrates for the study of phylogenetic relationships and population genetics of extinct plants and animals, including hominids. Because of extensive DNA degradation, these studies are limited to species that lived within the past 104–105 years (Late Pleistocene),

David Mitchell; Eske Willerslev; Anders Hansen

2005-01-01

195

Dynamics and pathway of electron tunneling in repair of damaged DNA by photolyase  

NASA Astrophysics Data System (ADS)

Through electron tunneling, photolyase, a photoenzyme, restores damaged DNA into normal bases. Here, we report our systematic characterization and analyses of three electron transfer processes in thymine dimer restoration by following the entire dynamical evolution during enzymatic repair with femtosecond resolution. Using (deoxy)uracil and thymine as dimer substrates, we unambiguously determined the electron tunneling pathways for the forward electron transfer to initiate repairing and for the final electron return to restore the active cofactor and complete the repair photocycle. Significantly, we found that the adenine moiety of the unusual bent cofactor is essential to mediating all electron-transfer dynamics through a super-exchange mechanism, leading to a delicate balance of time scales. The active-site structural integrity, unique electron tunneling pathways and the critical role of adenine assure these elementary dynamics in synergy in this complex photorepair machinery to achieve the maximum repair efficiency close to unity.

Liu, Zheyun; Guo, Xunmin; Tan, Chuang; Li, Jiang; Kao, Ya-Ting; Wang, Lijuan; Sancar, Aziz; Zhong, Dongping

2013-03-01

196

Involvement of DNA-PK(sub cs) in DSB Repair Following Fe-56 Ion Irradiation  

NASA Technical Reports Server (NTRS)

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.

O'Neill, Peter; Harper, Jane; Anderson, Jennifer a.; Cucinnota, Francis A.

2007-01-01

197

UV Radiation Damage and Bacterial DNA Repair Systems  

ERIC Educational Resources Information Center

This paper reports on a simple hands-on laboratory procedure for high school students in studying both radiation damage and DNA repair systems in bacteria. The sensitivity to ultra-violet (UV) radiation of both "Escherichia coli" and "Serratia marcescens" is tested by radiating them for varying time periods. Two growth temperatures are used in…

Zion, Michal; Guy, Daniel; Yarom, Ruth; Slesak, Michaela

2006-01-01

198

The Caenorhabditis elegans Homolog of Gen1/Yen1 Resolvases Links DNA Damage Signaling to DNA Double-Strand Break Repair  

PubMed Central

DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53–mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair. PMID:20661466

Bailly, Aymeric P.; Alpi, Arno; Lilley, David M. J.; Ahmed, Shawn; Gartner, Anton

2010-01-01

199

The USP1/UAF1 Complex Promotes Double-Strand Break Repair through Homologous Recombination ? †  

PubMed Central

Protein ubiquitination plays a key role in the regulation of a variety of DNA repair mechanisms. Protein ubiquitination is controlled by the coordinate activity of ubiquitin ligases and deubiquitinating enzymes (DUBs). The deubiquitinating enzyme USP1 regulates DNA repair and the Fanconi anemia pathway through its association with its WD40 binding partner, UAF1, and through its deubiquitination of two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. To investigate the function of USP1 and UAF1, we generated USP1?/?, UAF1?/?/?, and USP1?/? UAF1?/?/? chicken DT40 cell clones. These three clones showed similar sensitivities to chemical cross-linking agents, to a topoisomerase poison, camptothecin, and to an inhibitor of poly(ADP-ribose) polymerase (PARP), indicating that the USP1/UAF1 complex is a regulator of the cellular response to DNA damage. The hypersensitivity to both camptothecin and a PARP inhibitor suggests that the USP1/UAF1 complex promotes homologous recombination (HR)-mediated double-strand break (DSB) repair. To gain insight into the mechanism of the USP1/UAF1 complex in HR, we inactivated the nonhomologous end-joining (NHEJ) pathway in UAF1-deficient cells. Disruption of NHEJ in UAF1-deficient cells restored cellular resistance to camptothecin and the PARP inhibitor. Our results indicate that the USP1/UAF1 complex promotes HR, at least in part by suppressing NHEJ. PMID:21482670

Murai, Junko; Yang, Kailin; Dejsuphong, Donniphat; Hirota, Kouji; Takeda, Shunichi; D'Andrea, Alan D.

2011-01-01

200

Density functional theory study of the reaction mechanism of the DNA repairing enzyme alkylguanine alkyltransferase  

NASA Astrophysics Data System (ADS)

The reaction mechanism of human O6-alkylguanine-DNA alkyltransferase (AGT) is studied using density functional theory. AGT repairs alkylated DNA by directly removing the alkyl group from the O6 position of the guanine. A quantum chemical model of the active site was devised based on the recent crystal structure of the AGT-DNA complex. The potential energy curve is calculated and the stationary points are characterized. It is concluded that the previously proposed reaction mechanism is energetically plausible. In this mechanism, His146 first acts as a water-mediated general base to activate Cys145, which then performs a nucleophilic attack to dealkylate the guanine base.

Georgieva, Polina; Himo, Fahmi

2008-09-01

201

Functional interplay between ATM/ATR-mediated DNA damage response and DNA repair pathways in oxidative stress.  

PubMed

To maintain genome stability, cells have evolved various DNA repair pathways to deal with oxidative DNA damage. DNA damage response (DDR) pathways, including ATM-Chk2 and ATR-Chk1 checkpoints, are also activated in oxidative stress to coordinate DNA repair, cell cycle progression, transcription, apoptosis, and senescence. Several studies demonstrate that DDR pathways can regulate DNA repair pathways. On the other hand, accumulating evidence suggests that DNA repair pathways may modulate DDR pathway activation as well. In this review, we summarize our current understanding of how various DNA repair and DDR pathways are activated in response to oxidative DNA damage primarily from studies in eukaryotes. In particular, we analyze the functional interplay between DNA repair and DDR pathways in oxidative stress. A better understanding of cellular response to oxidative stress may provide novel avenues of treating human diseases, such as cancer and neurodegenerative disorders. PMID:24947324

Yan, Shan; Sorrell, Melanie; Berman, Zachary

2014-10-01

202

Spatiotemporal analysis of DNA repair using charged particle radiation.  

PubMed

Approaches to visualise the dynamics of the DNA lesion processing substantially contributes to the understanding of the hierarchical organisation of the DNA damage response pathways. Charged particle irradiation has recently emerged as a tool to generate discrete sites of subnuclear damage by its means of extremely localised dose deposition at low energies, thus facilitating the spatiotemporal analysis of repair events. In addition, they are of high interest for risk estimations of human space exploration (e.g. mars mission) in the high energy regime (HZE). In this short review we will give examples for the application of charged particle irradiation to study spatiotemporal aspects of DNA damage recognition and repair in the context of recent achievements in this field. Beamline microscopy allows determining the exact kinetics of repair-related proteins after irradiation with different charged particles that induce different lesion densities. The classification into fast recruited proteins like DNA-PK or XRCC1 or slower recruited ones like 53BP1 or MDC1 helps to establish the hierarchical organisation of damage recognition and subsequent repair events. Additionally, motional analysis of DNA lesions induced by traversing particles proved information about the mobility of DSBs. Increased mobility or the absence of large scale motion has direct consequences on the formation of chromosomal translocations and, thus, on mechanisms of cancer formation. Charged particle microbeams offer the interesting perspective of precise nuclear or subnuclear targeting with a defined number of ions, avoiding the Poisson distribution of traversals inherent to broad beam experiments. With the help of the microbeam, geometrical patterns of traversing ions can be applied facilitating the analysis of spatial organisation of repair. PMID:19944777

Tobias, F; Durante, M; Taucher-Scholz, G; Jakob, B

2010-01-01

203

Measurement of DNA repair deficiency in workers exposed to benzene.  

PubMed Central

We hypothesize that chronic exposure to environmental toxicants can induce genetic damage causing DNA repair deficiencies and leading to the postulated mutator phenotype of carcinogenesis. To test our hypothesis, a host cell reactivation (HCR) assay was used in which pCMVcat plasmids were damaged with UV light (175, 350 J/m2 UV light), inactivating the chloramphenicol acetyltransferase reporter gene, and then transfected into lymphocytes. Transfected lymphocytes were therefore challenged to repair the damaged plasmids, reactivating the reporter gene. Xeroderma pigmentosum (XP) and Gaucher cell lines were used as positive and negative controls for the HCR assay. The Gaucher cell line repaired normally but XP cell lines demonstrated lower repair activity. Additionally, the repair activity of the XP heterozygous cell line showed intermediate repair compared to the homozygous XP and Gaucher cells. We used HCR to measure the effects of benzene exposure on 12 exposed and 8 nonexposed workers from a local benzene plant. Plasmids 175 J/m2 and 350 J/m2 were repaired with a mean frequency of 66% and 58%, respectively, in control workers compared to 71% and 62% in exposed workers. Conversely, more of the exposed workers were grouped into the reduced repair category than controls. These differences in repair capacity between exposed and control workers were, however, not statistically significant. The lack of significant differences between the exposed and control groups may be due to extremely low exposure to benzene (< 0.3 ppm), small population size, or a lack of benzene genotoxicity at these concentrations. These results are consistent with a parallel hprt gene mutation assay. PMID:8781377

Hallberg, L M; el Zein, R; Grossman, L; Au, W W

1996-01-01

204

BLM–DNA2–RPA–MRN and EXO1–BLM–RPA–MRN constitute two DNA end resection machineries for human DNA break repair  

PubMed Central

Repair of dsDNA breaks requires processing to produce 3?-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, BLM and DNA2 physically and specifically interact to resect DNA in a process that is ATP-dependent and requires BLM helicase and DNA2 nuclease functions. RPA is essential for both DNA unwinding by BLM and enforcing 5? ? 3? resection polarity by DNA2. MRN accelerates processing by recruiting BLM to the end. In the other, EXO1 resects the DNA and is stimulated by BLM, MRN, and RPA. BLM increases the affinity of EXO1 for ends, and MRN recruits and enhances the processivity of EXO1. Our results establish two of the core machineries that initiate recombinational DNA repair in human cells. PMID:21325134

Nimonkar, Amitabh V.; Genschel, Jochen; Kinoshita, Eri; Polaczek, Piotr; Campbell, Judith L.; Wyman, Claire; Modrich, Paul; Kowalczykowski, Stephen C.

2011-01-01

205

Mre11 Dimers Coordinate DNA End Bridging and Nuclease Processing in Double-Strand-Break Repair  

PubMed Central

SUMMARY Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To de?ne the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of ?ssion yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD). PMID:18854158

Williams, R. Scott; Moncalian, Gabriel; Williams, Jessica S.; Yamada, Yoshiki; Limbo, Oliver; Shin, David S.; Groocock, Lynda M.; Cahill, Dana; Hitomi, Chiharu; Guenther, Grant; Moiani, Davide; Carney, James P.; Russell, Paul; Tainer, John A.

2008-01-01

206

Hypomorphic PCNA mutation underlies a human DNA repair disorder  

PubMed Central

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA’s interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration. PMID:24911150

Baple, Emma L.; Chambers, Helen; Cross, Harold E.; Fawcett, Heather; Nakazawa, Yuka; Chioza, Barry A.; Harlalka, Gaurav V.; Mansour, Sahar; Sreekantan-Nair, Ajith; Patton, Michael A.; Muggenthaler, Martina; Rich, Phillip; Wagner, Karin; Coblentz, Roselyn; Stein, Constance K.; Last, James I.; Taylor, A. Malcolm R.; Jackson, Andrew P.; Ogi, Tomoo; Lehmann, Alan R.; Green, Catherine M.; Crosby, Andrew H.

2014-01-01

207

Hypomorphic PCNA mutation underlies a human DNA repair disorder.  

PubMed

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA's interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration. PMID:24911150

Baple, Emma L; Chambers, Helen; Cross, Harold E; Fawcett, Heather; Nakazawa, Yuka; Chioza, Barry A; Harlalka, Gaurav V; Mansour, Sahar; Sreekantan-Nair, Ajith; Patton, Michael A; Muggenthaler, Martina; Rich, Phillip; Wagner, Karin; Coblentz, Roselyn; Stein, Constance K; Last, James I; Taylor, A Malcolm R; Jackson, Andrew P; Ogi, Tomoo; Lehmann, Alan R; Green, Catherine M; Crosby, Andrew H

2014-07-01

208

Cycling with BRCA2 from DNA repair to mitosis.  

PubMed

Genetic integrity in proliferating cells is guaranteed by the harmony of DNA replication, appropriate DNA repair, and segregation of the duplicated genome. Breast cancer susceptibility gene BRCA2 is a unique tumor suppressor that is involved in all three processes. Hence, it is critical in genome maintenance. The functions of BRCA2 in DNA repair and homology-directed recombination (HDR) have been reviewed numerous times. Here, I will briefly go through the functions of BRCA2 in HDR and focus on the emerging roles of BRCA2 in telomere homeostasis and mitosis, then discuss how BRCA2 exerts distinct functions in a cell-cycle specific manner in the maintenance of genomic integrity. PMID:25447315

Lee, Hyunsook

2014-11-15

209

The MRN complex in Double-Strand Break Repair and Telomere Maintenance  

PubMed Central

Genomes are subject to constant threat by damaging agents that generate DNA double-strand breaks (DSBs). The ends of linear chromosomes need to be protected from DNA damage recognition and end-joining, and this is achieved through protein-DNA complexes known as telomeres. The Mre11-Rad50-Nbs1 (MRN) complex plays important roles in detection and signaling of DSBs, as well as the repair pathways of homologous recombination (HR) and non-homologous end joining (NHEJ). In addition, MRN associates with telomeres and contributes to their maintenance. Here we provide an overview of MRN functions at DSBs, and examine its roles in telomere maintenance and dysfunction. PMID:20655309

Lamarche, Brandon J; Orazio, Nicole I; Weitzman, Matthew D

2010-01-01

210

Molecular Understanding of Efficient DNA Repair Machinery of Photolyase  

NASA Astrophysics Data System (ADS)

Photolyases repair the UV-induced pyrimidine dimers in damage DNA with high efficiency, through a cylic light-driven electron transfer radical mechanism. We report here our systematic studies of the repair dynamics in E. coli photolyase with mutation of five active-site residues. The significant loss of repair efficiency by the mutation indicates that those active-site residues play an important role in the DNA repair by photolyase. To understand how the active-site residues modulate the efficiency, we mapped out the entire evolution of each elementary step during the repair in those photolyase mutants with femtosecond resolution. We completely analyzed the electron transfer dynamics using the Sumi-Marcus model. The results suggest that photolyase controls the critical electron transfer and the ring-splitting of pyrimidine dimer through modulation of the redox potentials and reorganization energies, and stabilization of the anionic intermediates, maintaining the dedicated balance of all the reaction steps and achieving the maximum function activity.

Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Zhong, Dongping

2012-06-01

211

A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia  

PubMed Central

DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair. PMID:20613862

S?abicki, Miko?aj; Theis, Mirko; Krastev, Dragomir B.; Samsonov, Sergey; Mundwiller, Emeline; Junqueira, Magno; Paszkowski-Rogacz, Maciej; Teyra, Joan; Heninger, Anne-Kristin; Poser, Ina; Prieur, Fabienne; Truchetto, Jérémy; Confavreux, Christian; Marelli, Cécilia; Durr, Alexandra; Camdessanche, Jean Philippe; Brice, Alexis; Shevchenko, Andrej; Pisabarro, M. Teresa; Stevanin, Giovanni; Buchholz, Frank

2010-01-01

212

Large conformational changes in MutS during DNA scanning, mismatch recognition and repair signalling  

PubMed Central

MutS protein recognizes mispaired bases in DNA and targets them for mismatch repair. Little is known about the transient conformations of MutS as it signals initiation of repair. We have used single-molecule fluorescence resonance energy transfer (FRET) measurements to report the conformational dynamics of MutS during this process. We find that the DNA-binding domains of MutS dynamically interconvert among multiple conformations when the protein is free and while it scans homoduplex DNA. Mismatch recognition restricts MutS conformation to a single state. Steady-state measurements in the presence of nucleotides suggest that both ATP and ADP must be bound to MutS during its conversion to a sliding clamp form that signals repair. The transition from mismatch recognition to the sliding clamp occurs via two sequential conformational changes. These intermediate conformations of the MutS:DNA complex persist for seconds, providing ample opportunity for interaction with downstream proteins required for repair. PMID:22505031

Qiu, Ruoyi; DeRocco, Vanessa C; Harris, Credle; Sharma, Anushi; Hingorani, Manju M; Erie, Dorothy A; Weninger, Keith R

2012-01-01

213

Sumoylation Influences DNA Break Repair Partly by Increasing the Solubility of a Conserved End Resection Protein  

PubMed Central

Protein modifications regulate both DNA repair levels and pathway choice. How each modification achieves regulatory effects and how different modifications collaborate with each other are important questions to be answered. Here, we show that sumoylation regulates double-strand break repair partly by modifying the end resection factor Sae2. This modification is conserved from yeast to humans, and is induced by DNA damage. We mapped the sumoylation site of Sae2 to a single lysine in its self-association domain. Abolishing Sae2 sumoylation by mutating this lysine to arginine impaired Sae2 function in the processing and repair of multiple types of DNA breaks. We found that Sae2 sumoylation occurs independently of its phosphorylation, and the two modifications act in synergy to increase soluble forms of Sae2. We also provide evidence that sumoylation of the Sae2-binding nuclease, the Mre11-Rad50-Xrs2 complex, further increases end resection. These findings reveal a novel role for sumoylation in DNA repair by regulating the solubility of an end resection factor. They also show that collaboration between different modifications and among multiple substrates leads to a stronger biological effect. PMID:25569253

Sarangi, Prabha; Steinacher, Roland; Altmannova, Veronika; Fu, Qiong; Paull, Tanya T.; Krejci, Lumir; Whitby, Matthew C.; Zhao, Xiaolan

2015-01-01

214

Monitoring Repair of UV-Induced 6-4-Photoproducts with a Purified DDB2 Protein Complex  

PubMed Central

Because cells are constantly subjected to DNA damaging insults, DNA repair pathways are critical for genome integrity [1]. DNA damage recognition protein complexes (DRCs) recognize DNA damage and initiate DNA repair. The DNA-Damage Binding protein 2 (DDB2) complex is a DRC that initiates nucleotide excision repair (NER) of DNA damage caused by ultraviolet light (UV) [2]–[4]. Using a purified DDB2 DRC, we created a probe (“DDB2 proteo-probe”) that hybridizes to nuclei of cells irradiated with UV and not to cells exposed to other genotoxins. The DDB2 proteo-probe recognized UV-irradiated DNA in classical laboratory assays, including cyto- and histo-chemistry, flow cytometry, and slot-blotting. When immobilized, the proteo-probe also bound soluble UV-irradiated DNA in ELISA-like and DNA pull-down assays. In vitro, the DDB2 proteo-probe preferentially bound 6-4-photoproducts [(6-4)PPs] rather than cyclobutane pyrimidine dimers (CPDs). We followed UV-damage repair by cyto-chemistry in cells fixed at different time after UV irradiation, using either the DDB2 proteo-probe or antibodies against CPDs, or (6-4)PPs. The signals obtained with the DDB2 proteo-probe and with the antibody against (6-4)PPs decreased in a nearly identical manner. Since (6-4)PPs are repaired only by nucleotide excision repair (NER), our results strongly suggest the DDB2 proteo-probe hybridizes to DNA containing (6-4)PPs and allows monitoring of their removal during NER. We discuss the general use of purified DRCs as probes, in lieu of antibodies, to recognize and monitor DNA damage and repair. PMID:24489677

Dreze, Matija; Calkins, Anne S.; Gálicza, Judit; Echelman, Daniel J.; Schnorenberg, Mathew R.; Fell, Gillian L.; Iwai, Shigenori; Fisher, David E.; Szüts, David; Iglehart, J. Dirk; Lazaro, Jean-Bernard

2014-01-01

215

Impact of DNA3'pp5'G capping on repair reactions at DNA 3' ends.  

PubMed

Many biological scenarios generate "dirty" DNA 3'-PO4 ends that cannot be sealed by classic DNA ligases or extended by DNA polymerases. The noncanonical ligase RtcB can "cap" these ends via a unique chemical mechanism entailing transfer of GMP from a covalent RtcB-GMP intermediate to a DNA 3'-PO4 to form DNA3'pp5'G. Here, we show that capping protects DNA 3' ends from resection by Escherichia coli exonucleases I and III and from end-healing by T4 polynucleotide 3' phosphatase. By contrast, the cap is an effective primer for DNA synthesis. E. coli DNA polymerase I and Mycobacterium DinB1 extend the DNAppG primer to form an alkali-labile DNApp(rG)pDNA product. The addition of dNTP depends on pairing of the cap guanine with an opposing cytosine in the template strand. Aprataxin, an enzyme implicated in repair of A5'pp5'DNA ends formed during abortive ligation by classic ligases, is highly effective as a DNA 3' decapping enzyme, converting DNAppG to DNA3'p and GMP. We conclude that the biochemical impact of DNA capping is to prevent resection and healing of a 3'-PO4 end, while permitting DNA synthesis, at the price of embedding a ribonucleotide and a pyrophosphate linkage in the repaired strand. Aprataxin affords a means to counter the impact of DNA capping. PMID:25049385

Das, Ushati; Chauleau, Mathieu; Ordonez, Heather; Shuman, Stewart

2014-08-01

216

Requirement of the Saccharomyces cerevisiae APN1 Gene for the Repair of Mitochondrial DNA Alkylation Damage  

PubMed Central

The Saccharomyces cerevisiae APN1 gene that participates in base excision repair has been localized both in the nucleus and the mitochondria. APN1 deficient cells (apn1?) show increased mutation frequencies in mitochondrial DNA (mtDNA) suggesting that APN1 is also important for mtDNA stability. To understand APN1-dependent mtDNA repair processes we studied the formation and repair of mtDNA lesions in cells exposed to methyl methanesulfonate (MMS). We show that MMS induces mtDNA damage in a dose-dependent fashion and that deletion of the APN1 gene enhances the susceptibility of mtDNA to MMS. Repair kinetic experiments demonstrate that in wild-type cells (WT) it takes 4 hr to repair the damage induced by 0.1% MMS, whereas in the apn1? strain there is a lag in mtDNA repair that results in significant differences in the repair capacity between the two yeast strains. Analysis of lesions in nuclear DNA (nDNA) after treatment with 0.1% MMS shows a significant difference in the amount of nDNA lesions between WT and apn1? cells. Interestingly, comparisons between nDNA and mtDNA damage show that nDNA is more sensitive to the effects of MMS treatment. However, both strains are able to repair the nDNA lesions, contrary to mtDNA repair, which is compromised in the apn1? mutant strain. Therefore, although nDNA is more sensitive than mtDNA to the effects of MMS, deletion of APN1 has a stronger phenotype in mtDNA repair than in nDNA. These results highlight the prominent role of APN1 in the repair of environmentally induced mtDNA damage. PMID:19197988

Acevedo-Torres, Karina; Fonseca-Williams, Sharon; Ayala-Torres, Sylvette; Torres-Ramos, Carlos A.

2010-01-01

217

Deficient repair of chemical adducts in alpha DNA of monkey cells  

SciTech Connect

Researchers have examined excision repair of DNA damage in the highly repeated alpha DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in alpha DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for alpha DNA and bulk DNA. In cells treated with furocoumarins and long-wave-length ultraviolet light, however, repair synthesis in alpha DNA was only 30% of that in bulk DNA, although it followed the same time course. Researchers found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as researchers found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from alpha DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in alpha DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences.

Zolan, M.E.; Cortopassi, G.A.; Smith, C.A.; Hanawalt, P.C.

1982-03-01

218

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals  

PubMed Central

DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flash-cooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging. PMID:24101514

Hura, Greg L.; Tsai, Chi-Lin; Claridge, Shelley A.; Mendillo, Marc L.; Smith, Jessica M.; Williams, Gareth J.; Mastroianni, Alexander J.; Alivisatos, A. Paul; Putnam, Christopher D.; Kolodner, Richard D.; Tainer, John A.

2013-01-01

219

Low-Dose Formaldehyde Delays DNA Damage Recognition and DNA Excision Repair in Human Cells  

PubMed Central

Objective Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions. Methodology/Principal Findings The overall mobility of the DNA damage sensors UV-DDB (ultraviolet-damaged DNA-binding) and XPC (xeroderma pigmentosum group C) was analyzed by assessing real-time protein dynamics in the nucleus of cultured human cells exposed to non-cytotoxic (<100 ?M) formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair. Conclusions/Significance A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks. PMID:24722772

Luch, Andreas; Frey, Flurina C. Clement; Meier, Regula; Fei, Jia; Naegeli, Hanspeter

2014-01-01

220

Recruitment of the putative transcription-repair coupling factor CSB/ERCC6 to RNA polymerase II elongation complexes.  

