Forensic DNA databases in Western Balkan region: retrospectives, perspectives, and initiatives

PubMed Central

Marjanović, Damir; Konjhodžić, Rijad; Butorac, Sara Sanela; Drobnič, Katja; Merkaš, Siniša; Lauc, Gordan; Primorac, Damir; Anđelinović, Šimun; Milosavljević, Mladen; Karan, Željko; Vidović, Stojko; Stojković, Oliver; Panić, Bojana; Vučetić Dragović, Anđelka; Kovačević, Sandra; Jakovski, Zlatko; Asplen, Chris; Primorac, Dragan

2011-01-01

The European Network of Forensic Science Institutes (ENFSI) recommended the establishment of forensic DNA databases and specific implementation and management legislations for all EU/ENFSI members. Therefore, forensic institutions from Bosnia and Herzegovina, Serbia, Montenegro, and Macedonia launched a wide set of activities to support these recommendations. To assess the current state, a regional expert team completed detailed screening and investigation of the existing forensic DNA data repositories and associated legislation in these countries. The scope also included relevant concurrent projects and a wide spectrum of different activities in relation to forensics DNA use. The state of forensic DNA analysis was also determined in the neighboring Slovenia and Croatia, which already have functional national DNA databases. There is a need for a ‘regional supplement’ to the current documentation and standards pertaining to forensic application of DNA databases, which should include regional-specific preliminary aims and recommendations. PMID:21674821

Forensic DNA databases in Western Balkan region: retrospectives, perspectives, and initiatives.

PubMed

Marjanović, Damir; Konjhodzić, Rijad; Butorac, Sara Sanela; Drobnic, Katja; Merkas, Sinisa; Lauc, Gordan; Primorac, Damir; Andjelinović, Simun; Milosavljević, Mladen; Karan, Zeljko; Vidović, Stojko; Stojković, Oliver; Panić, Bojana; Vucetić Dragović, Andjelka; Kovacević, Sandra; Jakovski, Zlatko; Asplen, Chris; Primorac, Dragan

2011-06-01

The European Network of Forensic Science Institutes (ENFSI) recommended the establishment of forensic DNA databases and specific implementation and management legislations for all EU/ENFSI members. Therefore, forensic institutions from Bosnia and Herzegovina, Serbia, Montenegro, and Macedonia launched a wide set of activities to support these recommendations. To assess the current state, a regional expert