PubMed Central

Cockayne's syndrome (CS) is a disease characterized by developmental and growth defects, sunlight sensitivity, and a defect in transcription-coupled nucleotide excision repair. The two principle proteins involved in CS, CSA and CSB/ERCC6, have been hypothesized to bind RNA polymerase II (Pol II) and link transcription to DNA repair. We have tested CSA and CSB in assays designed to determine their role in transcription-coupled repair. Using a unique oligo(dC)-tailed DNA template, we provide biochemical evidence that CSB/ERCC6 interacts with Pol II molecules engaged in ternary complexes containing DNA and nascent RNA. CSB is a DNA-activated ATPase, and hydrolysis of the ATP beta-gamma phosphoanhydride bond is required for the formation of a stable Pol II-CSB-DNA-RNA complex. Unlike CSB, CSA does not directly bind Pol II. PMID:9372911

Tantin, D; Kansal, A; Carey, M

1997-01-01

221

Structural insights into recognition and repair of UV-DNA damage by Spore Photoproduct Lyase, a radical SAM enzyme  

PubMed Central

Bacterial spores possess an enormous resistance to ultraviolet (UV) radiation. This is largely due to a unique DNA repair enzyme, Spore Photoproduct Lyase (SP lyase) that repairs a specific UV-induced DNA lesion, the spore photoproduct (SP), through an unprecedented radical-based mechanism. Unlike DNA photolyases, SP lyase belongs to the emerging superfamily of radical S-adenosyl-l-methionine (SAM) enzymes and uses a [4Fe–4S]1+ cluster and SAM to initiate the repair reaction. We report here the first crystal structure of this enigmatic enzyme in complex with its [4Fe–4S] cluster and its SAM cofactor, in the absence and presence of a DNA lesion, the dinucleoside SP. The high resolution structures provide fundamental insights into the active site, the DNA lesion recognition and binding which involve a ?-hairpin structure. We show that SAM and a conserved cysteine residue are perfectly positioned in the active site for hydrogen atom abstraction from the dihydrothymine residue of the lesion and donation to the ?-thyminyl radical moiety, respectively. Based on structural and biochemical characterizations of mutant proteins, we substantiate the role of this cysteine in the enzymatic mechanism. Our structure reveals how SP lyase combines specific features of radical SAM and DNA repair enzymes to enable a complex radical-based repair reaction to take place. PMID:22761404

Benjdia, Alhosna; Heil, Korbinian; Barends, Thomas R. M.; Carell, Thomas; Schlichting, Ilme

2012-01-01

222

Oncogene-triggered suppression of DNA repair leads to DNA instability in cancer  

PubMed Central

DNA instability is an important contributor to cancer development. Previously, defects in the chromosome segregation and excessive DNA double strand breaks due to the replication or oxidative stresses were implicated in DNA instability in cancer. Here, we demonstrate that DNA instability can directly result from the oncogene-induced senescence signaling. Expression of the activated form of Her2 oncogene, NeuT, in immortalized breast epithelial cells led to downregulation of the major DNA repair factor histone H2AX and a number of other components of the HR and NHEJ double strand DNA breaks repair pathways. H2AX expression was regulated at the transcriptional level via a senescence pathway involving p21-mediated regulation of CDK and Rb1. The p21-dependent downregulation of H2AX was seen both in cell culture and the MMTV-neu mouse model of Her2-positive breast cancer. Importantly, downregulation of H2AX upon Her2/NeuT expression impaired repair of double strand DNA breaks. This impairment resulted in both increased DNA instability in the form of somatic copy number alterations, and in increased sensitivity to the chemotherapeutic drug doxorubicin. Overall, these findings indicate that the Her2/NeuT oncogene signaling directly potentiates DNA instability and increases sensitivity to DNA damaging treatments. PMID:25252808

Yaglom, Julia A.; McFarland, Christopher; Mirny, Leonid; Sherman, Michael Y.

2014-01-01

223

8-Oxoguanine DNA glycosylase-1-mediated DNA repair is associated with Rho GTPase activation and ?-smooth muscle actin polymerization  

PubMed Central

Reactive oxygen species (ROS) are activators of cell signaling and modify cellular molecules, including DNA. 8-Oxo-7,8-dihydroguanine (8-oxoG) is one of the prominent lesions in oxidatively damaged DNA, whose accumulation is causally linked to various diseases and aging processes, whereas its etiological relevance is unclear. 8-OxoG is repaired by the 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated DNA base excision repair (BER) pathway. OGG1 binds free 8-oxoG and this complex functions as an activator of Ras family GTPases. Here we examined whether OGG1-initiated BER is associated with the activation of Rho GTPase and mediates changes in the cytoskeleton. To test this possibility, we induced OGG1-initiated BER in cultured cells and mouse lungs and used molecular approaches such as active Rho pull-down assays, siRNA ablation of gene expression, immune blotting, and microscopic imaging. We found that OGG1 physically interacts with Rho GTPase and, in the presence of 8-oxoG base, increases Rho–GTP levels in cultured cells and lungs, which mediates ?-smooth muscle actin (?-SMA) polymerization into stress fibers and increases the level of ?-SMA in insoluble cellular/tissue fractions. These changes were absent in cells lacking OGG1. These unexpected data and those showing that 8-oxoG repair is a lifetime process suggest that, via Rho GTPase, OGG1 could be involved in the cytoskeletal changes and organ remodeling observed in various chronic diseases. PMID:24681335

Luo, Jixian; Hosoki, Koa; Bacsi, Attila; Radak, Zsolt; Hegde, Muralidhar L.; Sur, Sanjiv; Hazra, Tapas K.; Brasier, Allan R.; Ba, Xueqing; Boldogh, Istvan

2014-01-01

224

DNA mismatch repair: Molecular mechanism, cancer, and ageing  

PubMed Central

DNA mismatch repair (MMR) proteins are ubiquitous players in a diverse array of important cellular functions. In its role in post-replication repair, MMR safeguards the genome correcting base mispairs arising as a result of replication errors. Loss of MMR results in greatly increased rates of spontaneous mutation in organisms ranging from bacteria to humans. Mutations in MMR genes cause hereditary nonpolyposis colorectal cancer, and loss of MMR is associated with a significant fraction of sporadic cancers. Given its prominence in mutation avoidance and its ability to target a range of DNA lesions, MMR has been under investigation in studies of ageing mechanisms. This review summarizes what is known about the molecular details of the MMR pathway and the role of MMR proteins in cancer susceptibility and ageing. PMID:18406444

Hsieh, Peggy; Yamane, Kazuhiko

2008-01-01

225

DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide  

SciTech Connect

The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide.

Hagensee, M.E.; Bryan, S.K.; Moses, R.E.

1987-10-01

226

Metal complex interactions with DNA.  

PubMed

Increasing numbers of DNA structures are being revealed using biophysical, spectroscopic and genomic methods. The diversity of transition metal complexes is also growing, as the unique contributions that transition metals bring to the overall structure of metal complexes depend on the various coordination numbers, geometries, physiologically relevant redox potentials, as well as kinetic and thermodynamic characteristics. The vast range of ligands that can be utilised must also be considered. Given this diversity, a variety of biological interactions is not unexpected. Specifically, interactions with negatively-charged DNA can arise due to covalent/coordinate or subtle non-coordinate interactions such as electrostatic attraction, groove binding and intercalation as well as combinations of all of these modes. The potential of metal complexes as therapeutic agents is but one aspect of their utility. Complexes, both new and old, are currently being utilised in conjunction with spectroscopic and biological techniques to probe the interactions of DNA and its many structural forms. Here we present a review of metal complex-DNA interactions in which several binding modes and DNA structural forms are explored. PMID:25427534

Pages, Benjamin J; Ang, Dale L; Wright, Elisé P; Aldrich-Wright, Janice R

2015-02-10

227

A novel cell permeable DNA replication and repair marker.  

PubMed

Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells. PMID:25484186

Herce, Henry D; Rajan, Malini; Lättig-Tünnemann, Gisela; Fillies, Marion; Cardoso, M Cristina

2014-11-01

228

A novel cell permeable DNA replication and repair marker.  

PubMed

Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells. PMID:25184478

Herce, Henry D; Rajan, Malini; Lättig-Tünnemann, Gisela; Fillies, Marion; Cardoso, M Cristina

2014-09-01

229

Long-patch DNA repair synthesis during base excision repair in mammalian cells  

PubMed Central

The base excision repair (BER) process removes base damage such as oxidation, alkylation or abasic sites. Two BER sub-pathways have been characterized using in vitro methods, and have been classified according to the length of the repair patch as either 'short-patch' BER (one nucleotide) or 'long-patch' BER (LP-BER; more than one nucleotide). To investigate the occurrence of LP-BER in vivo, we developed an assay using a plasmid containing a single modified base in the transcribed strand of the enhanced green fluorescent protein (EGFP) gene and a stop codon, based on a single-nucleotide mismatch, at varying distances on the 3? side of the lesion. The reversion of the stop codon occurs after DNA repair synthesis and restores EGFP expression after transfection of mismatch-repair-deficient cells. Repair patches longer than one nucleotide were observed for 55–80% or 80–100% of the plasmids with a mean length of 2–6 or 6–12 nucleotides for 8-oxo-7,8-dihydroguanine or a synthetic abasic site, respectively. These data show the existence of LP-BER in vivo, and emphasize the effect of the type of BER substrate lesion on both the yield and the extent of the LP-BER sub-pathway. PMID:12671676

Sattler, Ulrike; Frit, Philippe; Salles, Bernard; Calsou, Patrick

2003-01-01

230

Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA  

Microsoft Academic Search

Repair of DNA damage is essential for maintaining genome integrity, and repair deficiencies in mammals are associated with cancer, neurological disease and developmental defects. Alkylation damage in DNA is repaired by at least three different mechanisms, including damage reversal by oxidative demethylation of 1-methyladenine and 3-methylcytosine by Escherichia coli AlkB. By contrast, little is known about consequences and cellular handling

Per Arne Aas; Marit Otterlei; Pål Ø. Falnes; Cathrine B. Vågbø; Frank Skorpen; Mansour Akbari; Ottar Sundheim; Magnar Bjørås; Geir Slupphaug; Erling Seeberg; Hans E. Krokan

2003-01-01

231

Mechanisms of DNA double strand break repair and chromosome aberration formation  

Microsoft Academic Search

It is widely accepted that unrepaired or misrepaired DNA double strand breaks (DSBs) lead to the formation of chromosome aberrations. DSBs induced in the DNA of higher eukaryotes by endogenous processes or exogenous agents can in principle be repaired either by non-homologous endjoining (NHEJ), or homology directed repair (HDR). The basis on which the selection of the DSB repair pathway

G. Iliakis; H. Wang; A. R. Perrault; W. Boecker; B. Rosidi; F. Windhofer; W. Wu; J. Guan; G. Terzoudi; G. Pantelias

2004-01-01

232

A potential copper-regulatory role for cytosolic expression of the DNA repair protein XRCC5.  

PubMed

Copper (Cu) has a critical role in the generation of oxidative stress during neurodegeneration and cancer. Reactive oxygen species generated through abnormal elevation or deficiency of Cu can lead to lipid, protein, and DNA damage. Oxidation of DNA can induce strand breaks and is associated with altered cell fate including transformation or death. DNA repair is mediated through the action of the multimeric DNA-PK repair complex. The components of this complex are the Ku autoantigens, XRCC5 and XRCC6 (Ku80 and Ku70, respectively). How this repair complex responds to perturbed Cu homeostasis and Cu-mediated oxidative stress has not been investigated. We previously reported that XRCC5 expression is altered in response to cellular Cu levels, with low Cu inhibiting XRCC5 expression and high Cu levels enhancing expression. In this study we further investigated the interaction between XRCC5 and Cu. We report that cytosolic XRCC5 is increased in response to Cu, but not zinc, iron, or nickel, and the level of cytosolic XRCC5 correlates with protection against oxidative damage to DNA. These observations were made in both HeLa cells and fibroblasts. Cytosolic XRCC5 interacted with the Cu chaperone and detoxification protein human Atox1 homologue (HAH), and down regulation of XRCC5 expression using siRNA led to enhanced HAH expression when cells were exposed to Cu. XRCC5 could also be purified from cytosolic extracts using a Cu-loaded column. These findings provide further evidence that cytosolic XRCC5 has a key role in protection against DNA oxidation from Cu, through either direct sequestration or signaling through other Cu-detoxification molecules. Our findings have important implications for the development of therapeutic treatments targeting Cu in neurodegeneration and/or cancer. PMID:21971347

Du, Tai; Caragounis, Aphrodite; Parker, Sarah J; Meyerowitz, Jodi; La Fontaine, Sharon; Kanninen, Katja M; Perreau, Victoria M; Crouch, Peter J; White, Anthony R

2011-12-01

233

A second DNA methyltransferase repair enzyme in Escherichia coli.  

PubMed Central

The Escherichia coli ada-alkB operon encodes a 39-kDa protein (Ada) that is a DNA-repair methyltransferase and a 27-kDa protein (AlkB) of unknown function. By DNA blot hybridization analysis we show that the alkylation-sensitive E. coli mutant BS23 [Sedgwick, B. & Lindahl, T. (1982) J. Mol. Biol. 154, 169-175] is a deletion mutant lacking the entire ada-alkB operon. Despite the absence of the ada gene and its product, the cells contain detectable levels of a DNA-repair methyltransferase activity. We conclude that the methyltransferase in BS23 cells is the product of a gene other than ada. A similar activity was detected in extracts of an ada-10::Tn10 insertion mutant of E. coli AB1157. This DNA methyltransferase has a molecular mass of about 19 kDa and transfers the methyl groups from O6-methylguanine and O4-methylthymine in DNA, but not those from methyl phosphotriester lesions. This enzyme was not induced by low doses of alkylating agent and is expressed at low levels in ada+ and a number of ada- E. coli strains. Images PMID:3283737

Rebeck, G W; Coons, S; Carroll, P; Samson, L

1988-01-01

234

Studying the organization of DNA repair by single-cell and single-molecule imaging  

PubMed Central

DNA repair safeguards the genome against a diversity of DNA damaging agents. Although the mechanisms of many repair proteins have been examined separately in vitro, far less is known about the coordinated function of the whole repair machinery in vivo. Furthermore, single-cell studies indicate that DNA damage responses generate substantial variation in repair activities across cells. This review focuses on fluorescence imaging methods that offer a quantitative description of DNA repair in single cells by measuring protein concentrations, diffusion characteristics, localizations, interactions, and enzymatic rates. Emerging single-molecule and super-resolution microscopy methods now permit direct visualization of individual proteins and DNA repair events in vivo. We expect much can be learned about the organization of DNA repair by linking cell heterogeneity to mechanistic observations at the molecular level. PMID:24629485

Uphoff, Stephan; Kapanidis, Achillefs N.

2014-01-01

235

Clinical Radiation Sensitivity With DNA Repair Disorders: An Overview  

SciTech Connect

Adverse reactions to radiotherapy represent a confounding phenomenon in radiation oncology. These reactions are rare, and many have been associated with individuals with DNA repair disorders such as ataxia-telangiectasia and Nijmegen Breakage syndrome. A paucity of published data is available detailing such circumstances. This overview describes four exemplary situations, a comprehensive list of 32 additional cases, and some insights gleaned from this overall experience. Fanconi anemia was associated with more than one-half of the reports. The lowest dose given to a patient that resulted in a reaction was 3 Gy, given to an ataxia-telangiectasia patient. Most patients died within months of exposure. It is clear that the patients discussed in this report had complicated illnesses, in addition to cancer, and the radiotherapy administered was most likely their best option. However, the underlying DNA repair defects make conventional radiation doses dangerous. Our findings support previous wisdom that radiotherapy should either be avoided or the doses should be selected with great care in the case of these radiosensitive genotypes, which must be recognized by their characteristic phenotypes, until more rapid, reliable, and functional assays of DNA repair become available.

Pollard, Julianne M. [Department of Radiation Physics, University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); Biomedical Physics Interdepartmental Graduate Program, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA (United States); Department of Pathology and Laboratory Medicine, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA (United States); Gatti, Richard A. [Department of Pathology and Laboratory Medicine, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA (United States); Department of Human Genetics, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA (United States)], E-mail: rgatti@mednet.ucla.edu

2009-08-01

236

DNA repair inhibition by UVA photoactivated fluoroquinolones and vemurafenib.  

PubMed

Cutaneous photosensitization is a common side effect of drug treatment and can be associated with an increased skin cancer risk. The immunosuppressant azathioprine, the fluoroquinolone antibiotics and vemurafenib-a BRAF inhibitor used to treat metastatic melanoma-are all recognized clinical photosensitizers. We have compared the effects of UVA radiation on cultured human cells treated with 6-thioguanine (6-TG, a DNA-embedded azathioprine surrogate), the fluoroquinolones ciprofloxacin and ofloxacin and vemurafenib. Despite widely different structures and modes of action, each of these drugs potentiated UVA cytotoxicity. UVA photoactivation of 6-TG, ciprofloxacin and ofloxacin was associated with the generation of singlet oxygen that caused extensive protein oxidation. In particular, these treatments were associated with damage to DNA repair proteins that reduced the efficiency of nucleotide excision repair. Although vemurafenib was also highly phototoxic to cultured cells, its effects were less dependent on singlet oxygen. Highly toxic combinations of vemurafenib and UVA caused little protein carbonylation but were nevertheless inhibitory to nucleotide excision repair. Thus, for three different classes of drugs, photosensitization by at least two distinct mechanisms is associated with reduced protection against potentially mutagenic and carcinogenic DNA damage. PMID:25414333

Peacock, Matthew; Brem, Reto; Macpherson, Peter; Karran, Peter

2014-12-16

237

DNA repair inhibition by UVA photoactivated fluoroquinolones and vemurafenib  

PubMed Central

Cutaneous photosensitization is a common side effect of drug treatment and can be associated with an increased skin cancer risk. The immunosuppressant azathioprine, the fluoroquinolone antibiotics and vemurafenib—a BRAF inhibitor used to treat metastatic melanoma—are all recognized clinical photosensitizers. We have compared the effects of UVA radiation on cultured human cells treated with 6-thioguanine (6-TG, a DNA-embedded azathioprine surrogate), the fluoroquinolones ciprofloxacin and ofloxacin and vemurafenib. Despite widely different structures and modes of action, each of these drugs potentiated UVA cytotoxicity. UVA photoactivation of 6-TG, ciprofloxacin and ofloxacin was associated with the generation of singlet oxygen that caused extensive protein oxidation. In particular, these treatments were associated with damage to DNA repair proteins that reduced the efficiency of nucleotide excision repair. Although vemurafenib was also highly phototoxic to cultured cells, its effects were less dependent on singlet oxygen. Highly toxic combinations of vemurafenib and UVA caused little protein carbonylation but were nevertheless inhibitory to nucleotide excision repair. Thus, for three different classes of drugs, photosensitization by at least two distinct mechanisms is associated with reduced protection against potentially mutagenic and carcinogenic DNA damage. PMID:25414333

Peacock, Matthew; Brem, Reto; Macpherson, Peter; Karran, Peter

2014-01-01

238

The complexities of DNA computation.  

PubMed

Over the past few years, a handful of insightful researchers have bridged the gap between biological computing theory and actual DNA-based computation. By using ingenious encoding techniques and clever molecular-biological manipulations, simple versions of computationally complex problems have been experimentally approached or resolved. However, the technical problems revealed during the execution of these scientific set pieces make it unlikely that DNA will ever rival silicon for the solution of any real-world problem. PMID:10203773

Cox, J C; Cohen, D S; Ellington, A D

1999-04-01

239

Visualization of a DNA-PK/PARP1 complex  

PubMed Central

The DNA-dependent protein kinase (DNA-PK) and Poly(ADP-ribose) polymerase-1 (PARP1) are critical enzymes that reduce genomic damage caused by DNA lesions. They are both activated by DNA strand breaks generated by physiological and environmental factors, and they have been shown to interact. Here, we report in vivo evidence that DNA-PK and PARP1 are equally necessary for rapid repair. We purified a DNA-PK/PARP1 complex loaded on DNA and performed electron microscopy and single particle analysis on its tetrameric and dimer-of-tetramers forms. By comparison with the DNA-PK holoenzyme and fitting crystallographic structures, we see that the PARP1 density is in close contact with the Ku subunit. Crucially, PARP1 binding elicits substantial conformational changes in the DNA-PK synaptic dimer assembly. Taken together, our data support a functional, in-pathway role for DNA-PK and PARP1 in double-strand break (DSB) repair. We also propose a NHEJ model where protein–protein interactions alter substantially the architecture of DNA-PK dimers at DSBs, to trigger subsequent interactions or enzymatic reactions. PMID:22223246

Spagnolo, Laura; Barbeau, Jody; Curtin, Nicola J.; Morris, Edward P.; Pearl, Laurence H.

2012-01-01

240

Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes  

PubMed Central

DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on >95% inhibition of 8-oxodeoxyguosine (8-oxodG) and other unidentified oxidative DNA adducts induced by 4-hydroxy-17ß-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 ?M) than that for 8-oxodG (100 ?M). In the in vivo study, female CD-1 mice (n=6) were fed either a control diet or diet supplemented with ellagic acid (400 ppm) and dehydrated berries (5% w/w) with varying ellagic acid contents – blueberry (low), strawberry (medium) and red raspberry (high), for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%). However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001) and 48% (p < 0.01), respectively. Both diets also resulted in a 3–8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA), DNA excision repair protein (ERCC5) and DNA ligase III (DNL3). These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair. PMID:19325752

Aiyer, Harini S.; Vadhanam, Manicka V.; Stoyanova, Radka; Caprio, Gerard D.; Clapper, Margie L.; Gupta, Ramesh C.

2008-01-01

241

Pathway choice in DNA double strand break repair: observations of a balancing act  

PubMed Central

Proper repair of DNA double strand breaks (DSBs) is vital for the preservation of genomic integrity. There are two main pathways that repair DSBs, Homologous recombination (HR) and Non-homologous end-joining (NHEJ). HR is restricted to the S and G2 phases of the cell cycle due to the requirement for the sister chromatid as a template, while NHEJ is active throughout the cell cycle and does not rely on a template. The balance between both pathways is essential for genome stability and numerous assays have been developed to measure the efficiency of the two pathways. Several proteins are known to affect the balance between HR and NHEJ and the complexity of the break also plays a role. In this review we describe several repair assays to determine the efficiencies of both pathways. We discuss how disturbance of the balance between HR and NHEJ can lead to disease, but also how it can be exploited for cancer treatment. PMID:23181949

2012-01-01

242

Mre11 nuclease activity has essential roles in DNA repair and genomic stability distinct from ATM activation.  

PubMed

The Mre11/Rad50/NBS1 (MRN) complex maintains genomic stability by bridging DNA ends and initiating DNA damage signaling through activation of the ATM kinase. Mre11 possesses DNA nuclease activities that are highly conserved in evolution but play unknown roles in mammals. To define the functions of Mre11, we engineered targeted mouse alleles that either abrogate nuclease activities or inactivate the entire MRN complex. Mre11 nuclease deficiency causes a striking array of phenotypes indistinguishable from the absence of MRN, including early embryonic lethality and dramatic genomic instability. We identify a crucial role for the nuclease activities in homology-directed double-strand-break repair and a contributing role in activating the ATR kinase. However, the nuclease activities are not required to activate ATM after DNA damage or telomere deprotection. Therefore, nucleolytic processing by Mre11 is an essential function of fundamental importance in DNA repair, distinct from MRN control of ATM signaling. PMID:18854157

Buis, Jeffrey; Wu, Yipin; Deng, Yibin; Leddon, Jennifer; Westfield, Gerwin; Eckersdorff, Mark; Sekiguchi, Joann M; Chang, Sandy; Ferguson, David O

2008-10-01

243

Mre11 Nuclease Activity has Essential Roles in DNA Repair and Genomic Stability Distinct from ATM Activation  

PubMed Central

Summary The Mre11/Rad50/NBS1 complex (MRN) maintains genomic stability by bridging DNA ends and initiating DNA damage signaling through activation of the ATM kinase. Mre11 possesses DNA nuclease activities that are highly conserved in evolution, but play unknown roles in mammals. To define functions of Mre11 we engineered targeted mouse alleles which either abrogate nuclease activities or inactivate the entire MRN complex. Mre11 nuclease deficiency causes a striking array of phenotypes indistinguishable from absence of MRN, including early embryonic lethality and dramatic genomic instability. We identify a crucial role for the nuclease activities in homology directed double strand break repair, and a contributing role in activating the ATR kinase. However, nuclease activities are not required to activate ATM after DNA damage or telomere deprotection. Therefore, nucleolytic processing by Mre11 is an essential function of fundamental importance in DNA repair distinct from MRN control of ATM signaling. PMID:18854157

Buis, Jeffrey; Wu, Yipin; Deng, Yibin; Leddon, Jennifer; Westfield, Gerwin; Eckersdorff, Mark; Sekiguchi, JoAnn M.; Chang, Sandy; Ferguson, David O.

2008-01-01

244

Resection is a major repair pathway of heavy ion-induced DNA lesions  

NASA Astrophysics Data System (ADS)

Space radiation include densely ionizing heavy ions, which can produce clustered DNA damage with high frequency in human cells. Repair of these complex lesions is generally assumed to be more difficult than for simple double-strand breaks. We show here that human cells use break resection with increasing frequency after exposure to heavy ions. Resection can lead to misrepair of the DNA lesion, via microhomology mediated end-joining. Resection can therefore be responsible for the increased effectiveness of heavy ions in the induction of mutations and genetic late effects.

Durante, Marco; Averbeck, Nicole; Taucher-Scholz, Gisela

245

Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin.  

PubMed

Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity. PMID:25066234

Kakarougkas, Andreas; Ismail, Amani; Chambers, Anna L; Riballo, Enriqueta; Herbert, Alex D; Künzel, Julia; Löbrich, Markus; Jeggo, Penny A; Downs, Jessica A

2014-09-01

246

Replication protein A: single-stranded DNA's first responder: dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair.  

PubMed

Replication protein A (RPA), the major single-stranded DNA-binding protein in eukaryotic cells, is required for processing of single-stranded DNA (ssDNA) intermediates found in replication, repair, and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high-affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies showed that while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error-prone repair of double-stranded breaks and promote error-free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis. PMID:25171654

Chen, Ran; Wold, Marc S

2014-12-01

247

A multiprotein complex necessary for both transcription and DNA replication at the ?-globin locus  

PubMed Central

DNA replication, repair, transcription and chromatin structure are intricately associated nuclear processes, but the molecular links between these events are often obscure. In this study, we have surveyed the protein complexes that bind at ?-globin locus control region, and purified and characterized the function of one such multiprotein complex from human erythroleukemic K562 cells. We further validated the existence of this complex in human CD34+ cell-derived normal erythroid cells. This complex contains ILF2/ILF3 transcription factors, p300 acetyltransferase and proteins associated with DNA replication, transcription and repair. RNAi knockdown of ILF2, a DNA-binding component of this complex, abrogates the recruitment of the complex to its cognate DNA sequence and inhibits transcription, histone acetylation and usage of the origin of DNA replication at the ?-globin locus. These results imply a direct link between mammalian DNA replication, transcription and histone acetylation mediated by a single multiprotein complex. PMID:20808282

Karmakar, Subhradip; Mahajan, Milind C; Schulz, Vincent; Boyapaty, Gokul; Weissman, Sherman M

2010-01-01

248

A DNA repair pathway score predicts survival in human multiple myeloma: the potential for therapeutic strategy  

PubMed Central

DNA repair is critical to resolve extrinsic or intrinsic DNA damage to ensure regulated gene transcription and DNA replication. These pathways control repair of double strand breaks, interstrand crosslinks, and nucleotide lesions occurring on single strands. Distinct DNA repair pathways are highly inter-linked for the fast and optimal DNA repair. A deregulation of DNA repair pathways may maintain and promote genetic instability and drug resistance to genotoxic agents in tumor cells by specific mechanisms that tolerate or rapidly bypass lesions to drive proliferation and abrogate cell death. Multiple Myeloma (MM) is a plasma cell disorder characterized by genetic instability and poor outcome for some patients, in which the compendium of DNA repair pathways has as yet not been assessed for a disease-specific prognostic relevance. We design a DNA repair risk score based on the expression of genes coding for proteins involved in DNA repair in MM cells. From a consensus list of 84 DNA repair genes, 17 had a bad prognostic value and 5 a good prognostic value for both event-free and overall survival of previously-untreated MM patients. The prognostic information provided by these 22 prognostic genes was summed within a global DNA repair score (DRScore) to take into account the tight linkage of repair pathways. DRscore was strongly predictive for both patients' event free and overall survivals. Also, DRscore has the potential to identify MM patients whose tumor cells are dependent on specific DNA repair pathways to design treatments that induce synthetic lethality by exploiting addiction to deregulated DNA repair pathways. PMID:24809299

Kassambara, Alboukadel; Gourzones-Dmitriev, Claire; Sahota, Surinder; Rème, Thierry; Moreaux, Jérôme; Goldschmidt, Hartmut; Constantinou, Angelos; Pasero, Philippe; Hose, Dirk; Klein, Bernard

2014-01-01

249

Disruption of Maternal DNA Repair Increases Sperm-DerivedChromosomal Aberrations  

SciTech Connect

The final weeks of male germ cell differentiation occur in aDNA repair-deficient environment and normal development depends on theability of the egg to repair DNA damage in the fertilizing sperm. Geneticdisruption of maternal DNA double-strand break repair pathways in micesignificantly increased the frequency of zygotes with chromosomalstructural aberrations after paternal exposure to ionizing radiation.These findings demonstrate that radiation-induced DNA sperm lesions arerepaired after fertilization by maternal factors and suggest that geneticvariation in maternal DNA repair can modulate the risk of early pregnancylosses and of children with chromosomal aberrations of paternalorigin.

Marchetti, Francesco; Essers, Jeroun; Kanaar, Roland; Wyrobek,Andrew J.

2007-02-07

250

Interplay between DNA repair and inflammation, and the link to cancer  

PubMed Central

DNA damage and repair are linked to cancer. DNA damage that is induced endogenously or from exogenous sources has the potential to result in mutations and genomic instability if not properly repaired, eventually leading to cancer. Inflammation is also linked to cancer. Reactive oxygen and nitrogen species (RONs) produced by inflammatory cells at sites of infection can induce DNA damage. RONs can also amplify inflammatory responses, leading to increased DNA damage. Here, we focus on the links between DNA damage, repair, and inflammation, as they relate to cancer. We examine the interplay between chronic inflammation, DNA damage and repair and review recent findings in this rapidly emerging field, including the links between DNA damage and the innate immune system, and the roles of inflammation in altering the microbiome, which subsequently leads to the induction of DNA damage in the colon. Mouse models of defective DNA repair and inflammatory control are extensively reviewed, including treatment of mouse models with pathogens, which leads to DNA damage. The roles of microRNAs in regulating inflammation and DNA repair are discussed. Importantly, DNA repair and inflammation are linked in many important ways, and in some cases balance each other to maintain homeostasis. The failure to repair DNA damage or to control inflammatory responses has the potential to lead to cancer. PMID:24410153

Kidane, Dawit; Chae, Wook Jin; Czochor, Jennifer; Eckert, Kristin A.; Glazer, Peter M.; Bothwell, Alfred L. M.; Sweasy, Joann B.

2015-01-01

251

DNA polymerases beta and lambda mediate overlapping and independent roles in base excision repair in mouse embryonic fibroblasts  

E-print Network

Base excision repair (BER) is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol ?) is known to be the main polymerase in the BER pathway, various studies have ...

Braithwaite, Elena K.

252

DNA repair as a biomarker in human biomonitoring studies; further applications of the comet assay.  

PubMed

DNA repair plays a major role in maintaining genetic stability, and so measurement of individual DNA repair capacity should be a valued tool in molecular epidemiology studies. The comet assay (single cell gel electrophoresis), in different versions, is commonly used to measure the repair pathways represented by strand break rejoining, removal of 8-oxoguanine, and repair of bulky adducts or UV-induced damage. Repair enzyme activity generally does not reflect the level of gene expression; but there is evidence - albeit piecemeal - that it is affected by polymorphisms in repair genes. There are mixed reports concerning the regulation of repair by environmental factors; several nutritional supplementation trials with phytochemical-rich foods have demonstrated increases in base excision repair of oxidation damage, while others have shown no effect. Exposure to genotoxic agents has in general not been found to stimulate repair. Crucial questions concerning the factors regulating repair and the causes of individual variation are as yet unanswered. PMID:21459100

Collins, Andrew R; Azqueta, Amaya

2012-08-01

253

RPA Antagonizes Microhomology-Mediated Repair of DNA Double-Strand Breaks  

PubMed Central

Microhomology-mediated end joining (MMEJ) is a Ku and Ligase IV independent mechanism for repair of DNA double-strand breaks, which contributes to chromosome rearrangements. Here we used a chromosomal end-joining assay to determine the genetic requirements for MMEJ in Saccharomyces cerevisiae. We found that end resection influences the ability to expose microhomologies; however, it is not rate limiting for MMEJ in wild-type cells. The frequency of MMEJ increased by up to 350-fold in rfa1 hypomorphic mutants, suggesting that replication protein A (RPA) bound to the ssDNA overhangs formed by resection prevents spontaneous annealing between microhomologies. In vitro, the mutant RPA complexes were unable to fully extend ssDNA and were compromised in their ability to prevent spontaneous annealing. We propose the helix-destabilizing activity of RPA channels ssDNA intermediates from mutagenic MMEJ to error-free homologous recombination, thus preserving genome integrity. PMID:24608368

Deng, Sarah K; Gibb, Bryan; de Almeida, Mariana Justino; Greene, Eric C; Symington, Lorraine S

2014-01-01

254

In vivo single-molecule imaging of bacterial DNA replication, transcription, and repair.  

PubMed

In vivo single-molecule experiments offer new perspectives on the behaviour of DNA binding proteins, from the molecular level to the length scale of whole bacterial cells. With technological advances in instrumentation and data analysis, fluorescence microscopy can detect single molecules in live cells, opening the doors to directly follow individual proteins binding to DNA in real time. In this review, we describe key technical considerations for implementing in vivo single-molecule fluorescence microscopy. We discuss how single-molecule tracking and quantitative super-resolution microscopy can be adapted to extract DNA binding kinetics, spatial distributions, and copy numbers of proteins, as well as stoichiometries of protein complexes. We highlight experiments which have exploited these techniques to answer important questions in the field of bacterial gene regulation and transcription, as well as chromosome replication, organisation and repair. Together, these studies demonstrate how single-molecule imaging is transforming our understanding of DNA-binding proteins in cells. PMID:24859634

Stracy, Mathew; Uphoff, Stephan; Garza de Leon, Federico; Kapanidis, Achillefs N

2014-10-01

255

Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene  

PubMed Central

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15–20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression. PMID:24137009

Morano, Annalisa; Angrisano, Tiziana; Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Bartollino, Silvia; Zuchegna, Candida; Babbio, Federica; Bonapace, Ian Marc; Allen, Brittany; Muller, Mark T.; Chiariotti, Lorenzo; Gottesman, Max E.; Porcellini, Antonio; Avvedimento, Enrico V.

2014-01-01

256

Host DNA repair proteins in response to Pseudomonas aeruginosa in lung epitehlial cells and in mice  

Technology Transfer Automated Retrieval System (TEKTRAN)

Host DNA damage and DNA repair response to bacterial infections and its significance are not fully understood. Here, we demonstrate that infection by Gram-negative bacterium P. aeruginosa significantly altered the expression and enzymatic activity of base excision DNA repair protein OGG1 in lung epi...

257

DNA Substrate Dependence of p53-Mediated Regulation of Double-Strand Break Repair  

Microsoft Academic Search

DNA double-strand breaks (DSBs) arise spontaneously after the conversion of DNA adducts or single-strand breaks by DNA repair or replication and can be introduced experimentally by expression of specific endo- nucleases. Correct repair of DSBs is central to the maintenance of genomic integrity in mammalian cells, since errors give rise to translocations, deletions, duplications, and expansions, which accelerate the multistep

Nuray Akyuz; Gisa S. Boehden; Silke Susse; Andreas Rimek; Ute Preuss; Karl-Heinz Scheidtmann; Lisa Wiesmuller

2002-01-01

258

DNA Repair 9 (2010) 11301141 Contents lists available at ScienceDirect  

E-print Network

DNA Repair 9 (2010) 1130­1141 Contents lists available at ScienceDirect DNA Repair journal homepage September 2010 Keywords: Polymerase Zinc finger Ubiquitin DNA damage Translesion synthesis a b s t r a c, and function. Here, we elucidate structural and functional features of the non-canonical UBZ motif

Zhou, Pei

259

Human DNA repair process recorded in action Published on Machines Like Us (http://  

E-print Network

Human DNA repair process recorded in action Published on Machines Like Us (http:// machineslikeus." University of California, Davis [1] Biology © Copyright MachinesLikeUs.com Source URL: http://machineslikeus.com/news/human-dna.com) Home > View content Human DNA repair process recorded in action By NLN Created 01/29/2009 - 15:35 A key

Kowalczykowski, Stephen C.

260

Peripheral tears of triangular fibrocartilage complex: results of primary repair  

Microsoft Academic Search

In 16 patients with ulnar wrist pain, we performed primary arthroscopic or open repair of the peripheral rim tears of the triangular fibrocartilage complex (TFCC) (14 ulnar, 4 volar, and 3 radial tears). The wrist function was assessed before and 1 year after the repair using the Mayo-modified wrist score. The average pain score improved from 9.1NJ.0 to 21.2Lj.5, the

Chen-Hsi Chou; Tu-Sheng Lee

2001-01-01

261

DNA Bending Propensity in the Presence of Base Mismatches: Implications for DNA Repair  

PubMed Central

DNA bending is believed to facilitate the initial recognition of the mismatched base for repair. The repair efficiencies are dependent on both the mismatch type and neighboring nucleotide sequence. We have studied bending of several DNA duplexes containing canonical matches: A:T, G:C, various mismatches: A:A, A:C, G:A, G:G, G:T, C:C, C:T, T:T, and a bis-abasic site: X:X. Free energy profiles were generated for DNA bending using umbrella sampling. The highest energetic cost associated with DNA bending is observed for canonical matches while bending free energies are lower in the presence of mismatches, with the lowest value for the abasic site. In all of the sequences, DNA duplexes bend towards the major groove with widening of the minor groove. For homoduplexes, DNA bending is observed to occur via smooth deformations, whereas for heteroduplexes, kinks are observed at the mismatch site during strong bending. In general, pyrimidine:pyrimidine mismatches are the most destabilizing, while purine:purine mismatches lead to intermediate destabilization and purine:pyrimidine mismatches are the least destabilizing. The ease of bending is partially correlated with the binding affinity of MutS to the mismatch pairs and subsequent repair efficiencies, indicating that intrinsic DNA bending propensities are a key factor of mismatch recognition. PMID:23621762

Sharma, Monika; Predeus, Alexander V.; Mukherjee, Shayantani; Feig, Michael

2013-01-01

262

PTIP associates with Artemis to dictate DNA repair pathway choice.  

PubMed

PARP inhibitors (PARPis) are being used in patients with BRCA1/2 mutations. However, doubly deficient BRCA1(-/-)53BP1(-/-) cells or tumors become resistant to PARPis. Since 53BP1 or its known downstream effectors, PTIP and RIF1 (RAP1-interacting factor 1 homolog), lack enzymatic activities directly implicated in DNA repair, we decided to further explore the 53BP1-dependent pathway. In this study, we uncovered a nuclease, Artemis, as a PTIP-binding protein. Loss of Artemis restores PARPi resistance in BRCA1-deficient cells. Collectively, our data demonstrate that Artemis is the major downstream effector of the 53BP1 pathway, which prevents end resection and promotes nonhomologous end-joining and therefore directly competes with the homologous recombination repair pathway. PMID:25512557

Wang, Jiadong; Aroumougame, Asaithamby; Lobrich, Markus; Li, Yujing; Chen, David; Chen, Junjie; Gong, Zihua

2014-12-15

263

Interactions between branched DNAs and peptide inhibitors of DNA repair  

PubMed Central

The RecG helicase of Escherichia coli unwinds both Holliday junction (HJ) and replication fork DNA substrates. Our lab previously identified and characterized peptides (WRWYCR and KWWCRW) that block the activity of RecG on these substrates. We determined that the peptides bind HJ DNA and prevent the binding of RecG. Herein, we present further evidence that the peptides are competitive inhibitors of RecG binding to its substrates. We have generated structural models of interactions between WRWYCR and a junction substrate. Using the fluorescent probe 2-aminopurine, we show that inhibitors interact with highest affinity with HJs (Kd = 14 nM) and ?4- to 9-fold more weakly with replication fork substrates. The fluorescence assay results agree with the structural model, and predict the molecular basis for interactions between HJ-trapping peptides and branched DNA molecules. Specifically, aromatic amino acids in the peptides stack with bases at the center of the DNA substrates. These interactions are stabilized by hydrogen bonds to the DNA and by intrapeptide interactions. These peptides inhibit several proteins involved in DNA repair in addition to RecG, have been useful as tools to dissect recombination, and possess antibiotic activity. Greater understanding of the peptides’ mechanism of action will further increase their utility. PMID:18689438

Kepple, Kevin V.; Patel, Namita; Salamon, Peter; Segall, Anca M.

2008-01-01

264

Interactions between branched DNAs and peptide inhibitors of DNA repair.  

PubMed

The RecG helicase of Escherichia coli unwinds both Holliday junction (HJ) and replication fork DNA substrates. Our lab previously identified and characterized peptides (WRWYCR and KWWCRW) that block the activity of RecG on these substrates. We determined that the peptides bind HJ DNA and prevent the binding of RecG. Herein, we present further evidence that the peptides are competitive inhibitors of RecG binding to its substrates. We have generated structural models of interactions between WRWYCR and a junction substrate. Using the fluorescent probe 2-aminopurine, we show that inhibitors interact with highest affinity with HJs (K(d) = 14 nM) and approximately 4- to 9-fold more weakly with replication fork substrates. The fluorescence assay results agree with the structural model, and predict the molecular basis for interactions between HJ-trapping peptides and branched DNA molecules. Specifically, aromatic amino acids in the peptides stack with bases at the center of the DNA substrates. These interactions are stabilized by hydrogen bonds to the DNA and by intrapeptide interactions. These peptides inhibit several proteins involved in DNA repair in addition to RecG, have been useful as tools to dissect recombination, and possess antibiotic activity. Greater understanding of the peptides' mechanism of action will further increase their utility. PMID:18689438

Kepple, Kevin V; Patel, Namita; Salamon, Peter; Segall, Anca M

2008-09-01

265

Analysis of DNA repair helicase UvrD from Arabidopsis thaliana and Oryza sativa.  

PubMed

Mismatch repair (MMR) proteins play important roles in maintaining genome stability in all the organisms. Studies of MMR genes in plants have identified several homologs of the Escherichia coli genes. Crop yield is directly related to genome stability, which is crucially required for optimal plant growth and development. Numerous genotoxic stresses such as UV light, radiations, pollutants and heavy metals cause DNA damage leading to genome instability, which can interfere with the plant growth and crop productivity. But the efficient repair mechanisms can help to overcome the deleterious effects of the damage. Therefore it is important to study the genes involved in various repair pathways in the plants in greater detail. UvrD helicase is a component of MMR complex and plays an essential role in the DNA repair by providing the unwinding function. In the present manuscript we present an in silico analysis of UvrD helicase from two plant species (Arabidopsis and rice). The Arabidopsis thaliana and Oryza sativa UvrD are 1149 (~129 kDa) and 1165 amino-acids (~130 kDa) proteins, respectively. These proteins contain all the conserved domains and are larger than the E. coli UvrD because they contain a longer N-terminal extension. In order to decipher the role of plant UvrD in various stresses it will be important to study the biochemical and functional properties of this enzyme. PMID:23974358

Tuteja, Renu; Tuteja, Narendra

2013-10-01

266

Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells  

SciTech Connect

The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior to their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. 62 references, 3 figures, 2 tables.

Gupta, P.K.; Sirover, M.A.

1984-10-01

267

DNA repair signature is associated with anthracycline response in triple negative breast cancer patients  

Microsoft Academic Search

We hypothesized that a subset of sporadic triple negative (TN) breast cancer patients whose tumors have defective DNA repair\\u000a similar to BRCA1-associated tumors are more likely to exhibit up-regulation of DNA repair-related genes, anthracycline-sensitivity,\\u000a and taxane-resistance. We derived a defective DNA repair gene expression signature of 334 genes by applying a previously published\\u000a BRCA1-associated expression pattern to three datasets of

A. A. RodriguezA; A. Makris; M. F. Wu; M. Rimawi; A. Froehlich; B. Dave; S. G. Hilsenbeck; G. C. Chamness; M. T. Lewis; L. E. Dobrolecki; D. Jain; S. Sahoo; C. K. Osborne; J. C. Chang

2010-01-01

268

The potential of exploiting DNA-repair defects for optimizing lung cancer treatment  

Microsoft Academic Search

The tumor genome is commonly aberrant as a consequence of mutagenic insult and incomplete DNA repair. DNA repair as a therapeutic target has recently received considerable attention owing to the promise of drugs that target tumor-specific DNA-repair enzymes and potentiate conventional cytotoxic therapy through mechanism-based approaches, such as synthetic lethality. Treatment for non-small-cell lung cancer (NSCLC) consists mainly of platinum-based

Sophie Postel-Vinay; Elsa Vanhecke; Ken A. Olaussen; Christopher J. Lord; Alan Ashworth; Jean-Charles Soria

2012-01-01

269

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

PubMed Central

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 ?-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

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

270

Roles of DNA adenine methylation in host-pathogen interactions: mismatch repair, transcriptional regulation, and more.  

PubMed

The DNA adenine methyltransferase (Dam methylase) of Gammaproteobacteria and the cell cycle-regulated methyltransferase (CcrM) methylase of Alphaproteobacteria catalyze an identical reaction (methylation of adenosine moieties using S-adenosyl-methionine as a methyl donor) at similar DNA targets (GATC and GANTC, respectively). Dam and CcrM are of independent evolutionary origin. Each may have evolved from an ancestral restriction-modification system that lost its restriction component, leaving an 'orphan' methylase devoted solely to epigenetic genome modification. The formation of 6-methyladenine reduces the thermodynamic stability of DNA and changes DNA curvature. As a consequence, the methylation state of specific adenosine moieties can affect DNA-protein interactions. Well-known examples include binding of the replication initiation complex to the methylated oriC, recognition of hemimethylated GATCs in newly replicated DNA by the MutHLS mismatch repair complex, and discrimination of methylation states in promoters and regulatory DNA motifs by RNA polymerase and transcription factors. In recent years, Dam and CcrM have been shown to play roles in host-pathogen interactions. These roles are diverse and have only partially been understood. Especially intriguing is the evidence that Dam methylation regulates virulence genes in Escherichia coli, Salmonella, and Yersinia at the posttranscriptional level. PMID:19175412

Marinus, Martin G; Casadesus, Josep

2009-05-01

271

DNA Repair Gene Polymorphisms and Risk of Pancreatic Cancer  

PubMed Central

Purpose The current research was undertaken to examine the association between genetic variations in DNA repair and pancreatic cancer risk. Experimental Design We analyzed nine single nucleotide polymorphisms (SNPs) of seven DNA repair genes (LIG3, LIG4, OGG1, ATM, POLB, RAD54L, and RECQL) in 734 patients with pancreatic adenocarcinoma and 780 healthy controls using the Taqman method. Information on cigarette smoking, alcohol consumption, medical history, and other risk factors was collected by personal interview. Results The homozygous mutant genotype of LIG3 G-39A (odds ratio [OR], 0.23; 95% confidence interval [CI] = 0.06-0.82; P = 0.027) and ATM D1853N (OR, 2.55; 95% CI = 1.08-6.00; P = 0.032) was significantly associated with altered risk for pancreatic cancer. A statistically significant interaction of ATM D1853N and LIG4 C54T genotype with diabetes on the risk of pancreatic cancer was also detected. Compared to non-diabetics with the ATM D1853N GG genotype, non-diabetics with the GA/AA, diabetics with the GG, and diabetics with the GA/AA genotypes, respectively, had ORs (95% CI) of 0.96 (0.74-1.24), 1.32 (0.89-1.95), and 3.23 (1.47-7.12) (Pinteraction = 0.032, likelihood ratio test). The OR (95% CI) was 0.91 (0.71-1.17), 1.11 (0.73-1.69), and 2.44 (1.34-4.46) for non-diabetics carrying the LIG4 CT/TT genotype, diabetics with the CC genotype, and diabetics carrying the CT/TT genotype, respectively, compared to non-diabetics carrying the CC genotype (Pinteraction= 0.02). Conclusions These observations suggest that genetic variations in DNA repair may act alone or in concert with other risk factors on modifying a patient's risk for pancreatic cancer. PMID:19147782

Li, Donghui; Suzuki, Hideo; Liu, Bingrong; Morris, Jeffrey; Liu, Jun; Okazaki, Taro; Li, Yanan; Chang, Ping; Abbruzzese, James L.

2008-01-01

272

C. elegans whole-genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency  

PubMed Central

Mutation is associated with developmental and hereditary disorders, aging, and cancer. While we understand some mutational processes operative in human disease, most remain mysterious. We used Caenorhabditis elegans whole-genome sequencing to model mutational signatures, analyzing 183 worm populations across 17 DNA repair-deficient backgrounds propagated for 20 generations or exposed to carcinogens. The baseline mutation rate in C. elegans was approximately one per genome per generation, not overtly altered across several DNA repair deficiencies over 20 generations. Telomere erosion led to complex chromosomal rearrangements initiated by breakage–fusion–bridge cycles and completed by simultaneously acquired, localized clusters of breakpoints. Aflatoxin B1 induced substitutions of guanines in a GpC context, as observed in aflatoxin-induced liver cancers. Mutational burden increased with impaired nucleotide excision repair. Cisplatin and mechlorethamine, DNA crosslinking agents, caused dose- and genotype-dependent signatures among indels, substitutions, and rearrangements. Strikingly, both agents induced clustered rearrangements resembling “chromoanasynthesis,” a replication-based mutational signature seen in constitutional genomic disorders, suggesting that interstrand crosslinks may play a pathogenic role in such events. Cisplatin mutagenicity was most pronounced in xpf-1 mutants, suggesting that this gene critically protects cells against platinum chemotherapy. Thus, experimental model systems combined with genome sequencing can recapture and mechanistically explain mutational signatures associated with human disease. PMID:25030888

Meier, Bettina; Cooke, Susanna L.; Weiss, Joerg; Bailly, Aymeric P.; Alexandrov, Ludmil B.; Marshall, John; Raine, Keiran; Maddison, Mark; Anderson, Elizabeth; Stratton, Michael R.; Campbell, Peter J.

2014-01-01

273

Complex reconfiguration of DNA nanostructures.  

PubMed

Nucleic acids have been used to create diverse synthetic structural and dynamic systems. Toehold-mediated strand displacement has enabled the construction of sophisticated circuits, motors, and molecular computers. Yet it remains challenging to demonstrate complex structural reconfiguration in which a structure changes from a starting shape to another arbitrarily prescribed shape. To address this challenge, we have developed a general structural-reconfiguration method that utilizes the modularly interconnected architecture of single-stranded DNA tile and brick structures. The removal of one component strand reveals a newly exposed toehold on a neighboring strand, thus enabling us to remove regions of connected component strands without the need to modify the strands with predesigned external toeholds. By using this method, we reconfigured a two-dimensional rectangular DNA canvas into diverse prescribed shapes. We also used this method to reconfigure a three-dimensional DNA cuboid. PMID:24899518

Wei, Bryan; Ong, Luvena L; Chen, Jeffrey; Jaffe, Alexander S; Yin, Peng

2014-07-14

274

Redox Regulation of DNA Repair: Implications for Human Health and Cancer Therapeutic Development  

PubMed Central

Abstract Redox reactions are known to regulate many important cellular processes. In this review, we focus on the role of redox regulation in DNA repair both in direct regulation of specific DNA repair proteins as well as indirect transcriptional regulation. A key player in the redox regulation of DNA repair is the base excision repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1) in its role as a redox factor. APE1 is reduced by the general redox factor thioredoxin, and in turn reduces several important transcription factors that regulate expression of DNA repair proteins. Finally, we consider the potential for chemotherapeutic development through the modulation of APE1's redox activity and its impact on DNA repair. Antioxid. Redox Signal. 12, 1247–1269. PMID:19764832

Luo, Meihua; He, Hongzhen; Kelley, Mark R.

2010-01-01

275

Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage-binding protein.  

PubMed Central

Cells from a subset of patients with the DNA-repair-defective disease xeroderma pigmentosum complementation group E (XP-E) are known to lack a DNA damage-binding (DDB) activity. Purified human DDB protein was injected into XP-E cells to test whether the DNA-repair defect in these cells is caused by a defect in DDB activity. Injected DDB protein stimulated DNA repair to normal levels in those strains that lack the DDB activity but did not stimulate repair in cells from other xeroderma pigmentosum groups or in XP-E cells that contain the activity. These results provide direct evidence that defective DDB activity causes the repair defect in a subset of XP-E patients, which in turn establishes a role for this activity in nucleotide-excision repair in vivo. Images PMID:8171034

Keeney, S; Eker, A P; Brody, T; Vermeulen, W; Bootsma, D; Hoeijmakers, J H; Linn, S

1994-01-01

276

Exploration of methods to identify polymorphisms associated with variation in DNA repair capacity phenotypes  

SciTech Connect

Elucidating the relationship between polymorphic sequences and risk of common disease is a challenge. For example, although it is clear that variation in DNA repair genes is associated with familial cancer, aging and neurological disease, progress toward identifying polymorphisms associated with elevated risk of sporadic disease has been slow. This is partly due to the complexity of the genetic variation, the existence of large numbers of mostly low frequency variants and the contribution of many genes to variation in susceptibility. There has been limited development of methods to find associations between genotypes having many polymorphisms and pathway function or health outcome. We have explored several statistical methods for identifying polymorphisms associated with variation in DNA repair phenotypes. The model system used was 80 cell lines that had been resequenced to identify variation; 191 single nucleotide substitution polymorphisms (SNPs) are included, of which 172 are in 31 base excision repair pathway genes, 19 in 5 anti-oxidation genes, and DNA repair phenotypes based on single strand breaks measured by the alkaline Comet assay. Univariate analyses were of limited value in identifying SNPs associated with phenotype variation. Of the multivariable model selection methods tested: the easiest that provided reduced error of prediction of phenotype was simple counting of the variant alleles predicted to encode proteins with reduced activity, which led to a genotype including 52 SNPs; the best and most parsimonious model was achieved using a two-step analysis without regard to potential functional relevance: first SNPs were ranked by importance determined by Random Forests Regression (RFR), followed by cross-validation in a second round of RFR modeling that included ever more SNPs in declining order of importance. With this approach 6 SNPs were found to minimize prediction error. The results should encourage research into utilization of multivariate analytical methods for epidemiological studies of the association of genetic variation in complex genotypes with risk of common diseases.

Jones, I M; Thomas, C B; Xi, T; Mohrenweiser, H W; Nelson, D O

2006-07-03

277

Repair of DNA Lesions by a Reductive Electron Tansfer  

NASA Astrophysics Data System (ADS)

Electron transfer phenomena in DNA are of fundamental importance for DNA damage[1] and DNA repair.[2] The movement of a positive charge (hole) through DNA[3-6] has been shown to proceed over significant distances. Two mechanisms, namely coherent superexchange for small transfer distances and hole, or polaron hopping for long range transfer are used to describe this phenomenon. In contrast to hole transfer, little is known about the transport of excess electrons (negative charges) through a DNA duplex. Such an excess electron transfer, however, is important in biology because DNA photolyase enzymes repair UV-induced cyclobutane pyrimidine dimer lesions (T=T) in the DNA duplex by an electron transfer from a reduced an deprotonated FADH-cofactor to the dimer lesion. The presentation covers recent results obtained in our group about the distance and sequence dependence of an excess electron transfer in a defined donor-DNA-acceptor system.[7-9] The prepared DNA double strands contain a reduced flavin electron donor and a thymine dimer acceptor, separated by adenine:thymine (A:T)n bridges of various lengths. The electron injection is initiated by irradiation of the DNA-double strand at 360 nm, which causes excitation of the reduced and deprotonated flavin donor. The injected electron, if captured by the dimer (T=T), triggers subsequently a cycloreversion, which is detectable by HPLC. A plot of the observed splitting yields against the distance between the flavin donor and the dimer gave a straight line with a small beta'-value of beta' = 0.1 Å-1. Such small beta'-values were determined for long range hole transfer as well. Our data show that excess electron transfer proceeds similarly efficient. Plotting of the yield data according to the hopping model ln(yield per minute) against ln(N) by assuming that every T between the flavin donor and the dimer acceptor can function as a discrete charge carrier (N), gives a straight line with a reasonable eta-value of close to 2. The result indicates that the negative charge transfer may proceed by hopping. [1] J. P. Pouget, T. Douki, M. J. Richard, J. Cadet, Chem. Res. Toxicol. 2000, 13, 541. [2] E. C. Friedberg, DNA repair and mutagenesis, ASM Press, Washington, D.C., 1995. [3] R. E. Holmlin, P. J. Dandlicker, J. K. Barton, Angew. Chem. Int. Ed. 1997, 36, 2715. [4] B. Giese, Acc. Chem. Res. 2000, 33, 631. [5] F. D. Lewis, R. L. Letsinger, M. R. Wasielewski, Acc. Chem. Res. 2001, 34, 159. [6] G. B. Schuster, Acc. Chem. Res. 2000, 33, 253. [7] A. Schwögler, L. T. Burgdorf, T. Carell, Angew. Chem. Int. Ed. 2000, 39, 3918. [8] A. Schwögler, T. Carell, Org. Lett. 2000, 2, 1415. [9] M. K. Cichon, C. H. Haas, F. Grolle, A. Mees, T. Carell.J. Am. Chem. Soc. 2002, 124, 13984-13985.

Carell, Thomas

2003-03-01

278

ATM-mediated phosphorylation of the chromatin remodeling enzyme BRG1 modulates DNA double-strand break repair.  

PubMed

ATP-dependent chromatin remodeling complexes such as SWI/SNF (SWItch/Sucrose NonFermentable) have been implicated in DNA double-strand break (DSB) repair and damage responses. However, the regulatory mechanisms that control the function of chromatin remodelers in DNA damage response are largely unknown. Here, we show that ataxia telangiectasia mutated (ATM) mediates the phosphorylation of BRG1, the catalytic ATPase of the SWI/SNF complex that contributes to DSB repair by binding ?-H2AX-containing nucleosomes via interaction with acetylated histone H3 and stimulating ?-H2AX formation, at Ser-721 in response to DNA damage. ATM-mediated phosphorylation of BRG1 occurs rapidly and transiently after DNA damage. Phosphorylated BRG1 binds ?-H2AX-containing nucleosomes to form the repair foci. The Ser-721 phosphorylation of BRG1 is critical for binding ?-H2AX-containing nucleosomes and stimulating ?-H2AX formation and DSB repair. BRG1 binds to acetylated H3 peptides much better after phosphorylation at Ser-721 by DNA damage. However, the phosphorylation of Ser-721 does not significantly affect the ATPase and transcriptional activities of BRG1. These results, establishing BRG1 as a novel and functional ATM substrate, suggest that the ATM-mediated phosphorylation of BRG1 facilitates DSB repair by stimulating the association of this remodeler with ?-H2AX nucleosomes via enhancing the affinity to acetylated H3. Our work also suggests that the mechanism of BRG1 stimulation of DNA repair is independent of the remodeler's enzymatic or transcriptional activities. PMID:24413084

Kwon, S-J; Park, J-H; Park, E-J; Lee, S-A; Lee, H-S; Kang, S W; Kwon, J

2015-01-15

279

Structural complexity of DNA sequence.  

PubMed

In modern bioinformatics, finding an efficient way to allocate sequence fragments with biological functions is an important issue. This paper presents a structural approach based on context-free grammars extracted from original DNA or protein sequences. This approach is radically different from all those statistical methods. Furthermore, this approach is compared with a topological entropy-based method for consistency and difference of the complexity results. PMID:23662161

Liou, Cheng-Yuan; Tseng, Shen-Han; Cheng, Wei-Chen; Tsai, Huai-Ying

2013-01-01

280

Structural Complexity of DNA Sequence  

PubMed Central

In modern bioinformatics, finding an efficient way to allocate sequence fragments with biological functions is an important issue. This paper presents a structural approach based on context-free grammars extracted from original DNA or protein sequences. This approach is radically different from all those statistical methods. Furthermore, this approach is compared with a topological entropy-based method for consistency and difference of the complexity results. PMID:23662161

Liou, Cheng-Yuan; Cheng, Wei-Chen; Tsai, Huai-Ying

2013-01-01

281

Relationship of DNA repair and chromosome aberrations to potentially lethal damage repair in X-irradiated mammalian cells  

SciTech Connect

By the alkaline elution technique, the repair of x-ray-induced DNA single strand breaks and DNA-protein cross-links was investigated in stationary phase, contact-inhibited mouse cells. During the first hour of repair, approximately 90% of x-ray induced single strand breaks were rejoined whereas most of the remaining breaks were rejoined more slowly during the next 5 h. The number of residual non-rejoined single strand breaks was approximately proportional to the x-ray dose at early repair times. DNA-protein cross-links were removed at a slower rate - T 1/2 approximately 10 to 12 h. Cells were subcultured at low density at various times after irradiation and scored for colony survival, and chromosome aberrations in the first mitosis after sub-culture. Both cell lethality and the frequency of chromosome aberrations decreased during the first several hours of repair, reaching a minimum level by 6 h; this decrease correlated temporally with the repair of the slowly rejoining DNA strand breaks. The possible relationship of DNA repair to changes in survival and chromosome aberrations is discussed.

Fornace, A.J. Jr.; Nagasawa, H.; Little, J.B.

1980-01-01

282

Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins  

PubMed Central

Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase ?. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a “cowcatcher” and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function. PMID:23898192

Hegde, Muralidhar L.; Hegde, Pavana M.; Bellot, Larry J.; Mandal, Santi M.; Hazra, Tapas K.; Li, Guo-Min; Boldogh, Istvan; Tomkinson, Alan E.; Mitra, Sankar

2013-01-01

283

Mechanism of Cluster DNA Damage Repair in Response to High-Atomic Number and Energy Particles Radiation  

PubMed Central

Low-linear energy transfer (LET) radiation (i.e., ?- and X-rays) induces DNA double-strand breaks (DSBs) that are rapidly repaired (rejoined). In contrast, DNA damage induced by the dense ionizing track of high-atomic number and energy (HZE) particles are slowly repaired or are irreparable. These unrepaired and/or misrepaired DNA lesions may contribute to the observed higher relative biological effectiveness for cell killing, chromosomal aberrations, mutagenesis, and carcinogenesis in HZE particle irradiated cells compared to those treated with low-LET radiation. The types of DNA lesions induced by HZE particles have been characterized in vitro and usually consist of two or more closely spaced strand breaks, abasic sites, or oxidized bases on opposing strands. It is unclear why these lesions are difficult to repair. In this review, we highlight the potential of a new technology allowing direct visualization of different types of DNA lesions in human cells and document the emerging significance of live-cell imaging for elucidation of the spatio-temporal characterization of complex DNA damage. We focus on the recent insights into the molecular pathways that participate in the repair of HZE particle-induced DSBs. We also discuss recent advances in our understanding of how different end-processing nucleases aid in repair of DSBs with complicated ends generated by HZE particles. Understanding the mechanism underlying the repair of DNA damage induced by HZE particles will have important implications for estimating the risks to human health associated with HZE particle exposure. PMID:21126526

Asaithamby, Aroumougame; Chen, David J.

2012-01-01

284

A rapid, comprehensive system for assaying DNA repair activity and cytotoxic effects of DNA-damaging reagents.  

PubMed

DNA repair systems protect cells from genomic instability and carcinogenesis. Therefore, assays for measuring DNA repair activity are valuable, not only for clinical diagnoses of DNA repair deficiency disorders but also for basic research and anticancer drug development. Two commonly used assays are UDS (unscheduled DNA synthesis, requiring a precise measurement of an extremely small amount of repair DNA synthesis) and RRS (recovery of RNA synthesis after DNA damage). Both UDS and RRS are major endpoints for assessing the activity of nucleotide excision repair (NER), the most versatile DNA repair process. Conventional UDS and RRS assays are laborious and time-consuming, as they measure the incorporation of radiolabeled nucleosides associated with NER. Here we describe a comprehensive protocol for monitoring nonradioactive UDS and RRS by studying the incorporation of alkyne-conjugated nucleoside analogs followed by a fluorescent azide-coupling click-chemistry reaction. The system is also suitable for quick measurement of cell sensitivity to DNA-damaging reagents and for lentivirus-based complementation assays, which can be used to systematically determine the pathogenic genes associated with DNA repair deficiency disorders. A typical UDS or RRS assay using primary fibroblasts, including a virus complementation test, takes 1 week to complete. PMID:25474029

Jia, Nan; Nakazawa, Yuka; Guo, Chaowan; Shimada, Mayuko; Sethi, Mieran; Takahashi, Yoshito; Ueda, Hiroshi; Nagayama, Yuji; Ogi, Tomoo

2015-01-01

285

Exploiting DNA mismatch repair deficiency as a therapeutic strategy.  

PubMed

The DNA Mismatch repair (MMR) pathway is critical for the maintenance of genomic stability. It is primarily responsible for the recognition and repair of mismatches that occur during DNA replication, but accumulating evidence suggest additional non-canonical roles for MMR proteins. MMR deficiency is a common feature of many tumor types. Germline mutations in MMR genes gives rise to the familial disorder, Lynch syndrome, which is associated with an increased predisposition to numerous cancers, including colorectal and endometrial. MMR deficiency has been associated with resistance to a wide range of standard therapeutic agents such as methylating agents, platinum compounds and fluoropyrimidine agents. Therefore, there is critical clinical need to identify new therapies for these resistant tumors. Recent studies, focussing on synthetic lethal interactions with MMR loss and emerging data identifying novel regulators of MMR may enable more successful treatment for MMR deficient patients. This review focuses on MMR loss in cancer and how exploiting both the canonical and non-canonical roles of MMR proteins may aid future therapeutic strategies. PMID:25017099

Guillotin, Delphine; Martin, Sarah A

2014-11-15

286

Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions  

NASA Astrophysics Data System (ADS)

Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the repair machinery, and it is thought to be involved in the initial step as a DNA damage recognition and/or confirmation factor. Human replication protein A (RPA) and XPA have been reported to interact to form a DNA damage recognition complex with greater specificity for damaged DNA than XPA alone. The mechanism by which these two proteins recognize such a wide array of structures resulting from different types of DNA damage is not known. One possibility is that they recognize a common feature of the lesions, such as distortions of the helical backbone. We have tested this idea by determining whether human XPA and RPA proteins can recognize the helical distortions induced by a DNA triple helix, a noncanonical DNA structure that has been shown to induce DNA repair, mutagenesis, and recombination. We measured binding of XPA and RPA, together or separately, to substrates containing triplexes with three, two, or no strands covalently linked by psoralen conjugation and photoaddition. We found that RPA alone recognizes all covalent triplex structures, but also forms multivalent nonspecific DNA aggregates at higher concentrations. XPA by itself does not recognize the substrates, but it binds them in the presence of RPA. Addition of XPA decreases the nonspecific DNA aggregate formation. These results support the hypothesis that the NER machinery is targeted to helical distortions and demonstrate that RPA can recognize damaged DNA even without XPA.

Vasquez, Karen M.; Christensen, Jesper; Li, Lei; Finch, Rick A.; Glazer, Peter M.

2002-04-01

287

DNA-PK target identification reveals novel links between DNA repair signaling and cytoskeletal regulation.  

PubMed

The DNA-dependent protein kinase (DNA-PK) may function as a key signaling kinase in various cellular pathways other than DNA repair. Using a two-dimensional gel electrophoresis approach and stable DNA double-strand break-mimicking molecules (Dbait32Hc) to activate DNA-PK in the nucleus and cytoplasm, we identified 26 proteins that were highly phosphorylated following DNA-PK activation. Most of these proteins are involved in protein stability and degradation, cell signaling and the cytoskeleton. We investigated the relationship between DNA-PK and the cytoskeleton and found that the intermediate filament (IF) vimentin was a target of DNA-PK in vitro and in cells. Vimentin was phosphorylated at Ser459, by DNA-PK, in cells transfected with Dbait32Hc. We produced specific antibodies and showed that Ser459-P-vimentin was mostly located at cell protrusions. In migratory cells, the vimentin phosphorylation induced by Dbait32Hc was associated with a lower cellular adhesion and migration capacity. Thus, this approach led to the identification of downstream cytoplasmic targets of DNA-PK and revealed a connection between DNA damage signaling and the cytoskeleton. PMID:24282534

Kotula, Ewa; Faigle, Wolfgang; Berthault, Nathalie; Dingli, Florent; Loew, Damarys; Sun, Jian-Sheng; Dutreix, Marie; Quanz, Maria

2013-01-01

288

DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation  

PubMed Central

The DNA-dependent protein kinase (DNA-PK) may function as a key signaling kinase in various cellular pathways other than DNA repair. Using a two-dimensional gel electrophoresis approach and stable DNA double-strand break-mimicking molecules (Dbait32Hc) to activate DNA-PK in the nucleus and cytoplasm, we identified 26 proteins that were highly phosphorylated following DNA-PK activation. Most of these proteins are involved in protein stability and degradation, cell signaling and the cytoskeleton. We investigated the relationship between DNA-PK and the cytoskeleton and found that the intermediate filament (IF) vimentin was a target of DNA-PK in vitro and in cells. Vimentin was phosphorylated at Ser459, by DNA-PK, in cells transfected with Dbait32Hc. We produced specific antibodies and showed that Ser459-P-vimentin was mostly located at cell protrusions. In migratory cells, the vimentin phosphorylation induced by Dbait32Hc was associated with a lower cellular adhesion and migration capacity. Thus, this approach led to the identification of downstream cytoplasmic targets of DNA-PK and revealed a connection between DNA damage signaling and the cytoskeleton. PMID:24282534

Kotula, Ewa; Faigle, Wolfgang; Berthault, Nathalie; Dingli, Florent; Loew, Damarys; Sun, Jian-Sheng; Dutreix, Marie; Quanz, Maria

2013-01-01

289

Use of hydroxyurea in the measurement of DNA repair by the BND cellulose method  

SciTech Connect

Hydroxyurea inhibition is a convenient method of suppressing replicative DNA synthesis for DNA excision-repair measurement by the BND cellulose technique. Nonetheless, hydroxyurea can introduce artefacts by direct reaction with repair-inducing compounds and by long-term inhibition of the overall repair process. A simple technique of overcoming these problems is described. Cells are reacted with repair-inducing compounds in the absence of hydroxyurea, the cells are washed free of inducer, hydroxyurea is added to 2 mM, and after a short period to establish replication inhibition, /sup 3/H dThd is added and repair measured over a one-hour incubation period.

Irwin, J.; Strauss, B.

1980-01-01

290

Repair of DNA treated with. gamma. -irradiation and chemical carcinogens. Progress report, 1980-1983  

SciTech Connect

We have studied in vitro DNA repair with the isolation and characterization of DNA glycosylases active in the removable of 3-methyladenine and the problem of repair of DNA in chromatin. The second area of focus has been on transposable elements and carcinogen action. The work on DNA adducts with ..beta..-propiolactone was done to define potential new substrates useful in a search for new glycosylases.

Goldthwait, D.A.

1984-02-01

291

Repair of DNA Strand Breaks in a Minichromosome In Vivo: Kinetics, Modeling, and Effects of Inhibitors  

PubMed Central

To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ?170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with ? photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20–30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair. PMID:23382828

Kumala, Slawomir; Fujarewicz, Krzysztof; Jayaraju, Dheekollu; Rzeszowska-Wolny, Joanna; Hancock, Ronald

2013-01-01

292

Topical liposomal DNA-repair enzymes in polymorphic light eruption.  

PubMed

Polymorphic light eruption (PLE) is a very frequent photodermatosis in Europe whose pathogenesis may involve resistance to UV-induced immune suppression and simultaneous immune reactions against skin photoneoantigens. We performed a randomized, double-blind, placebo-controlled intra-individual half-body trial to investigate the protective effect of an after-sun (AS) lotion containing DNA-repair enzymes (photolyase from Anacystis nidulans and Micrococcus luteus extract with endonuclease activity). Fourteen PLE patients were exposed to suberythemal doses of solar-simulated UV radiation on 4 consecutive days at 4 symmetrically located PLE-prone test fields per patient. The test fields were treated with (i) active AS lotion or (ii) a placebo lotion immediately after each UV exposure, or (iii) an SPF30 sunscreen before UV exposure or left untreated. All test fields were exposed to photoactivating blue light 1 h after each UV exposure. As shown by a newly established specific PLE test score (AA + SI + 0.4P [range, 0-12], where AA is affected area score [range, 0-4], SI is skin infiltration score [range, 0-4], and P is pruritus score on a visual analogue scale [range, 0-10]), PLE symptoms were significantly fewer on test sites treated with active AS lotion than on untreated (P = 0.00049) or placebo-treated test sites (P = 0.024). At 144 h after first UV exposure (the time point of maximal PLE symptoms), the mean test scores for untreated, active AS lotion-treated, and placebo-treated test fields were 4.39, 1.73 (61% reduction; 95% confidence interval (CI), 36% to 85%), and 3.20 (27% reduction; 95% CI, 3% to 51%), respectively. Pretreatment with SPF30 sunscreen completely prevented PLE symptoms in all patients. The present results indicate that DNA damage may trigger PLE and that the application of topical liposomes containing DNA repair enzymes to increase DNA repair may effectively prevent PLE. PMID:21437317

Hofer, Angelika; Legat, Franz J; Gruber-Wackernagel, Alexandra; Quehenberger, Franz; Wolf, Peter

2011-07-01

293

Orchestration of cooperative events in DNA synthesis and repair mechanism unraveled by transition path sampling of DNA polymerase ?'s closing  

PubMed Central

Our application of transition path sampling to a complex biomolecular system in explicit solvent, the closing transition of DNA polymerase ?, unravels atomic and energetic details of the conformational change that precedes the chemical reaction of nucleotide incorporation. The computed reaction profile offers detailed mechanistic insights into, as well as kinetic information on, the complex process essential for DNA synthesis and repair. The five identified transition states extend available experimental and modeling data by revealing highly cooperative dynamics and critical roles of key residues (Arg-258, Phe-272, Asp-192, and Tyr-271) in the enzyme's function. The collective cascade of these sequential conformational changes brings the DNA/DNA polymerase ? system to a state nearly competent for the chemical reaction and suggests how subtle residue motions and conformational rate-limiting steps affect reaction efficiency and fidelity; this complex system of checks and balances directs the system to the chemical reaction and likely helps the enzyme discriminate the correct from the incorrect incoming nucleotide. Together with the chemical reaction, these conformational features may be central to the dual nature of polymerases, requiring specificity (for correct nucleotide selection) as well as versatility (to accommodate different templates at every step) to maintain overall fidelity. Besides leading to these biological findings, our developed protocols open the door to other applications of transition path sampling to long-time, large-scale biomolecular reactions. PMID:15069184

Radhakrishnan, Ravi; Schlick, Tamar

2004-01-01

294

Comparison of phosphorylation kinetics in DNA repair proteins after exposure to high and low LET radiations  

NASA Astrophysics Data System (ADS)

We irradiated plateau phase normal human fibroblasts with 2 Gy X-rays 70 keV um carbon 290MeV n and 200 keV um iron ions 500 MeV n and observed the kinetics of phosphorylation in various proteins associated with DNA double strand break DSB repair GammaH2AX foci a marker for DSBs were detected immediately after irradiation and the peak of phosphorylation was seen 30 to 60 min post-irradiation for three kinds of radiations Disappearance of gamma-H2AX foci was much faster for X-irradiated samples than that for heavy ion irradiated samples the phosphorylation kinetics for carbon and iron ions are similar for gamma-H2AX foci In contrast phosphorylation of an NHEJ protein DNA-PKcs threonine 2609 was significantly delayed in carbon and iron irradiated cells when compared to X-irradiated cells Disappearance of DNA-PKcs sites was much faster in X-irradiated samples than carbon and iron samples which showed a similar pattern as in the case of gamma-H2AX Furthermore in the case of ATM protein phosphorylation serine 1981 iron irradiation alone caused a significant initial delay but the kinetics of disappearance is similar for iron and carbon samples with much higher remaining number of foci in iron samples than those for X-rays and carbon ions These results suggest that 1 high LET irradiation induces complex and or severe DNA DSB damage which affects the function of DSB repair proteins 2 Both ATM and DNA-PKcs may recognize the complexity of DSBs but ATM may be more sensitive to detecting the complexity of DSB damage 3 gamma-H2AX may

Okayasu, R.; Okabe, A.; Takakura, K.

295

Molecular pathways: exploiting tumor-specific molecular defects in DNA repair pathways for precision cancer therapy.  

PubMed

Disabling mutations in genome maintenance and DNA repair pathways are frequently observed in cancer. These DNA repair defects represent genetic aberrations that are specific to cancer cells and not present in healthy tissues. It is thought that these molecular defects produce a "mutator phenotype," which allows incipient cancer cells to accumulate additional cancer-promoting mutations. In recent years, our molecular understanding of DNA double-strand break (DSB) repair mechanisms has led to the development of targeted therapeutic approaches to selectively eradicate cancer cells that display defects in homologous recombination-mediated DNA DSB repair. These regimens for the treatment of homologous recombination-defective tumors predominantly aim at pharmacologically repressing the activity of PARP1, which is crucial for base excision repair, or to inhibit the nonhomologous end joining kinase DNA-PKcs (DNA-dependent protein kinase, catalytic subunit). Normal tissue can bypass PARP1- or DNA-PKcs inhibitor-induced genotoxic lesions via homologous recombination-mediated DNA DSB repair. In contrast, homologous recombination-defective cancer cells are unable to properly repair DNA DSBs, in the presence of PARP1 or DNA-PKcs inhibitors, ultimately leading to apoptotic cancer cell death. Clin Cancer Res; 20(23); 5882-7. ©2014 AACR. PMID:25451105

Dietlein, Felix; Reinhardt, H Christian

2014-12-01

296

Chromosomal Aberrations in DNA Repair Defective Cell Lines: Comparisons of Dose Rate and Radiation Quality  

NASA Technical Reports Server (NTRS)

Chromosome aberration yields were assessed in DNA double-strand break repair (DSB) deficient cells after acute doses of gamma-rays or high-LET iron nuclei, or low dose-rate (0.018 Gy/hr) gamma-rays. We studied several cell lines including fibroblasts deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase, DNA-PK activity. Chromosomes were analyzed using the fluorescence in-situ hybridization (FISH) chromosome painting method in cells at the first division post-irradiation and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma radiation induced higher yields of both simple and complex exchanges in the DSB repair defective cells than in the normal cells. The quadratic dose-response terms for both chromosome exchange types were significantly higher for the ATM and NBS defective lines than for normal fibroblasts. However, the linear dose-response term was significantly higher only for simple exchanges in the NBS cells. Large increases in the quadratic dose response terms indicate the important roles of ATM and NBS in chromatin modifications that facilitate correct DSB repair and minimize aberration formation. Differences in the response of AT and NBS deficient cells at lower doses suggests important questions about the applicability of observations of radiation sensitivity at high dose to low dose exposures. For all iron nuclei irradiated cells, regression models preferred purely linear and quadratic dose responses for simple and complex exchanges, respectively. All the DNA repair defective cell lines had lower Relative biological effectiveness (RBE) values than normal cells, the lowest being for the DNA-PK-deficient cells, which was near unity. To further investigate the sensitivity differences for low and low high doses, we performed chronic low dose-rate irradiation, and have begun studies with ATM and Nibrin inhibitors and siRNA knockout of these proteins. Results support the conclusion that for the endpoint of simple chromosomal aberrations (translocation or dicentrics), the increased radiation sensitivity of AT cells found at high doses (>1 Gy) does not carry over to low doses or doserates, while NBS cells show increased sensitivity for both high and low dose exposures.

George, K. A.; Hada, M.; Patel, Z.; Huff, J.; Pluth, J. M.; Cucinotta, F. A.

2009-01-01

297

Replication-coupled DNA Interstrand Crosslink repair in Xenopus egg extracts  

PubMed Central

Summary Interstrand crosslinks (ICL) are one of the most hazardous types of DNA damage as they form a roadblock to all processes that involve strand separation. Repair of these lesions involves several different DNA repair pathways but the molecular mechanism is unclear. Here we describe a system that allows the examination of ICL repair, via a physiological mechanism, in vitro. This system, which uses Xenopus egg extracts in combination with a DNA template that contains a site-specific ICL, represents a unique tool to study the molecular mechanism of ICL repair. PMID:22941607

Knipscheer, Puck; Räschle, Markus; Schärer, Orlando D.; Walter, Johannes C.

2014-01-01

298

Emergence of rationally designed therapeutic strategies for breast cancer targeting DNA repair mechanisms  

PubMed Central

Accumulating evidence suggests that many cancers, including BRCA1- and BRCA2-associated breast cancers, are deficient in DNA repair processes. Both hereditary and sporadic breast cancers have been found to have significant downregulation of repair factors. This has provided opportunities to exploit DNA repair deficiencies, whether acquired or inherited. Here, we review efforts to exploit DNA repair deficiencies in tumors, with a focus on breast cancer. A variety of agents, including PARP (poly [ADP-ribose] polymerase) inhibitors, are currently under investigation in clinical trials and available results will be reviewed. PMID:20459590

2010-01-01

299

Dynamics and Mechanism of (6-4) Photoproduct Repair in Damaged DNA by Photolyase  

NASA Astrophysics Data System (ADS)

(6-4) photoproduct, the second major DNA lesion induced by UV irradiation, is repaired by (6-4) photolyase using light energy. The molecular mechanism of enzymatic repair is poorly understood. Here we report the direct observation of catalytic processes by synchronizing the enzymatic dynamics with the repair function through femtosecond spectroscopy. We observed forward electron transfer from the excited flavin cofactor to damaged DNA at 225 ps, backward electron transfer from unrepaired DNA to flavin at 50 ps, and electron returns from repaired DNA to flavin at tens of nanoseconds. Strikingly, a 425-ps electron-induced proton transfer was observed for the first time, which is crucial for repair efficiency by competing with the non-repair backward electron transfer channel.

Li, J.; Liu, Z.; Tan, C.; Guo, X.; Wang, L.; Zhong, D.; Sancar, A.

2010-06-01

300

Spatiotemporal Dynamics of Early DNA Damage Response Proteins on Complex DNA Lesions  

PubMed Central

The response of cells to ionizing radiation-induced DNA double-strand breaks (DSB) is determined by the activation of multiple pathways aimed at repairing the injury and maintaining genomic integrity. Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. FRAP measurements revealed two different binding modalities of NBS1 to damage sites and a direct impact of lesion complexity on the binding. Faster recruitment with increasing lesion complexity was also observed for the mediator MDC1, but mobility was limited at very high damage densities due to nuclear-wide binding. We constructed a minimal computer model of the initial response to DSB based on known protein interactions only. By fitting all measured data using the same set of parameters, we can reproduce the experimentally characterized steps of the DNA damage response over a wide range of damage densities. The model suggests that the influence of increasing lesion density accelerating NBS1 recruitment is only dependent on the different binding modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This elucidates an impact of damage clustering on repair without the need of invoking extra processing steps. PMID:23469115

Tobias, Frank; Löb, Daniel; Lengert, Nicor; Durante, Marco; Drossel, Barbara; Taucher-Scholz, Gisela; Jakob, Burkhard

2013-01-01

301

Calculation of complex DNA damage induced by ions  

E-print Network

This paper is devoted to the analysis of the complex damage of DNA irradiated by ions. The analysis and assessment of complex damage is important because cells in which it occurs are less likely to survive because the DNA repair mechanisms may not be sufficiently effective. We studied the flux of secondary electrons through the surface of nucleosomes and calculated the radial dose and the distribution of clustered damage around the ion's track. The calculated radial dose distribution is compared to simulations. The radial distribution of the complex damage is found to be different from that of the dose. Comparison with experiments may solve the question of what is more lethal for the cell, damage complexity or absorbed energy. We suggest a way to calculate the probability of cell death based on the complexity of the damage. This work is done within the framework of the phenomenon-based multiscale approach to radiation damage by ions.

Eugene Surdutovich; David C. Gallagher; Andrey V. Solov'yov

2012-01-27

302

Intelligent Design | Overwhelming Evidence Human DNA repair process video -by chance?  

E-print Network

Intelligent Design | Overwhelming Evidence Human DNA repair process video - by chance? Uncommon process recorded in action (Video) (PhysOrg.com) -- A key phase in the repair process of damaged human DNA natural and man-made. Because damage can lead to cancer, cell death and mutations, an army of proteins

Kowalczykowski, Stephen C.

303

XERODERMA PIGMENTOSUM: A HUMAN DISEASE IN WHICH AN INITIAL STAGE OF DNA REPAIR IS DEFECTIVE*  

PubMed Central

Homozygous xeroderma pigmentosum fibroblasts cannot repair damage to DNA bases, but can repair damage that involves chain breaks. In xeroderma pigmentosum, therefore, there is a defect in an early step in repair at which base damage is recognized and the polynucleotide chain broken enzymatically (by an endonuclease). Heterozygous fibroblasts repair base damage to normal extents. Carcinogenesis in xeroderma pigmentosum, and perhaps in some normal individuals, may be the result of somatic mutations caused by unrepaired damage. Images PMID:5257133

Cleaver, J. E.

1969-01-01

304

Structural Basis of O6-Alkylguanine Recognition by a Bacterial Alkyltransferase-like DNA Repair Protein*  

PubMed Central

Alkyltransferase-like proteins (ATLs) are a novel class of DNA repair proteins related to O6-alkylguanine-DNA alkyltransferases (AGTs) that tightly bind alkylated DNA and shunt the damaged DNA into the nucleotide excision repair pathway. Here, we present the first structure of a bacterial ATL, from Vibrio parahaemolyticus (vpAtl). We demonstrate that vpAtl adopts an AGT-like fold and that the protein is capable of tightly binding to O6-methylguanine-containing DNA and disrupting its repair by human AGT, a hallmark of ATLs. Mutation of highly conserved residues Tyr23 and Arg37 demonstrate their critical roles in a conserved mechanism of ATL binding to alkylated DNA. NMR relaxation data reveal a role for conformational plasticity in the guanine-lesion recognition cavity. Our results provide further evidence for the conserved role of ATLs in this primordial mechanism of DNA repair. PMID:20212037

Aramini, James M.; Tubbs, Julie L.; Kanugula, Sreenivas; Rossi, Paolo; Ertekin, Asli; Maglaqui, Melissa; Hamilton, Keith; Ciccosanti, Colleen T.; Jiang, Mei; Xiao, Rong; Soong, Ta-Tsen; Rost, Burkhard; Acton, Thomas B.; Everett, John K.; Pegg, Anthony E.; Tainer, John A.; Montelione, Gaetano T.

2010-01-01

305

Electron Tunneling Pathway and Role of Adenine in Repair of Damaged DNA by Photolyase  

NASA Astrophysics Data System (ADS)

Through electron tunneling, photolyase, a photoenzyme, restores damaged DNA into normal bases. Here, we report our systematic characterization and analyses of three electron transfer processes in thymine dimer restoration by following the entire dynamical evolution during enzymatic repair with femtosecond resolution. We observed the complete dynamics of the reactants, all intermediates and final products, and determined their reaction time scales. Using (deoxy)uracil and thymine as dimer substrates, we unambiguously determined the electron tunneling pathways for the forward electron transfer to initiate repairing and for the final electron return to restore the active cofactor and complete the repair photocycle. Significantly, we found that the adenine moiety of the unusual bent cofactor is essential to mediating all electron transfer dynamics through a super-exchange mechanism, leading to a delicate balance of time scales. The active-site structural integrity, unique electron tunneling pathways and the critical role of adenine assure these elementary dynamics in synergy in this complex photorepair machinery to achieve the maximum repair efficiency close to unity. Z. Liu, C. Tan, X. Guo, Y.-T. Kao, J. Li, L. Wang, A. Sancar, and D. Zhong, Proc. Natl. Acad. Sci. USA 108, 14831 (2011) J. Li, Z. Liu, C. Tan, X. Guo, L. Wang, A. Sancar, and D. Zhong, Nature 466, 887 (2010)

Liu, Zheyun; Tan, Chuang; Guo, Xunmin; Kao, Ya-Ting; Li, Jiang; Wang, Lijuan; Zhong, Dongping

2012-06-01

306

Laser microbeam - kinetic studies combined with molecule - structures reveal mechanisms of DNA repair  

NASA Astrophysics Data System (ADS)

Kinetic studies on double strand DNA damages induced by a laser microbeam have allowed a precise definition of the temporal order of recruitment of repair molecules. The order is KU70 / KU80 - XRCC4 --NBS1 -- RAD51. These kinetic studies are now complemented by studies on molecular structures of the repair proteins, using the program YASARA which does not only give molecular structures but also physicochemical details on forces involved in binding processes. It turns out that the earliest of these repair proteins, the KU70 / KU80 heterodimer, has a hole with high DNA affinity. The next molecule, XRCC4, has a body with two arms, the latter with extremely high DNA affinity at their ends and a binding site for Ligase 4. Together with the laser microbeam results this provides a detailed view on the early steps of DNA double strand break repair. The sequence of DNA repair events is presented as a movie.

Altenberg, B.; Greulich, K. O.

2011-10-01

307

SOS repair and DNA supercoiling influence the genetic stability of DNA triplet repeats in Escherichia coli.  

PubMed

Molecular mechanisms responsible for the genetic instability of DNA trinucleotide sequences (TRS) account for at least 20 human hereditary disorders. Many aspects of DNA metabolism influence the frequency of length changes in such repeats. Herein, we demonstrate that expression of Escherichia coli SOS repair proteins dramatically decreases the genetic stability of long (CTG/CAG)n tracts contained in plasmids. Furthermore, the growth characteristics of the bacteria are affected by the (CTG/CAG)n tract, with the effect dependent on the length of the TRS. In an E. coli host strain with constitutive expression of the SOS regulon, the frequency of deletions to the repeat is substantially higher than that in a strain with no SOS response. Analyses of the topology of reporter plasmids isolated from the SOS+ and SOS- strains revealed higher levels of negative supercoiling in strains with the constitutively expressed SOS network. Hence, we used strains with mutations in topoisomerases to examine the effect of DNA topology upon the TRS instability. Higher levels of negative DNA supercoiling correlated with increased deletions in long (CTG/CAG)n, (CGG/CCG)n and (GAA/TTC)n. These observations suggest a link between the induction of bacterial SOS repair, changes in DNA topology and the mechanisms leading to genetic instability of repetitive DNA sequences. PMID:17028021

Majchrzak, Marta; Bowater, Richard P; Staczek, Pawel; Parniewski, Pawel

2006-12-01

308

Toll-like receptor-4 deficiency enhances repair of UVR-induced cutaneous DNA damage by nucleotide excision repair mechanism.  

PubMed

UVB-induced DNA damage has a critical role in the development of photoimmunosuppression. The purpose of this study was to determine whether repair of UVB-induced DNA damage is regulated by Toll-like receptor-4 (TLR4). When TLR4 gene knockout (TLR4(-/-)) and TLR4-competent (TLR4(+/+)) mice were subjected to 90?mJ?cm(-2) UVB radiation locally, DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) was repaired more efficiently in the skin and bone marrow-derived dendritic cells (BMDCs) of TLR4(-/-) mice in comparison to TLR4(+/+) mice. Expression of DNA repair gene XPA (xeroderma pigmentosum complementation group A) was significantly lower in skin and BMDCs of TLR4(+/+) mice than TLR4(-/-) mice after UVB exposure. When cytokine levels were compared in these strains after UVB exposure, BMDCs from UV-irradiated TLR4(-/-) mice produced significantly more interleukin (IL)-12 and IL-23 cytokines (P<0.05) than BMDCs from TLR4(+/+) mice. Addition of anti-IL-12 and anti-IL-23 antibodies to BMDCs of TLR4(-/-) mice (before UVB exposure) inhibited repair of CPDs, with a concomitant decrease in XPA expression. Addition of TLR4 agonist to TLR4(+/+) BMDC cultures decreased XPA expression and inhibited CPD repair. Thus, strategies to inhibit TLR4 may allow for immunopreventive and immunotherapeutic approaches for managing UVB-induced cutaneous DNA damage and skin cancer. PMID:24326454

Ahmad, Israr; Simanyi, Eva; Guroji, Purushotham; Tamimi, Iman A; delaRosa, Hillary J; Nagar, Anusuiya; Nagar, Priyamvada; Katiyar, Santosh K; Elmets, Craig A; Yusuf, Nabiha

2014-06-01

309

Toll-Like Receptor-4 deficiency enhances repair of ultraviolet radiation induced cutaneous DNA damage by nucleotide excision repair mechanism  

PubMed Central

UVB-induced DNA damage plays a critical role in development of photoimmunosuppression. The purpose of this study was to determine whether repair of UVB-induced DNA damage is regulated by Toll-like receptor-4 (TLR4). When TLR4 gene knockout (TLR4-/-) and TLR4 competent (TLR4+/+) mice were subjected to 90 mJ/cm2 UVB radiation locally, DNA damage in the form of CPD, were repaired more efficiently in the skin and bone marrow dendritic cells (BMDC) of TLR4-/- mice in comparison to TLR4+/+ mice. Expression of DNA repair gene XPA (Xeroderma pigmentosum complementation group A) was significantly lower in skin and BMDC of TLR4+/+ mice than TLR4-/- mice after UVB exposure. When cytokine levels were compared in these strains after UVB exposure, BMDC from UV-irradiated TLR4-/- mice produced significantly more interleukin (IL)-12 and IL-23 cytokines (p<0.05) than BMDC from TLR4+/+ mice. Addition of anti-IL-12 and anti-IL-23 antibodies to BMDC of TLR4-/- mice (before UVB exposure) inhibited repair of CPD, with a concomitant decrease in XPA expression. Addition of TLR4 agonist to TLR4+/+ BMDC cultures decreased XPA expression and inhibited CPD repair. Thus, strategies to inhibit TLR4 may allow for immunopreventive and immunotherapeutic approaches for managing UVB-induced cutaneous DNA damage and skin cancer. PMID:24326454

Ahmad, Israr; Simanyi, Eva; Guroji, Purushotham; Tamimi, Iman A; delaRosa, Hillary J; Nagar, Anusuiya; Nagar, Priyamvada; Katiyar, Santosh K; Elmets, Craig A; Yusuf, Nabiha

2014-01-01

310

Involvement of oxidatively damaged DNA and repair in cancer development and aging  

PubMed Central

DNA damage and DNA repair may mediate several cellular processes, like replication and transcription, mutagenesis and apoptosis and thus may be important factors in the development and pathology of an organism, including cancer. DNA is constantly damaged by reactive oxygen species (ROS) and reactive nitrogen species (RNS) directly and also by products of lipid peroxidation (LPO), which form exocyclic adducts to DNA bases. A wide variety of oxidatively-generated DNA lesions are present in living cells. 8-oxoguanine (8-oxoGua) is one of the best known DNA lesions due to its mutagenic properties. Among LPO-derived DNA base modifications the most intensively studied are ethenoadenine and ethenocytosine, highly miscoding DNA lesions considered as markers of oxidative stress and promutagenic DNA damage. Although at present it is impossible to directly answer the question concerning involvement of oxidatively damaged DNA in cancer etiology, it is likely that oxidatively modified DNA bases may serve as a source of mutations that initiate carcinogenesis and are involved in aging (i.e. they may be causal factors responsible for these processes). To counteract the deleterious effect of oxidatively damaged DNA, all organisms have developed several DNA repair mechanisms. The efficiency of oxidatively damaged DNA repair was frequently found to be decreased in cancer patients. The present work reviews the basis for the biological significance of DNA damage, particularly effects of 8-oxoGua and ethenoadduct occurrence in DNA in the aspect of cancer development, drawing attention to the multiplicity of proteins with repair activities. PMID:20589166

Tudek, Barbara; Winczura, Alicja; Janik, Justyna; Siomek, Agnieszka; Foksinski, Marek; Oli?ski, Ryszard

2010-01-01

311

ARCH domain of XPD, an anchoring platform for CAK that conditions TFIIH DNA repair and transcription activities.  

PubMed

The xeroderma pigmentosum group D (XPD) helicase is a subunit of transcription/DNA repair factor, transcription factor II H (TFIIH) that catalyzes the unwinding of a damaged DNA duplex during nucleotide excision repair. Apart from two canonical helicase domains, XPD is composed of a 4Fe-S cluster domain involved in DNA damage recognition and a module of uncharacterized function termed the "ARCH domain." By investigating the consequences of a mutation found in a patient with trichothiodystrophy, we show that the ARCH domain is critical for the recruitment of the cyclin-dependent kinase (CDK)-activating kinase (CAK) complex. Indeed, this mutation not only affects the interaction with the MAT1 CAK subunit, thereby decreasing the in vitro basal transcription activity of TFIIH itself and impeding the efficient recruitment of the transcription machinery on the promoter of an activated gene, but also impairs the DNA unwinding activity of XPD and the nucleotide excision repair activity of TFIIH. We further demonstrate the role of CAK in downregulating the XPD helicase activity within TFIIH. Taken together, our results identify the ARCH domain of XPD as a platform for the recruitment of CAK and as a potential molecular switch that might control TFIIH composition and play a key role in the conversion of TFIIH from a factor active in transcription to a factor involved in DNA repair. PMID:23382212

Abdulrahman, Wassim; Iltis, Izarn; Radu, Laura; Braun, Cathy; Maglott-Roth, Anne; Giraudon, Christophe; Egly, Jean-Marc; Poterszman, Arnaud

2013-02-19

312

Conformational Analysis of DNA Repair Intermediates by Time-Resolved Fluorescence Spectroscopy  

NASA Astrophysics Data System (ADS)

DNA repair enzymes are essential for maintaining the integrity of the DNA sequence. Unfortunately, very little is known about how these enzymes recognize damaged regions along the helix. Structural analysis of cellular repair enzymes bound to DNA reveals that these enzymes are able to recognize DNA in a variety of conformations. However, the prevalence of these deformations in the absence of enzymes remains unclear, as small populations of DNA conformations are often difficult to detect by NMR and X-ray crystallography. Here, we used time-resolved fluorescence spectroscopy to examine the conformational dynamics of linear, nicked, gapped, and bulged DNA in the absence of protein enzymes. This analysis reveals that damaged DNA is polymorphic in nature and able to adopt multiple individual conformations. We show that DNA repair intermediates that contain a one-nucleotide gap and bulge have a significant propensity to adopt conformations in which the orphan base resides outside the DNA helix, while DNA structures damaged by a nick or two-nucleotide gap favor intrahelical conformations. Because changes in DNA conformation appear to guide the recognition of DNA repair enzymes, we suggest that the current approach could be used to study the mechanism of DNA repair.

Lin, Su; Horning, David P.; Szostak, Jack W.; Chaput, John C.

2009-08-01

313

BRCA2: One Small Step for DNA Repair, One Giant Protein Purified  

PubMed Central

DNA damage, malfunctions in DNA repair, and genomic instability are processes that intersect at the crossroads of carcinogenesis. Underscoring the importance of DNA repair in breast and ovarian tumorigenesis is the familial inherited cancer predisposition gene BRCA2. The role of BRCA2 in DNA double-strand break repair was first revealed based on its interaction with RAD51, a central player in homologous recombination. The RAD51 protein forms a nucleoprotein filament on single-stranded DNA, invades a DNA duplex, and initiates a search for homology. Once a homologous DNA sequence is found, the DNA is used as a template for the high-fidelity repair of the DNA break. Many of the biochemical features that allow BRCA2 to choreograph the activities of RAD51 have been elucidated and include: targeting RAD51 to single-stranded DNA while inhibiting binding to dsDNA, reducing the ATPase activity of RAD51, and facilitating the displacement of the single-strand DNA binding protein, Replication Protein A. These reinforcing activities of BRCA2 culminate in the correct positioning of RAD51 onto a processed DNA double-strand break and initiate its faithful repair by homologous recombination. In this review, I will address current biochemical data concerning the BRCA2 protein and highlight unanswered questions regarding BRCA2 function in homologous recombination and cancer. PMID:24348212

Jensen, Ryan B.

2013-01-01

314

A Rapid, Simple DNA Mismatch Repair Substrate Construction Method  

PubMed Central

A more flexible and higher-yielding in vitro DNA mismatch repair (MMR) substrate construction method, which was developed initially by Wang and Hays, is described for the construction of a nucleotide-based chemical mismatch (G/IU) and a G/T mismatch. Our modifications use the combination of two endonuclease enzymes (NheI and BciVI) and two new redesigned plasmids (pWDAH1A and pWDSH1B). In our modified methodology, plasmids are initially digested with the nicking endonucleases, followed by the streptavidin treatment. The mismatch-containing oligo is then annealed to the gap DNA and finally ligated to produce a mismatch-containing DNA substrate. We report a high efficiency (up to 90%) of these mismatch substrates and confirm recognition using a functional assay. These modifications, coupled with the use of the redesigned plasmids, can be applied for the construction of other types of chemically induced mismatches as well as insertion-deletion loops for future in vitro studies of MMR processing by our group and others. PMID:22655228

Du, Weinan; Kinsella, Timothy J.

2011-01-01

315

Insights into protein -- DNA interactions, stability and allosteric communications: A computational study of MutS-DNA recognition complexes  

NASA Astrophysics Data System (ADS)

DNA mismatch repair proteins (MMR) maintain genetic stability by recognizing and repairing mismatched bases and insertion/deletion loops mistakenly incorporated during DNA replication, and initiate cellular response to certain types of DNA damage. The most abundant MMR mismatch-binding factor in eukaryotes, MutS, recognizes and initiates the repair of base-base mismatches and small insertion/deletions. We performed molecular dynamics simulations on mismatched and damaged MutS-DNA complexes. A comprehensive DNA binding site analysis of relevant conformations shows that MutS proteins recognize the mismatched and platinum cross-linked DNA substrates in significantly different modes. Distinctive conformational changes associated with MutS binding to mismatched and damaged DNA have been identified and they provide insight into the involvement of MMR proteins in DNA-repair and DNA-damage pathways. Stability and allosteric interactions at the heterodimer interface associated with the mismatch and damage recognition step allow for prediction of key residues in MMR cancer-causing mutations. A rigorous hydrogen bonding analysis for ADP molecules at the ATPase binding sites is also presented. A large number of known MMR cancer causing mutations among the residues were found.

Negureanu, Lacramioara; Salsbury, Freddie

2012-02-01

316

DNA Damage Response Factors from Diverse Pathways, Including DNA Crosslink Repair, Mediate Alternative End Joining  

PubMed Central

Alternative end joining (Alt-EJ) chromosomal break repair involves bypassing classical non-homologous end joining (c-NHEJ), and such repair causes mutations often with microhomology at the repair junction. Since the mediators of Alt-EJ are not well understood, we have sought to identify DNA damage response (DDR) factors important for this repair event. Using chromosomal break reporter assays, we surveyed an RNAi library targeting known DDR factors for siRNAs that cause a specific decrease in Alt-EJ, relative to an EJ event that is a composite of Alt-EJ and c-NHEJ (Distal-EJ between two tandem breaks). From this analysis, we identified several DDR factors that are specifically important for Alt-EJ relative to Distal-EJ. While these factors are from diverse pathways, we also found that most of them also promote homologous recombination (HR), including factors important for DNA crosslink repair, such as the Fanconi Anemia factor, FANCA. Since bypass of c-NHEJ is likely important for both Alt-EJ and HR, we disrupted the c-NHEJ factor Ku70 in Fanca-deficient mouse cells and found that Ku70 loss significantly diminishes the influence of Fanca on Alt-EJ. In contrast, an inhibitor of poly ADP-ribose polymerase (PARP) causes a decrease in Alt-EJ that is enhanced by Ku70 loss. Additionally, the helicase/nuclease DNA2 appears to have distinct effects from FANCA and PARP on both Alt-EJ, as well as end resection. Finally, we found that the proteasome inhibitor Bortezomib, a cancer therapeutic that has been shown to disrupt FANC signaling, causes a significant reduction in both Alt-EJ and HR, relative to Distal-EJ, as well as a substantial loss of end resection. We suggest that several distinct DDR functions are important for Alt-EJ, which include promoting bypass of c-NHEJ and end resection. PMID:25629353

Howard, Sean M.; Yanez, Diana A.; Stark, Jeremy M.

2015-01-01

317

[Functions of PALB2 and BRCA2 tumor suppressors in DNA double-strand break repair].  

PubMed

Cancer is now the leading cause of mortality in France. It has been clearly demonstrated that mutations in the genetic information is the initiating event of cancer. DNA damage such as DNA double-strand breaks leads to genomic instability and cancer development. Cells can repair DNA double-strand breaks through several mechanisms. Nevertheless, only homologous recombination repair is faithful and repairs DNA without creating mutations. Here, we review the roles of PALB2 and BRCA2 in homologous recombination and genome stability. PMID:23544385

Buisson, Rémi; Masson, Jean-Yves

2013-03-01

318

DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks  

PubMed Central

Nonhomologous end joining (NHEJ) is the major pathway for the repair of DNA double strand breaks (DSBs) in human cells. NHEJ requires the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku70, Ku80, XRCC4, DNA ligase IV and Artemis, as well as DNA polymerases ? and ? and polynucleotide kinase. Recent studies have identified an additional participant, XLF, for XRCC4-like factor (also called Cernunnos), which interacts with the XRCC4-DNA ligase IV complex and stimulates its activity in vitro, however, its precise role in the DNA damage response is not fully understood. Since the protein kinase activity of DNA-PKcs is required for NHEJ, we asked whether XLF might be a physiological target of DNA-PK. Here, we have identified two major in vitro DNA-PK phosphorylation sites in the C-terminal region of XLF, serines 245 and 251. We show that these represent the major phosphorylation sites in XLF in vivo and that serine 245 is phosphorylated in vivo by DNA-PK, while serine 251 is phosphorylated by Ataxia-Telangiectasia Mutated (ATM). However, phosphorylation of XLF did not have a significant effect on the ability of XLF to interact with DNA in vitro or its recruitment to laser-induced DSBs in vivo. Similarly, XLF in which the identified in vivo phosphorylation sites were mutated to alanine was able to complement the DSB repair defect as well as radiation sensitivity in XLF-deficient 2BN cells. We conclude that phosphorylation of XLF at these sites does not play a major role in the repair of IR-induced DSBs in vivo. PMID:18644470

Yu, Yaping; Mahaney, Brandi L.; Yano, Ken-Ichi; Ye, Ruiqiong; Fang, Shujuan; Douglas, Pauline; Chen, David J.; Lees-Miller, Susan P.

2012-01-01

319

Electrostatic interactions play an essential role in DNA repair and cold-adaptation of uracil DNA glycosylase.  

PubMed

Life has adapted to most environments on earth, including low and high temperature niches. The increased catalytic efficiency and thermoliability observed for enzymes from organisms living in constantly cold regions when compared to their mesophilic and thermophilic cousins are poorly understood at the molecular level. Uracil DNA glycosylase (UNG) from cod (cUNG) catalyzes removal of uracil from DNA with an increased k(cat) and reduced K(m) relative to its warm-active human (hUNG) counterpart. Specific issues related to DNA repair and substrate binding/recognition (K(m)) are here investigated by continuum electrostatics calculations, MD simulations and free energy calculations. Continuum electrostatic calculations reveal that cUNG has surface potentials that are more complementary to the DNA potential at and around the catalytic site when compared to hUNG, indicating improved substrate binding. Comparative MD simulations combined with free energy calculations using the molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) method show that large opposing energies are involved when forming the enzyme-substrate complexes. Furthermore, the binding free energies obtained reveal that the Michaelis-Menten complex is more stable for cUNG, primarily due to enhanced electrostatic properties, suggesting that energetic fine-tuning of electrostatics can be utilized for enzymatic temperature adaptation. Energy decomposition pinpoints the residual determinants responsible for this adaptation. PMID:18196298

Olufsen, Magne; Smalås, Arne O; Brandsdal, Bjørn O

2008-03-01

320

Temple scientists target DNA repair to eradicate leukemia stem cells  

Cancer.gov

Despite treatment with imatinib, a successful drug that targets chronic myeloid leukemia (CML), a deadly type of cancer, some patients may continue to be at risk for relapse because a tiny pool of stem cells is resistant to treatment and may even accumulate additional genetic aberrations, eventually leading to disease progression and relapse. These leukemia stem cells are full of genetic errors, loaded with potentially lethal breaks in DNA, and are in a state of constant self-repair. Now, scientists at Temple University School of Medicine (home to the Fox Chase Cancer Center) may have figured out a way to corral this stem cell activity and stunt further cancer development. In a series of experiments in mice with cancer and in cancer cells, they have shown that they can block the process by which leukemia stem cells repair themselves by targeting a particular protein, RAD52, which the cells depend on to fix genetic mistakes. The findings may lead to a new strategy to help overcome drug resistance that hinges on cancer stem cells gone awry.

321

Interfacial instability and DNA fork reversal by repair proteins  

NASA Astrophysics Data System (ADS)

A repair protein like RecG moves the stalled replication fork in the direction from the zipped to the unzipped state of DNA. It is proposed here that a softening of the zipped-unzipped interface at the fork results in the front propagating towards the unzipped side. In this scenario, an ordinary helicase destabilizes the zipped state locally near the interface and the fork propagates towards the zipped side. The softening of the interface can be produced by the aromatic interaction, predicted from the crystal structure, between RecG and the nascent broken base pairs at the Y-fork. A numerical analysis of the model also reveals the possibility of a stop and go type motion.

Bhattacharjee, Somendra M.

2010-04-01

322

The role of DNA repair pathways in cisplatin resistant lung cancer.  

PubMed

Platinum chemotherapeutic agents such as cisplatin are currently used in the treatment of various malignancies such as lung cancer. However, their efficacy is significantly hindered by the development of resistance during treatment. While a number of factors have been reported that contribute to the onset of this resistance phenotype, alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon. The mode of action of cisplatin has been linked to its ability to crosslink purine bases on the DNA, thereby interfering with DNA repair mechanisms and inducing DNA damage. Following DNA damage, cells respond by activating a DNA-damage response that either leads to repair of the lesion by the cell thereby promoting resistance to the drug, or cell death via activation of the apoptotic response. Therefore, DNA repair is a vital target to improving cancer therapy and reduce the resistance of tumour cells to DNA damaging agents currently used in the treatment of cancer patients. To date, despite the numerous findings that differential expression of components of the various DNA repair pathways correlate with response to cisplatin, translation of such findings in the clinical setting are still warranted. The identification of alterations in specific proteins and pathways that contribute to these unique DNA repair pathways in cisplatin resistant cancer cells may potentially lead to a renewed interest in the development of rational novel therapies for cisplatin resistant cancers, in particular, lung cancer. PMID:25458603

O'Grady, Shane; Finn, Stephen P; Cuffe, Sinead; Richard, Derek J; O'Byrne, Kenneth J; Barr, Martin P

2014-12-01

323

TAT-mediated delivery of a DNA repair enzyme to skin cells rapidly initiates repair of UV-induced DNA damage  

PubMed Central

Ultraviolet (UV) light causes DNA damage in skin cells, leading to more than one million cases of non-melanoma skin cancer diagnosed annually in the United States. Although human cells possess a mechanism (Nucleotide Excision Repair, NER) to repair UV-induced DNA damage, mutagenesis still occurs when DNA is replicated prior to repair of these photoproducts. While human cells have all the enzymes necessary to complete an alternate repair pathway, Base Excision Repair (BER), they lack a DNA glycosylase that can initiate BER of dipyrimidine photoproducts. Certain prokaryotes and viruses produce pyrimidine dimer-specific DNA glycosylases (pdgs) that initiate BER of cyclobutane pyrimidine dimers (CPDs), the predominant UV-induced lesions. Such a pdg was identified in the Chlorella virus PBCV-1 and termed Cv-pdg. The Cv-pdg protein was engineered to contain a nuclear localization sequence (NLS) and a membrane permeabilization peptide (TAT). Here, we demonstrate that the Cv-pdg-NLS-TAT protein was delivered to repair-proficient keratinocytes and fibroblasts, and to a human skin model, where it rapidly initiated removal of CPDs. These data suggest a potential strategy for prevention of human skin cancer. PMID:20927123

Johnson, Jodi L.; Lowell, Brian C.; Ryabinina, Olga P.; Lloyd, R. Stephen; McCullough, Amanda K.

2011-01-01

324

GENETIC AND MOLECULAR ANALYSIS OF DNA DAMAGE REPAIR AND TOLERANCE PATHWAYS.  

SciTech Connect

Radiation can damage cellular components, including DNA. Organisms have developed a panoply of means of dealing with DNA damage. Some repair paths have rather narrow substrate specificity (e.g. photolyases), which act on specific pyrimidine photoproducts in a specific type (e.g., DNA) and conformation (double-stranded B conformation) of nucleic acid. Others, for example, nucleotide excision repair, deal with larger classes of damages, in this case bulky adducts in DNA. A detailed discussion of DNA repair mechanisms is beyond the scope of this article, but one can be found in the excellent book of Friedberg et al. [1] for further detail. However, some DNA damages and paths for repair of those damages important for photobiology will be outlined below as a basis for the specific examples of genetic and molecular analysis that will be presented below.

SUTHERLAND, B.M.

2001-07-26

325

HSF4 is involved in DNA damage repair through regulation of Rad51.  

PubMed

Heat shock factor protein 4 (HSF4) is expressed exclusively in the ocular lens and plays a critical role in the lens formation and differentiation. Mutations in the HSF4 gene lead to congenital and senile cataract. However, the molecular mechanisms causing this disease have not been well characterized. DNA damage in lens is a crucial risk factor in senile cataract formation, and its timely repair is essential for maintaining the lens' transparency. Our study firstly found evidence that HSF4 contributes to the repair of DNA strand breaks. Yet, this does not occur with cataract causative mutations in HSF4. We verify that DNA damage repair is mediated by the binding of HSF4 to a heat shock element in the Rad51 promoter, a gene which assists in the homologous recombination (HR) repair of DNA strand breaks. HSF4 up-regulates Rad51 expression while mutations in HSF4 fail, and DNA does not get repaired. Camptothecin, which interrupts the regulation of Rad51 by HSF4, also affects DNA damage repair. Additionally, with HSF4 knockdown in the lens of Zebrafish, DNA damage was observed and the protein level of Rad51 was significantly lower. Our study presents the first evidence demonstrating that HSF4 plays a role in DNA damage repair and may contribute a better understanding of congenital cataract formation. PMID:22587838

Cui, Xiukun; Zhang, Jing; Du, Rong; Wang, Lei; Archacki, Stephen; Zhang, Yuexuan; Yuan, Mingxiong; Ke, Tie; Li, Hui; Li, Duanzhuo; Li, Chang; Li, David Wan-Cheng; Tang, Zhaohui; Yin, Zhan; Liu, Mugen

2012-08-01

326

Emerging roles of Jab1/CSN5 in DNA damage response, DNA repair, and cancer.  

PubMed

Jab1/CSN5 is a multifunctional protein that plays an important role in integrin signaling, cell proliferation, apoptosis, and the regulation of genomic instability and DNA repair. Dysregulation of Jab1/CSN5 activity has been shown to contribute to oncogenesis by functionally inactivating several key negative regulatory proteins and tumor suppressors. In this review, we discuss our current understanding of the relationship between Jab1/CSN5 and DNA damage and summarize recent findings regarding opportunities for and challenges to therapeutic intervention. PMID:24495954

Pan, Yunbao; Yang, Huiling; Claret, Francois X

2014-03-01

327

Genetic Variation in DNA Repair Pathway Genes and Melanoma Risk  

PubMed Central

Reduced DNA repair capacity has been proposed as a predisposing factor for melanoma. We comprehensively evaluated 1,463 genetic variants across 60 DNA repair–related pathway genes in relation to melanoma risk in a nested case-control study of 218 melanoma cases (20% on head and neck) and 218 matched controls within the Nurses' Health Study (NHS). We then genotyped the two variants with the smallest P value in two replication sets: 184 melanoma cases (28% on head and neck) and 184 matched controls in the Health Professionals Follow-Up Study (HPFS); and 183 melanoma cases (10% on head and neck) and 183 matched controls in the NHS. The SNP rs3219125 in the PARP1 gene was significantly associated with melanoma risk in the discovery set (odds ratio (OR) 3.14; 95% confidence interval (CI) 1.70–5.80) and in the HPFS replication set (OR, 1.92; 95%CI, 1.05–3.54) but not in the NHS replication set (OR, 1.07; 95%CI, 0.58–1.97). In the joint analysis, the OR was 1.89 (95%CI, 1.34–2.68) for this polymorphism, and this increased risk was more pronounced among patients with lesions in head/neck (OR, 3.19; 95% CI, 1.77–5.73 for head/neck, and OR, 1.54; 95% CI, 1.03–2.30 for other sites, P value for heterogeneity test = 0.036). Our findings suggest the possible involvement of the PARP1 variant in melanoma development, especially for sites with high sun exposure. Further work on fine-mapping and on the functional characterization of this and linked SNPs in this region is required. PMID:20837404

Zhang, Mingfeng; Qureshi, Abrar A.; Guo, Qun; Han, Jiali

2010-01-01

328

Polymorphism of the DNA Base Excision Repair Genes in Keratoconus  

PubMed Central

Keratoconus (KC) is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER). Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase ? (POLG) gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1) were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1) nor the c.2285T>C polymorphism of the poly(ADP-ribose) polymerase-1 (PARP-1) was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease. PMID:25356504

Wojcik, Katarzyna A.; Synowiec, Ewelina; Sobierajczyk, Katarzyna; Izdebska, Justyna; Blasiak, Janusz; Szaflik, Jerzy; Szaflik, Jacek P.

2014-01-01

329

New Tools to Study DNA Double-Strand Break Repair Pathway Choice  

PubMed Central

A broken DNA molecule is difficult to repair, highly mutagenic, and extremely cytotoxic. Such breaks can be repaired by homology-independent or homology-directed mechanisms. Little is known about the network that controls the repair pathway choice except that a licensing step for homology-mediated repair exists, called DNA-end resection. The choice between these two repair pathways is a key event for genomic stability maintenance, and an imbalance of the ratio is directly linked with human diseases, including cancer. Here we present novel reporters to study the balance between both repair options in human cells. In these systems, a double-strand break can be alternatively repaired by homology-independent or -dependent mechanisms, leading to the accumulation of distinct fluorescent proteins. These reporters thus allow the balance between both repair pathways to be analyzed in different experimental setups. We validated the reporters by analyzing the effect of protein downregulation of the DNA end resection and non-homologous end-joining pathways. Finally, we analyzed the role of the DNA damage response on double-strand break (DSB) repair mechanism selection. Our reporters could be used in the future to understand the roles of specific factors, whole pathways, or drugs in DSB repair pathway choice, or for genome-wide screening. Moreover, our findings can be applied to increase gene-targeting efficiency, making it a beneficial tool for a broad audience in the biological sciences. PMID:24155929

Gomez-Cabello, Daniel; Jimeno, Sonia; Fernández-Ávila, María Jesús; Huertas, Pablo

2013-01-01

330

Methylation of histone H3 lysine 36 enhances DNA repair by nonhomologous end-joining.  

PubMed

Given its significant role in the maintenance of genomic stability, histone methylation has been postulated to regulate DNA repair. Histone methylation mediates localization of 53BP1 to a DNA double-strand break (DSB) during homologous recombination repair, but a role in DSB repair by nonhomologous end-joining (NHEJ) has not been defined. By screening for histone methylation after DSB induction by ionizing radiation we found that generation of dimethyl histone H3 lysine 36 (H3K36me2) was the major event. Using a novel human cell system that rapidly generates a single defined DSB in the vast majority of cells, we found that the DNA repair protein Metnase (also SETMAR), which has a SET histone methylase domain, localized to an induced DSB and directly mediated the formation of H3K36me2 near the induced DSB. This dimethylation of H3K36 improved the association of early DNA repair components, including NBS1 and Ku70, with the induced DSB, and enhanced DSB repair. In addition, expression of JHDM1a (an H3K36me2 demethylase) or histone H3 in which K36 was mutated to A36 or R36 to prevent H3K36me2 formation decreased the association of early NHEJ repair components with an induced DSB and decreased DSB repair. Thus, these experiments define a histone methylation event that enhances DNA DSB repair by NHEJ. PMID:21187428

Fnu, Sheema; Williamson, Elizabeth A; De Haro, Leyma P; Brenneman, Mark; Wray, Justin; Shaheen, Montaser; Radhakrishnan, Krishnan; Lee, Suk-Hee; Nickoloff, Jac A; Hromas, Robert

2011-01-11

331

Contrasting enantioselective DNA preference: chiral helical macrocyclic lanthanide complex binding to DNA  

PubMed Central

There is great interest in design and synthesis of small molecules which selectively target specific genes to inhibit biological functions in which particular DNA structures participate. Among these studies, chiral recognition has been received much attention because more evidences have shown that conversions of the chirality and diverse conformations of DNA are involved in a series of important life events. Here, we report that a pair of chiral helical macrocyclic lanthanide (III) complexes, (M)-Yb[LSSSSSS]3+ and (P)-Yb[LRRRRRR]3+, can enantioselectively bind to B-form DNA and show remarkably contrasting effects on GC-rich and AT-rich DNA. Neither of them can influence non-B-form DNA, nor quadruplex DNA stability. Our results clearly show that P-enantiomer stabilizes both poly(dG-dC)2 and poly(dA-dT)2 while M-enantiomer stabilizes poly(dA-dT)2, however, destabilizes poly(dG-dC)2. To our knowledge, this is the best example of chiral metal compounds with such contrasting preference on GC- and AT-DNA. Ligand selectively stabilizing or destabilizing DNA can interfere with protein–DNA interactions and potentially affect many crucial biological processes, such as DNA replication, transcription and repair. As such, bearing these unique capabilities, the chiral compounds reported here may shed light on the design of novel enantiomers targeting specific DNA with both sequence and conformation preference. PMID:22675072

Zhao, Chuanqi; Ren, Jinsong; Gregoli?ski, Janusz; Lisowski, Jerzy; Qu, Xiaogang

2012-01-01

332

RAD18 transmits DNA damage signalling to elicit homologous recombination repair  

Microsoft Academic Search

To maintain genome stability, cells respond to DNA damage by activating signalling pathways that govern cell-cycle checkpoints and initiate DNA repair. Cell-cycle checkpoint controls should connect with DNA repair processes, however, exactly how such coordination occurs in vivo is largely unknown. Here we describe a new role for the E3 ligase RAD18 as the integral component in translating the damage

Jun Huang; Michael S. Y. Huen; Hongtae Kim; Charles Chung Yun Leung; J N Mark Glover; Xiaochun Yu; Junjie Chen

2009-01-01

333

In vivo DNA repair after N-methyl-N-nitrosourea administration to rats of different ages  

Microsoft Academic Search

Summary DNA repair time-course was studied after injury by N-methyl-N-nitrosourea (MNU) in rat liver cells of animals of different ages and in fetuses using hydroxyurea (HU) as inhibitor of scheduled DNA synthesis. DNA repair was a rapid phenomenon, more so in young adults than in newborns, and was not detectable in fetuses. A correlation seems to exist among organ sensitivity

G. Arfellini; S. Grilli; G. Prodi

1978-01-01

334

DNA repair in bacterial cultures and plasmid DNA exposed to infrared laser for treatment of pain  

NASA Astrophysics Data System (ADS)

Biostimulation of tissues by low intensity lasers has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases, but their effects on DNA are controversial. The objective of this work was to evaluate effects of low intensity infrared laser exposure on survival and bacterial filamentation in Escherichia coli cultures, and induction of DNA lesions in bacterial plasmids. In E. coli cultures and plasmids exposed to an infrared laser at fluences used to treat pain, bacterial survival and filamentation and DNA lesions in plasmids were evaluated by electrophoretic profile. Data indicate that the infrared laser (i) increases survival of E. coli wild type in 24 h of stationary growth phase, (ii) induces bacterial filamentation, (iii) does not alter topological forms of plasmids and (iv) does not alter the electrophoretic profile of plasmids incubated with exonuclease III or formamidopyrimidine DNA glycosylase. A low intensity infrared laser at the therapeutic fluences used to treat pain can alter survival of E. coli wild type, induce filamentation in bacterial cells, depending on physiologic conditions and DNA repair, and induce DNA lesions other than single or double DNA strand breaks or alkali-labile sites, which are not targeted by exonuclease III or formamidopyrimidine DNA glycosylase.

Canuto, K. S.; Sergio, L. P. S.; Marciano, R. S.; Guimarães, O. R.; Polignano, G. A. C.; Geller, M.; Paoli, F.; Fonseca, A. S.

2013-06-01

335

The comet assay, DNA damage, DNA repair and cytotoxicity: hedgehogs are not always dead.  

PubMed

DNA damage is commonly measured at the level of individual cells using the so-called comet assay (single-cell gel electrophoresis). As the frequency of DNA breaks increases, so does the fraction of the DNA extending towards the anode, forming the comet tail. Comets with almost all DNA in the tail are often referred to as 'hedgehog' comets and are widely assumed to represent apoptotic cells. We review the literature and present theoretical and empirical arguments against this interpretation. The level of DNA damage in these comets is far less than the massive fragmentation that occurs in apoptosis. 'Hedgehog' comets are formed after moderate exposure of cells to, for example, H2O2, but if the cells are incubated for a short period, 'hedgehogs' are no longer seen. We confirm that this is not because DNA has degraded further and been lost from the gel, but because the DNA is repaired. The comet assay may detect the earliest stages of apoptosis, but as it proceeds, comets disappear in a smear of unattached DNA. It is clear that 'hedgehogs' can correspond to one level on a continuum of genotoxic damage, are not diagnostic of apoptosis and should not be regarded as an indicator of cytotoxicity. PMID:23630247

Lorenzo, Yolanda; Costa, Solange; Collins, Andrew R; Azqueta, Amaya

2013-07-01

336

Spatio-temporal analysis of DNA damage repair using the X-ray microbeam  

NASA Astrophysics Data System (ADS)

Cellular response to radiation damage is made by a complex network of pathways and feedback loops whose spatiotemporal organization is still unclear despite its decisive role in determining the fate of the damaged cell. The single-cell approach and the high spatial resolution offered by microbeams provide the perfect tool to study and quantify the dynamic processes associated with the induction and repair of DNA damage. The soft X-ray microbeam has been used to follow the development of radiation induced foci in live cells by monitoring their size and intensity as a function of dose and time using yellow fluorescent protein (YFP) tagging techniques. Preliminary data indicate a delayed and linear rising of the intensity signal indicating a slow kinetic for the accumulation of DNA repair protein 53BP1. A slow and limited foci diffusion has also been observed. Further investigations are required to assess whatever such diffusion is consistent with a random walk pattern or if it is the result of a more structured lesion processing phenomenon. In conclusion, our data indicates that the use of microbeams coupled to live cell microscopy represent a sophisticated approach for visualizing and quantifying the dynamics changes of DNA proteins at the damaged sites.

Schettino, G.; Ghita, M.; Prise, K. M.

2010-10-01

337

A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA  

E-print Network

A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA Paul C. Blainey*, Antoine M. van Oijen* , Anirban Banerjee*, Gregory L. Verdine*§¶ , and X. Sunney in the function of site-specific DNA-binding proteins is the detailed mechanism for rapid location and binding

338

1999 Gordon Research Conference on Mammalian DNA Repair. Final Progress Report  

SciTech Connect

This Conference will examine DNA repair as the key component in genomic surveillance that is so crucial to the overall integrity and function of mammalian cells. Recent discoveries have catapulted the field of DNA repair into a pivotal position for fundamental investigations into oncology, aging, environmental health, and developmental biology. We hope to highlight the most promising and exciting avenues of research in robust discussions at this conference. This Mammalian DNA Repair Gordon Conference differs from the past conferences in this series, in which the programs were broader in scope, with respect to topics and biological systems covered. A conference sponsored by the Genetics Society in April 1998 emphasized recombinational mechanisms for double-strand break repair and the role of mismatch repair deficiency in colorectal cancer. These topics will therefore receive somewhat less emphasis in the upcoming Conference. In view of the recent mechanistic advances in mammalian DNA repair, an upcoming comprehensive DNA repair meeting next autumn at Hilton Head; and the limited enrollment for Gordon Conferences we have decided to focus session-by-session on particular areas of controversy and/or new developments specifically in mammalian systems. Thus, the principal presentations will draw upon results from other cellular systems only to the extent that they impact our understanding of mammalian DNA repair.

NONE

1999-02-12

339

Studies on the relationship between the cancer chemotherapeutic agent, hydroxyurea, and DNA repair in mammalian cells  

SciTech Connect

To examine the possibility that manipulating DNA repair might lessen drug resistance, we investigated whether depletion of the thymidine triphosphate (TTP) pool or administration of hydroxyurea could interfere with the ability of confluent normal human skin fibroblasts to repair ultraviolet irradiation-induced DNA damage. A method was developed for the quantitation of cellular TTP pools by labeling them with (/sup 3/H)thymidine. The addition of hydroxyurea, either simultaneously with (/sup 3/H)thymidine or two hours later, resulted in a dose- and time-dependent increase in the (/sup 3/H)TTP pool. The capacity of these cells to carry out DNA repair was quantitated by their ability to perform repair replication synthesis of DNA after exposure to ultraviolet irradiation. This radiation produces thymine dimers in DNA, which are repaired by the nucleotide excision repair pathway. The experimental protocol resulted in an 8-10-fold reduction in the (/sup 3/H)TTP pool. Saturating levels of DNA repair synthesis were observed under these conditions, with no further diminution of the already reduced (/sup 3/H)TTP pool. Repair replication and (/sup 3/H)TTP pool measurements were identical in cultures treated with 10 mM hydroxyurea and in those not exposed to the drug.

Katz, E.J.

1988-01-01

340

Inhibition of zygotic DNA repair: transcriptome analysis of the offspring in trout (Oncorhynchus mykiss).  

PubMed

Zygotic repair of the paternal genome is a key event after fertilization. Spermatozoa accumulate DNA strand breaks during spermatogenesis and can suffer additional damage by different factors, including cryopreservation. Fertilization with DNA-damaged spermatozoa (DDS) is considered to promote implantation failures and abortions, but also long-term effects on the progeny that could be related with a defective repair. Base excision repair (BER) pathway is considered the most active in zygotic DNA repair, but healthy oocytes contain enzymes for all repairing pathways. In this study, the effects of the inhibition of the BER pathway in the zygote were analyzed on the progeny obtained after fertilization with differentially DDS. Massive gene expression (GE; 61?657 unique probes) was analyzed after hatching using microarrays. Trout oocytes are easily fertilized with DDS and the high prolificacy allows live progeny to be obtained even with a high rate of abortions. Nevertheless, the zygotic inhibition of Poly (ADP-ribose) polymerase, upstream of BER pathway, resulted in 810 differentially expressed genes (DEGs) after hatching. DEGs are related with DNA repair, apoptosis, telomere maintenance, or growth and development, revealing a scenario of impaired DNA damage signalization and repair. Downregulation of the apoptotic cascade was noticed, suggesting a selection of embryos tolerant to residual DNA damage during embryo development. Our results reveal changes in the progeny from defective repairing zygotes including higher malformations rate, weight gain, longer telomeres, and lower caspase 3/7 activity, whose long-term consequences should be analyzed in depth. PMID:25433028

Fernández-Díez, C; González-Rojo, S; Montfort, J; Le Cam, A; Bobe, J; Robles, V; Pérez-Cerezales, S; Herráez, M P

2015-01-01

341

INO80 chromatin remodeling complex promotes the removal of UV lesions by the nucleotide excision repair pathway.  

PubMed

The creation of accessible DNA in the context of chromatin is a key step in many DNA functions. To reveal how ATP-dependent chromatin remodeling activities impact DNA repair, we constructed mammalian genetic models for the INO80 chromatin remodeling complex and investigated the impact of loss of INO80 function on the repair of UV-induced photo lesions. We showed that deletion of two core components of the INO80 complex, INO80 and ARP5, significantly hampered cellular removal of UV-induced photo lesions but had no significant impact on the transcription of nucleotide excision repair (NER) factors. Loss of INO80 abolished the assembly of NER factors, suggesting that prior chromatin relaxation is important for the NER incision process. Ino80 and Arp5 are enriched to UV-damaged DNA in an NER-incision-independent fashion, suggesting that recruitment of the remodeling activity likely takes place during the initial stage of damage recognition. These results demonstrate a critical role of INO80 in creating DNA accessibility for the NER pathway and provide direct evidence that repair of UV lesions and perhaps most bulky adduct lesions requires chromatin reconfiguration. PMID:20855601

Jiang, Yingjun; Wang, Xin; Bao, Shilai; Guo, Ruifeng; Johnson, David G; Shen, Xuetong; Li, Lei

2010-10-01

342

Quantifying clustered DNA damage induction and repair by gel electrophoresis, electronic imaging and number average length analysis  

NASA Technical Reports Server (NTRS)

Assessing DNA damage induction, repair and consequences of such damages requires measurement of specific DNA lesions by methods that are independent of biological responses to such lesions. Lesions affecting one DNA strand (altered bases, abasic sites, single strand breaks (SSB)) as well as damages affecting both strands (clustered damages, double strand breaks) can be quantified by direct measurement of DNA using gel electrophoresis, gel imaging and number average length analysis. Damage frequencies as low as a few sites per gigabase pair (10(9)bp) can be quantified by this approach in about 50ng of non-radioactive DNA, and single molecule methods may allow such measurements in DNA from single cells. This review presents the theoretical basis, biochemical requirements and practical aspects of this approach, and shows examples of their applications in identification and quantitation of complex clustered damages.

Sutherland, Betsy M.; Georgakilas, Alexandros G.; Bennett, Paula V.; Laval, Jacques; Sutherland, John C.; Gewirtz, A. M. (Principal Investigator)

2003-01-01

343

Defective DNA damage response and repair in liver cells expressing hepatitis B virus surface antigen.  

PubMed

Hepatitis B virus (HBV) is implicated in liver cancer. The aim of this study was to find out whether HBV or its components [HBV surface antigen (HBsAg), HBV core protein (HBc), and HBV X protein (HBx)] could interfere with the host DNA damage response and repair pathway. The full HBV genome or individual HBV open-reading frame (ORF) was introduced into HepG2 cells to examine the effect on host genomic stability, DNA repair efficacy in response to double-strand DNA damage, and DNA damage-induced cell death. Responses to apoptosis induction in the HBV ORF-transfected HepG2 cells were also compared with those in HBV-positive and HBV-negative human hepatocellular carcinoma (HCC) cells. In the absence of HBV replication, accumulation of HBsAg in liver cells without other HBV proteins enhanced DNA repair protein and tumor suppressor promyelocytic leukemia (PML) degradation, which resulted in resistance to apoptosis induction and deficient double-strand DNA repair. However, HBsAg-positive cells exhibited increased cell death with exposure to the poly(ADP-ribose) polymerase inhibitor that blocks single-strand DNA repair. These results indicate that suppression of PML by HBsAg disrupts cellular mechanisms that respond to double-strand DNA damage for DNA repair or apoptosis induction, which may facilitate hepatocarcinogenesis and open up a synthetic lethality strategy for HBsAg-positive HCC treatment. PMID:23444429

Chung, Yih-Lin

2013-06-01

344

Psoralen-plus-light damage and repair in transforming DNA of Bacillus subtilis  

SciTech Connect

The relative contributions of excision and recombination in the repair of damage by 8-methoxypsoralen (8-MOP) plus black light to Bacillus subtilis were studied. The results indicate that the pyrimidine dimer excision system and a recombination pathway are probably both involved in repair of lethal damage to cells exposed in vivo to 8-MOP plus black light, but repair is not very efficient. Transforming DNA exposed in vitro to 8-MOP plus black light was inactivated mainly by crosslinks rather than by monoadducts, and was repaired predominantly by an incision-dependent process. There was very little demonstrable damage-induced recombination in transforming DNA.

Hadden, C.T.

1981-01-01

345

Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe.  

PubMed Central

Schizosaccharomyces pombe Rhp55 and Rhp57 are RecA-like proteins involved in double-strand break (DSB) repair. Here we demonstrate that Rhp55 and Rhp57 proteins strongly interact in vivo, similar to Saccharomyces cerevisiae Rad55p and Rad57p. Mutations in the conserved ATP-binding/hydrolysis folds of both the Rhp55 and Rhp57 proteins impaired their function in DNA repair but not in cell proliferation. However, when combined, ATPase fold mutations in Rhp55p and Rhp57p resulted in severe defects of both functions, characteristic of the deletion mutants. Yeast two-hybrid analysis also revealed other multiple in vivo interactions among S. pombe proteins involved in recombinational DNA repair. Similar to S. cerevisiae Rad51p-Rad54p, S. pombe Rhp51p and Rhp54p were found to interact. Both putative Rad52 homologs in S. pombe, Rad22p and Rti1p, were found to interact with the C-terminal region of Rhp51 protein. Moreover, Rad22p and Rti1p exhibited mutual, as well as self-, interactions. In contrast to the S. cerevisiae interacting pair Rad51p-Rad55p, S. pombe Rhp51 protein strongly interacted with Rhp57 but not with Rhp55 protein. In addition, the Rti1 and Rad22 proteins were found to form a complex with the large subunit of S. pombe RPA. Our data provide compelling evidence that most, but not all, of the protein-protein interactions found in S. cerevisiae DSB repair are evolutionarily conserved. PMID:11560889

Tsutsui, Y; Khasanov, F K; Shinagawa, H; Iwasaki, H; Bashkirov, V I

2001-01-01

346

Protein oxidation and DNA repair inhibition by 6-thioguanine and UVA radiation.  

PubMed

Damage to skin DNA by solar UV is largely unavoidable, and an optimal cellular response to it requires the coordinated operation of proteins in numerous pathways. A fully functional DNA repair proteome for removing harmful DNA lesions is a prerequisite for an appropriate DNA damage response. Genetically determined failure to repair UV-induced DNA damage is associated with skin photosensitivity and increased skin cancer risk. Patients treated with immunosuppressant/anti-inflammatory thiopurines are also photosensitive and have high rates of sun-related skin cancer. Their DNA contains the base analog 6-thioguanine (6-TG), which acts as a UVA photosensitizer to generate reactive oxygen species (ROS), predominantly singlet oxygen ((1)O2). ROS damage both DNA and proteins. Here we show that UVA irradiation of cultured human cells containing DNA 6-TG causes significant protein oxidation and damages components of the DNA repair proteome, including the Ku, OGG-1, MYH, and RPA proteins. Assays of DNA repair in intact cells or in cell extracts indicate that this protein damage compromises DNA break rejoining and base and nucleotide excision repair. As these experimental conditions simulate those in the skin of patients taking thiopurines, our findings suggest a mechanism whereby UVA in sunlight may contribute to skin carcinogenesis in immunosuppressed patients. PMID:24284422

Gueranger, Quentin; Li, Feng; Peacock, Matthew; Larnicol-Fery, Annabel; Brem, Reto; Macpherson, Peter; Egly, Jean-Marc; Karran, Peter

2014-05-01

347

Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways.  

PubMed Central

Ku, a heterodimer of polypeptides of approximately 70 kDa and 80 kDa (Ku70 and Ku80, respectively), binds avidly to DNA double-strand breaks (DSBs). Mammalian cells defective in Ku are hypersensitive to ionizing radiation due to a deficiency in DSB repair. Here, we show that the simple inactivation of the Saccharomyces cerevisiae Ku70 homologue (Yku70p), does not lead to increased radiosensitivity. However, yku70 mutations enhance the radiosensitivity of rad52 strains, which are deficient in homologous recombination. Through establishing a rapid and reproducible in vivo plasmid rejoining assay, we show that Yku70p plays a crucial role in the repair of DSBs bearing cohesive termini. Whereas this damage is repaired accurately in YKU70 backgrounds, in yku70 mutant strains terminal deletions of up to several hundred bp occur before ligation ensues. Interestingly, this error-prone DNA repair pathway utilizes short homologies between the two recombining molecules and is thus highly reminiscent of a predominant form of DSB repair that operates in vertebrates. These data therefore provide evidence for two distinct and evolutionarily conserved illegitimate recombination pathways. One of these is accurate and Yku70p-dependent, whereas the other is error-prone and Yku70-independent. Furthermore, our studies suggest that Yku70 promotes genomic stability both by promoting accurate DNA repair and by serving as a barrier to error-prone repair processes. Images PMID:8890183

Boulton, S J; Jackson, S P

1996-01-01

348

DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O6-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy  

PubMed Central

O6-alkylguanine-DNA alkyltransferase (AGT) is a crucial target both for the prevention of cancer and for chemotherapy, since it repairs mutagenic lesions in DNA, and it limits the effectiveness of alkylating chemotherapies. AGT catalyzes the unique, single-step, direct damage reversal repair of O6-alkylguanines by selectively transferring the O6-alkyl adduct to an internal cysteine residue. Recent crystal structures of human AGT alone and in complex with substrate DNA reveal a two-domain a/? fold and a bound zinc ion. AGT uses its helix-turn-helix motif to bind substrate DNA via the minor groove. The alkylated guanine is then flipped out from the base stack into the AGT active site for repair by covalent transfer of the alkyl adduct to Cys145. An asparagine hinge (Asn137) couples the helix-turn-helix DNA binding and active site motifs. An arginine finger (Arg128) stabilizes the extrahelical DNA conformation. With this newly improved structural understanding of AGT and its interactions with biologically relevant substrates, we can now begin to unravel the role it plays in preserving genetic integrity and discover how it promotes resistance to anticancer therapies. PMID:17485252

Tubbs, Julie L.; Pegg, Anthony E.; Tainer, John A.

2007-01-01

349

Photoreactivation is the main repair pathway for UV-induced DNA damage in coral planulae.  

PubMed

The larvae of most coral species spend some time in the plankton, floating just below the surface and hence exposed to high levels of ultraviolet radiation (UVR). The high levels of UVR are potentially stressful and damaging to DNA and other cellular components, such as proteins, reducing survivorship. Consequently, mechanisms to either shade (prevent) or repair damage potentially play an important role. In this study, the role of photoreactivation in the survival of coral planulae was examined. Photoreactivation is a light-stimulated response to UV-damaged DNA in which photolyase proteins repair damaged DNA. Photoreactivation rates, as well as the localization of photolyase, were explored in planulae under conditions where photoreactivation was or was not inhibited. The results indicate that photoreactivation is the main DNA repair pathway in coral planulae, repairing UV-induced DNA damage swiftly (K=1.75 h(-1) and a half-life of repair of 23 min), with no evidence of any light-independent DNA repair mechanisms, such as nucleotide excision repair (NER), at work. Photolyase mRNA was localized to both the ectoderm and endoderm of the larvae. The amount of cell death in the coral planulae increased significantly when photoreactivation was inhibited, by blocking photoreactivating light. We found that photoreactivation, along with additional UV shielding in the form of five mycosporine-like amino acids, are sufficient for survival in surface tropical waters and that planulae do not accumulate DNA damage despite being exposed to high UVR. PMID:19684208

Reef, Ruth; Dunn, Simon; Levy, Oren; Dove, Sophie; Shemesh, Eli; Brickner, Itzchak; Leggat, William; Hoegh-Guldberg, Ove

2009-09-01

350

DNA repair enables sex identification in genetic material from human teeth  

PubMed Central

Background: The purpose of this study was to test the effectiveness of a DNA repair protocol in improving genetic testing in compromised samples, frequently encountered in Forensic Medicine. Methods: In order to stretch the experiment conditions to the limits, as far as quality of samples and DNA is concerned, we tried the repair protocol on ten ancient human teeth obtained from an equal number of skeletons from a burial site in Lerna, Middle Helladic Greece (2100 - 1700 BC). For these samples, sex was previously determined morphologically, serving as a reference to compare our molecular data with. The samples were analysed using the DNA amelogenin sex test assay prior and after DNA polymerase repair. For every individual, two molecular sex determinations were obtained by visualising PCR products on an agarose gel. Results: DNA repair enabled genetic testing in these samples. Successful amplification of the amelogenin gene was obtained only from the repaired DNA in eight out of ten samples. Prior to the repair treatment, none of these samples yielded any PCR products, thus attesting to the authenticity of the amplified sequence. The concordance between morphological and molecular analysis was in reasonable agreement (71%). Conclusions: These results reveal the impact of the repair process in studying single copy genes from low quality DNA. This protocol could facilitate molecular analysis in compromised samples, encountered in forensic medicine, as well as enable genetic studies in ancient remnants. PMID:19918305

Kovatsi, L; Nikou, D; Triantaphyllou, S; Njau, S N; Voutsaki, S; Kouidou, S

2009-01-01

351

Unbalanced restriction impairs SOS-induced DNA repair effects.  

PubMed

The contribution of a type II restriction-modification system (R-M system) to genome integrity and cell viability was investigated. We established experimental conditions which enabled the achievement of hemimethylated and unmethylated states for the specific bases of the recognition sequences of the host's DNA. To achieve this, we constructed the MboII R-M system containing only one (i.e. M2.MboII) out of two functional MboII methyltransferases found in Moraxella bovis. Using the incomplete R-M system we were able to perturb the balance between methylation and restriction in an inducible manner. We demonstrate that upon the SOS-induced DNA repair in the mitomycin C treated cells, restriction significantly reduces cell viability. Similar results for the well-studied wild type EcoRI R-M system, expressed constitutively in Escherichia coli, were obtained. Our data provide further insights into the benefits and disadvantages of maintaining of a type II R-M system, highlighting its impact on host cell fitness. PMID:20134230

Katna, Anna; Boratynski, Robert; Furmanek-Blaszk, Beata; Zolcinska, Natalia; Sektas, Marian

2010-01-01

352

Fluorescence detection of cellular nucleotide excision repair of damaged DNA  

PubMed Central

To maintain genetic integrity, ultraviolet light-induced photoproducts in DNA must be removed by the nucleotide excision repair (NER) pathway, which is initiated by damage recognition and dual incisions of the lesion-containing strand. We intended to detect the dual-incision step of cellular NER, by using a fluorescent probe. A 140-base pair linear duplex containing the (6–4) photoproduct and a fluorophore–quencher pair was prepared first. However, this type of DNA was found to be degraded rapidly by nucleases in cells. Next, a plasmid was used as a scaffold. In this case, the fluorophore and the quencher were attached to the same strand, and we expected that the dual-incision product containing them would be degraded in cells. At 3?h after transfection of HeLa cells with the plasmid-type probes, fluorescence emission was detected at the nuclei by fluorescence microscopy only when the probe contained the (6–4) photoproduct, and the results were confirmed by flow cytometry. Finally, XPA fibroblasts and the same cells expressing the XPA gene were transfected with the photoproduct-containing probe. Although the transfer of the probe into the cells was slow, fluorescence was detected depending on the NER ability of the cells. PMID:24993089

Toga, Tatsuya; Kuraoka, Isao; Watanabe, Shun; Nakano, Eiji; Takeuchi, Seiji; Nishigori, Chikako; Sugasawa, Kaoru; Iwai, Shigenori

2014-01-01

353

Dependence of u. v. -induced DNA excision repair on deoxyribonucleoside triphosphate concentrations in permeable human fibroblasts: a model for the inhibition of repair by hydroxyurea  

SciTech Connect

We have tested the hypothesis that the inhibition by hydroxyurea of repair patch ligation and chromatin rearrangement during u.v.-induced DNA excision repair results from a reduction in cellular deoxyribonucleotide concentrations and not from a direct effect of hydroxyurea on the repair process. Using permeable human fibroblasts, we have shown that hydroxyurea has no direct effect on either repair synthesis or repair patch ligation. We also have shown that by reducing the deoxyribonucleoside triphosphate concentrations in the permeable cell reaction mixture, we can mimic the inhibition of repair patch ligation and chromatin rearrangement seen when u.v.-damaged intact confluent fibroblasts are treated with hydroxyurea. Our results are consistent with the concept that hydroxyurea inhibits DNA repair in intact cells by inhibiting deoxyribonucleotide synthesis through its effect on ribonucleotide reductase and, conversely, that continued deoxyribonucleotide synthesis is required for the excision repair of u.v.-induced DNA damage even in resting cells.

Hunting, D.J.; Dresler, S.L.

1985-10-01

354

Homologous recombination is a primary pathway to repair DNA double-strand breaks generated during DNA rereplication.  

PubMed

Re-initiation of DNA replication at origins within a given cell cycle would result in DNA rereplication, which can lead to genome instability and tumorigenesis. DNA rereplication can be induced by loss of licensing control at cellular replication origins, or by viral protein-driven multiple rounds of replication initiation at viral origins. DNA double-strand breaks (DSBs) are generated during rereplication, but the mechanisms of how these DSBs are repaired to maintain genome stability and cell viability are poorly understood in mammalian cells. We generated novel EGFP-based DSB repair substrates, which specifically monitor the repair of rereplication-associated DSBs. We demonstrated that homologous recombination (HR) is an important mechanism to repair rereplication-associated DSBs, and sister chromatids are used as templates for such HR-mediated DSB repair. Micro-homology-mediated non-homologous end joining (MMEJ) can also be used but to a lesser extent compared to HR, whereas Ku-dependent classical non-homologous end joining (C-NHEJ) has a minimal role to repair rereplication-associated DSBs. In addition, loss of HR activity leads to severe cell death when rereplication is induced. Therefore, our studies identify HR, the most conservative repair pathway, as the primary mechanism to repair DSBs upon rereplication. PMID:25160628

Truong, Lan N; Li, Yongjiang; Sun, Emily; Ang, Katrina; Hwang, Patty Yi-Hwa; Wu, Xiaohua

2014-10-17

355

Photoactivated excited states of DNA repair photolyase: Dynamical and semiempircal identification  

NASA Astrophysics Data System (ADS)

DNA damage caused by UV light radiation is often naturally repaired in a process initiated by excited state electron transfer from the photoactivated photolyase enzyme to the DNA cyclobutane pyrimidine dimer lesion. The active cofactor in the excited state electron transfer in the photolyase is the two-electron fully reduced form of the flavin adenine dinucleotide (FADH-). To calculate electron tunneling matrix element and model the DNA binding with photolyase, the LUMO of the FADH- calculated using extended Huckel method was previously chosen from the SCF wavefunctions. Recently, the DNA-photolyase complex was crystallized in its bound form, in good agreement with our previous model in even minute details at the active site. Here we carry out molecular dynamics simulation of the entire complex using the new experimental structure of Anacystis nidulans and identify the low-lying photoactivated states of the enzyme for the dynamical confirmations. Our results from ZINDO/S CIS calculations are compared with experimental UV spectra, and their implications for excited state electron transfer and energy transfer are discussed.0

Zheng, Xuehe; Ly, Ngan M.; Stuchebrukhov, Alexei A.

356

Influence of XRCC1 Genetic Polymorphisms on Ionizing Radiation-Induced DNA Damage and Repair.  

PubMed

It is well known that ionizing radiation (IR) can damage DNA through a direct action, producing single- and double-strand breaks on DNA double helix, as well as an indirect effect by generating oxygen reactive species in the cells. Mammals have evolved several and distinct DNA repair pathways in order to maintain genomic stability and avoid tumour cell transformation. This review reports important data showing a huge interindividual variability on sensitivity to IR and in susceptibility to developing cancer; this variability is principally represented by genetic polymorphisms, that is, DNA repair gene polymorphisms. In particular we have focussed on single nucleotide polymorphisms (SNPs) of XRCC1, a gene that encodes for a scaffold protein involved basically in Base Excision Repair (BER). In this paper we have reported and presented recent studies that show an influence of XRCC1 variants on DNA repair capacity and susceptibility to breast cancer. PMID:20798883

Sterpone, Silvia; Cozzi, Renata

2010-01-01

357

Influence of XRCC1 Genetic Polymorphisms on Ionizing Radiation-Induced DNA Damage and Repair  

PubMed Central

It is well known that ionizing radiation (IR) can damage DNA through a direct action, producing single- and double-strand breaks on DNA double helix, as well as an indirect effect by generating oxygen reactive species in the cells. Mammals have evolved several and distinct DNA repair pathways in order to maintain genomic stability and avoid tumour cell transformation. This review reports important data showing a huge interindividual variability on sensitivity to IR and in susceptibility to developing cancer; this variability is principally represented by genetic polymorphisms, that is, DNA repair gene polymorphisms. In particular we have focussed on single nucleotide polymorphisms (SNPs) of XRCC1, a gene that encodes for a scaffold protein involved basically in Base Excision Repair (BER). In this paper we have reported and presented recent studies that show an influence of XRCC1 variants on DNA repair capacity and susceptibility to breast cancer. PMID:20798883

Sterpone, Silvia; Cozzi, Renata

2010-01-01

358

DNA Damage Induced Hyperphosphorylation of Replication Protein A. 2. Characterization of DNA Binding Activity, Protein Interactions, and Activity in DNA Replication and Repair  

PubMed Central

Replication protein A (RPA) is a heterotrimeric protein consisting of 70-, 34-, and 14- kDa subunits that is required for many DNA metabolic processes including DNA replication and DNA repair. Using a purified hyperphosphorylated form of RPA protein prepared in vitro, we have addressed the effects of hyperphosphorylation on steady-state and pre-steady-state DNA binding activity, the ability to support DNA repair and replication reactions, and the effect on the interaction with partner proteins. Equilibrium DNA binding activity measured by fluorescence polarization reveals no difference in ssDNA binding to pyrimidine-rich DNA sequences. However, RPA hyperphosphorylation results in a decreased affinity for purine-rich ssDNA and duplex DNA substrates. Pre-steady-state kinetic analysis is consistent with the equilibrium DNA binding and demonstrates a contribution from both the kon and koff to achieve these differences. The hyperphosphorylated form of RPA retains damage-specific DNA binding, and, importantly, the affinity of hyperphosphorylated RPA for damaged duplex DNA is 3-fold greater than the affinity of unmodified RPA for undamaged duplex DNA. The ability of hyperphosphorylated RPA to support DNA repair showed minor differences in the ability to support nucleotide excision repair (NER). Interestingly, under reaction conditions in which RPA is maintained in a hyperphosphorylated form, we also observed inhibition of in vitro DNA replication. Analyses of protein–protein interactions bear out the effects of hyperphosphorylated RPA on DNA metabolic pathways. Specifically, phosphorylation of RPA disrupts the interaction with DNA polymerase ? but has no significant effect on the interaction with XPA. These results demonstrate that the effects of DNA damage induced hyperphosphorylation of RPA on DNA replication and DNA repair are mediated through alterations in DNA binding activity and protein–protein interactions. PMID:15938633

Dixon, Kathleen; Turchi, John J.

2015-01-01

359

Elevated level of DNA damage and impaired repair of oxidative DNA damage in patients with recurrent depressive disorder.  

PubMed

Background Depressive disorder (DD), including recurrent DD (rDD), is a severe psychological disease, which affects a large percentage of the world population. Although pathogenesis of the disease is not known, a growing body of evidence shows that inflammation together with oxidative stress may contribute to development of DD. Since reactive oxygen species produced during stress may damage DNA, we wanted to evaluate the extent of DNA damage and efficiency of DNA repair in patients with depression. Material and Methods We measured and compared the extent of endogenous DNA damage - single- and double-strand breaks, alkali-labile sites, and oxidative damage of the pyrimidines and purines - in peripheral blood mononuclear cells isolated from rDD patients (n=40) and healthy controls (n=46) using comet assay. We also measured DNA damage evoked by hydrogen peroxide and monitored changes in DNA damage during repair incubation. Results We found an increased number DNA breaks, alkali-labile sites, and oxidative modification of DNA bases in the patients compared to the controls. Exposure to hydrogen peroxide evoked the same increased damage in both groups. Examination of the repair kinetics of both groups revealed that the lesions were more efficiently repaired in the controls than in the patients. Conclusions For the first time we showed that patients with depression, compared with non-depresses individuals, had more DNA breaks, alkali-labile sites, and oxidative DNA damage, and that those lesions may be accumulated by impairments of the DNA repair systems. More studies must be conducted to elucidate the role of DNA damage and repair in depression. PMID:25656523

Czarny, Piotr; Kwiatkowski, Dominik; Kacperska, Dagmara; Kawczy?ska, Daria; Talarowska, Monika; Orzechowska, Agata; Bielecka-Kowalska, Anna; Szemraj, Janusz; Ga?ecki, Piotr; ?liwi?ski, Tomasz

2015-01-01

360

Elevated Level of DNA Damage and Impaired Repair of Oxidative DNA Damage in Patients with Recurrent Depressive Disorder  

PubMed Central

Background Depressive disorder (DD), including recurrent DD (rDD), is a severe psychological disease, which affects a large percentage of the world population. Although pathogenesis of the disease is not known, a growing body of evidence shows that inflammation together with oxidative stress may contribute to development of DD. Since reactive oxygen species produced during stress may damage DNA, we wanted to evaluate the extent of DNA damage and efficiency of DNA repair in patients with depression. Material/Methods We measured and compared the extent of endogenous DNA damage – single- and double-strand breaks, alkali-labile sites, and oxidative damage of the pyrimidines and purines – in peripheral blood mononuclear cells isolated from rDD patients (n=40) and healthy controls (n=46) using comet assay. We also measured DNA damage evoked by hydrogen peroxide and monitored changes in DNA damage during repair incubation. Results We found an increased number DNA breaks, alkali-labile sites, and oxidative modification of DNA bases in the patients compared to the controls. Exposure to hydrogen peroxide evoked the same increased damage in both groups. Examination of the repair kinetics of both groups revealed that the lesions were more efficiently repaired in the controls than in the patients. Conclusions For the first time we showed that patients with depression, compared with non-depresses individuals, had more DNA breaks, alkali-labile sites, and oxidative DNA damage, and that those lesions may be accumulated by impairments of the DNA repair systems. More studies must be conducted to elucidate the role of DNA damage and repair in depression. PMID:25656523

Czarny, Piotr; Kwiatkowski, Dominik; Kacperska, Dagmara; Kawczy?ska, Daria; Talarowska, Monika; Orzechowska, Agata; Bielecka-Kowalska, Anna; Szemraj, Janusz; Gaandlstrokecki, Piotr; ?liwi?ski, Tomasz

2015-01-01

361

Unusual DNA binding modes for metal anticancer complexes  

PubMed Central

DNA is believed to be the primary target for many metal-based drugs. For example, platinum-based anticancer drugs can form specific lesions on DNA that induce apoptosis. New platinum drugs can be designed that have novel modes of interaction with DNA, such as the trinuclear platinum complex BBR3464. Also it is possible to design inert platinum(IV) pro-drugs which are non-toxic in the dark, but lethal when irradiated with certain wavelengths of light. This gives rise to novel DNA lesions which are not as readily repaired as those induced by cisplatin, and provides the basis for a new type of photoactivated chemotherapy. Finally, newly emerging ruthenium(II) organometallic complexes not only bind to DNA coordinatively, but also by H-bonding and hydrophibic interactions triggered by the introduction of extended arene rings into their versatile structures. Intriguingly osmium (the heavier congener of ruthenium) reacts differently with DNA but can also give rise to highly cytotoxic organometallic complexes. PMID:19344743

Pizarro, Ana M.; Sadler, Peter J.

2010-01-01

362

RPA antagonizes microhomology-mediated repair of DNA double-strand breaks.  

PubMed

Microhomology-mediated end joining (MMEJ) is a Ku- and ligase IV-independent mechanism for the repair of DNA double-strand breaks that contributes to chromosome rearrangements. Here we used a chromosomal end-joining assay to determine the genetic requirements for MMEJ in Saccharomyces cerevisiae. We found that end resection influences the ability to expose microhomologies; however, it is not rate limiting for MMEJ in wild-type cells. The frequency of MMEJ increased by up to 350-fold in rfa1 hypomorphic mutants, suggesting that replication protein A (RPA) bound to the single-stranded DNA (ssDNA) overhangs formed by resection prevents spontaneous annealing between microhomologies. In vitro, the mutant RPA complexes were unable to fully extend ssDNA and were compromised in their ability to prevent spontaneous annealing. We propose that the helix-destabilizing activity of RPA channels ssDNA intermediates from mutagenic MMEJ to error-free homologous recombination, thus preserving genome integrity. PMID:24608368

Deng, Sarah K; Gibb, Bryan; de Almeida, Mariana Justino; Greene, Eric C; Symington, Lorraine S

2014-04-01

363

Recent progress with the DNA repair mutants of Chinese hamster ovary cells  

SciTech Connect

Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs.

Thompson, L.H.; Salazar, E.P.; Brookman, K.W.; Collins, C.C.; Stewart, S.A.; Busch, D.B.; Weber, C.A.

1986-04-02

364

Metabolic modulation of chromatin: implications for DNA repair and genomic integrity  

PubMed Central

The maintenance of genomic integrity in response to DNA damage is tightly linked to controlled changes in the damage-proximal chromatin environment. Many of the chromatin modifying enzymes involved in DNA repair depend on metabolic intermediates as cofactors, suggesting that changes in cellular metabolism can have direct consequences for repair efficiency and ultimately, genome stability. Here, we discuss how metabolites may contribute to DNA double-strand break repair, and how alterations in cellular metabolism associated with both aging and tumorigenesis may affect the integrity of our genomes. PMID:24065984

Liu, Jinping; Kim, Jeongkyu; Oberdoerffer, Philipp

2013-01-01

365

Nick translation - a new assay for monitoring DNA damage and repair in cultured human fibroblasts  

SciTech Connect

An in vitro assay has been developed to detect DNA damage and repair following chemical treatment of human diploid fibroblasts. DNA damage is measured by following the Escherichia coli DNA polymerase I-catalyzed incorporation of radiolabeled deoxycytidine triphosphate (dCTP) into the DNA of lysolecithin-permeabilized cells. DNA strand breaks with free 3' OH termini serve as template sites for incorporation, and decrease of this incorporation with time, following removal of the test chemical, indicates loss (repair) of initial damage. Inhibition of the DNA excision repair process by the addition of the repair inhibitors arabinofuranosyl cytosine (ara-C) and hydroxyurea (HU) during the incubation period gives rise to an increased number of template sites, manifesting itself in increased incorporation and indicating the induction of long-patch excision repair. Results presented demonstrate that all 14 direct-acting carcinogens tested and 8 of 14 carcinogens requiring metabolic activation give positive indication of DNA damage, repair, or both. Eleven of 14 noncarcinogens tested were scored as negative, the other 3 having previously been shown to interact with cellular DNA. This assay is shown to have predictive capability at least equal to that of UDS assays but to allow a broader spectrum of genotoxic effects to be analyzed.

Snyder, R.D.; Matheson, D.W.

1985-01-01

366

Genetic manipulation in Sulfolobus islandicus and functional analysis of DNA repair genes.  

PubMed

Recently, a novel gene-deletion method was developed for the crenarchaeal model Sulfolobus islandicus, which is a suitable tool for addressing gene essentiality in depth. Using this technique, we have investigated functions of putative DNA repair genes by constructing deletion mutants and studying their phenotype. We found that this archaeon may not encode a eukarya-type of NER (nucleotide excision repair) pathway because depleting each of the eukaryal NER homologues XPD, XPB and XPF did not impair the DNA repair capacity in their mutants. However, among seven homologous recombination proteins, including RadA, Hel308/Hjm, Rad50, Mre11, HerA, NurA and Hjc, only the Hjc nuclease is dispensable for cell viability. Sulfolobus encodes redundant BER (base excision repair) enzymes such as two uracil DNA glycosylases and two putative apurinic/apyrimidinic lyases, but inactivation of one of the redundant enzymes already impaired cell growth, highlighting their important roles in archaeal DNA repair. Systematically characterizing these mutants and generating mutants lacking two or more DNA repair genes will yield further insights into the genetic mechanisms of DNA repair in this model organism. PMID:23356319

Zhang, Changyi; Tian, Bin; Li, Suming; Ao, Xiang; Dalgaard, Kevin; Gökce, Serkan; Liang, Yunxiang; She, Qunxin

2013-02-01

367

A potential copper-regulatory role for cytosolic expression of the DNA repair protein XRCC5  

Microsoft Academic Search

Copper (Cu) has a critical role in the generation of oxidative stress during neurodegeneration and cancer. Reactive oxygen species generated through abnormal elevation or deficiency of Cu can lead to lipid, protein, and DNA damage. Oxidation of DNA can induce strand breaks and is associated with altered cell fate including transformation or death. DNA repair is mediated through the action

Tai Du; Aphrodite Caragounis; Sarah J. Parker; Jodi Meyerowitz; Sharon La Fontaine; Katja M. Kanninen; Victoria M. Perreau; Peter J. Crouch; Anthony R. White

2011-01-01

368

Coupling of human DNA excision repair and the DNA damage checkpoint in a defined in vitro system.  

PubMed

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5' to 3' exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5' to 3' exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response. PMID:24403078

Lindsey-Boltz, Laura A; Kemp, Michael G; Reardon, Joyce T; DeRocco, Vanessa; Iyer, Ravi R; Modrich, Paul; Sancar, Aziz

2014-02-21

369

Analysis of Actively Transcribed DNA Repair Using a Transfection-Based System  

PubMed Central

Host cell reactivation (HCR) is a transfection-based assay in which intact cells repair damage localized to exogenous DNA. This chapter provides instructions for the application of this technique, using as an exemplar UV irradiation as a source of damage to a luciferase reporter plasmid. Through measurement of the activity of a successfully transcribed and translated reporter enzyme, the amount of damaged plasmid that a cell can “reactivate” or repair and express can be quantitated. Different DNA repair pathways can be analyzed by this technique by damaging the reporter plasmid in different ways. Since it involves repair of a transcriptionally active gene, when applied to UV damage the HCR assay measures the capacity of the host cells to perform transcription-coupled repair, a subset of the overall nucleotide excision repair pathway that specifically targets transcribed gene sequences. PMID:24623251

Latimer, Jean J.

2015-01-01

370

Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression  

NASA Technical Reports Server (NTRS)

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.

Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

2008-01-01

371

Noncanonical MMS2Encoded Ubiquitin-Conjugating Enzyme Functions in Assembly of Novel Polyubiquitin Chains for DNA Repair  

Microsoft Academic Search

Ubiquitin-conjugating enzyme variant (UEV) proteins resemble ubiquitin-conjugating enzymes (E2s) but lack the defining E2 active-site residue. The MMS2-encoded UEV protein has been genetically implicated in error-free postreplicative DNA repair in Saccharomyces cerevisiae. We show that Mms2p forms a specific heteromeric complex with the UBC13-encoded E2 and is required for the Ubc13p-dependent assembly of polyubiquitin chains linked through lysine 63. A

Roseanne M Hofmann; Cecile M Pickart

1999-01-01

372

Regulation of NuA4 Histone Acetyltransferase Activity in Transcription and DNA Repair by Phosphorylation of Histone H4  

PubMed Central

The NuA4 complex is a histone H4/H2A acetyltransferase involved in transcription and DNA repair. While histone acetylation is important in many processes, it has become increasingly clear that additional histone modifications also play a crucial interrelated role. To understand how NuA4 action is regulated, we tested various H4 tail peptides harboring known modifications in HAT assays. While dimethylation at arginine 3 (R3M) had little effect on NuA4 activity, phosphorylation of serine 1 (S1P) strongly decreased the ability of the complex to acetylate H4 peptides. However, R3M in combination with S1P alleviates the repression of NuA4 activity. Chromatin from cells treated with DNA damage-inducing agents shows an increase in phosphorylation of serine 1 and a concomitant decrease in H4 acetylation. We found that casein kinase 2 phosphorylates histone H4 and associates with the Rpd3 deacetylase complex, demonstrating a physical connection between phosphorylation of serine 1 and unacetylated H4 tails. Chromatin immunoprecipitation experiments also link local phosphorylation of H4 with its deacetylation, during both transcription and DNA repair. Time course chromatin immunoprecipitation data support a model in which histone H4 phosphorylation occurs after NuA4 action during double-strand break repair at the step of chromatin restoration and deacetylation. These findings demonstrate that H4 phospho-serine 1 regulates chromatin acetylation by the NuA4 complex and that this process is important for normal gene expression and DNA repair. PMID:16135807

Utley, Rhea T.; Lacoste, Nicolas; Jobin-Robitaille, Olivier; Allard, Stéphane; Côté, Jacques

2005-01-01

373

Genome analysis of DNA repair genes in the alpha proteobacterium Caulobacter crescentus  

PubMed Central

Background The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria. Results In this study, we employed a bioinformatic approach, to analyse and describe the open reading frames potentially related to DNA repair from the genome of the alpha-proteobacterium Caulobacter crescentus. This was performed by comparison with known DNA repair related genes found in public databases. As expected, although C. crescentus and E. coli bacteria belong to separate phylogenetic groups, many of their DNA repair genes are very similar. However, some important DNA repair genes are absent in the C. crescentus genome and other interesting functionally related gene duplications are present, which do not occur in E. coli. These include DNA ligases, exonuclease III (xthA), endonuclease III (nth), O6-methylguanine-DNA methyltransferase (ada gene), photolyase-like genes, and uracil-DNA-glycosylases. On the other hand, the genes imuA and imuB, which are involved in DNA damage induced mutagenesis, have recently been described in C. crescentus, but are absent in E. coli. Particularly interesting are the potential atypical phylogeny of one of the photolyase genes in alpha-proteobacteria, indicating an origin by horizontal transfer, and the duplication of the Ada orthologs, which have diverse structural configurations, including one that is still unique for C. crescentus. Conclusion The absence and the presence of certain genes are discussed and predictions are made considering the particular aspects of the C. crescentus among other known DNA repair pathways. The observed differences enlarge what is known for DNA repair in the Bacterial world, and provide a useful framework for further experimental studies in this organism. PMID:17352799

Martins-Pinheiro, Marinalva; Marques, Regina CP; Menck, Carlos FM

2007-01-01

374

The Yin and Yang of Repair Mechanisms in DNA Structure-induced Genetic Instability  

PubMed Central

DNA can adopt a variety of secondary structures that deviate from the canonical Watson-Crick B-DNA form. More than 10 types of non-canonical or non-B DNA secondary structures have been characterized, and the sequences that have the capacity to adopt such structures are very abundant in the human genome. Non-B DNA structures have been implicated in many important biological processes and can serve as sources of genetic instability, implicating them in disease and evolution. Non-B DNA conformations interact with a wide variety of proteins involved in replication, transcription, DNA repair, and chromatin architectural regulation. In this review, we will focus on the interactions of DNA repair proteins with non-B DNA and their roles in genetic instability, as the proteins and DNA involved in such interactions may represent plausible targets for selective therapeutic intervention. PMID:23219604

Vasquez, Karen M.; Wang, Guliang

2013-01-01

375

Interaction of DNA and DNA-anti-DNA complexes to fibronectin  

SciTech Connect

Fibronectin (Fn) is a large multidomain glycoprotein found in the basement membrane, on cell surface and in plasma. The interactions of Fn with DNA may be significant in glomerular deposition of DNA-anti-DNA complexes in patients with systemic lupus erythematosus (SLE). The authors examined the binding of DNA and DNA-anti-DNA complexes to Fn by a solid phase assay in which Fn was coated to microtiter plates and reacted with (/sup 3/H)DNA or DNA complexes with a monoclonal anti-DNA antibody. The optimal interaction of DNA with Fn occurs at <0.1M NaCl suggesting that the binding is charge dependent; the specificity of this binding was shown by competitive inhibition and locking experiments using anti-Fn. The binding was maximum at pH 6.5 and in the absence of Ca/sup 2 +/. The addition of Clq enhanced the binding of DNA and DNA-anti-DNA complexes to Fn, whereas heparan sulfate inhibited such binding. The monomeric or aggregated IgC did not bind Fn but aggregated IgG bound to Fn in the presence of Clq. Furthermore, DNA-anti-DNA complexes in sera from active SLE patients bound Fn which was enhanced in the presence of Clq; DNase abolished this bindi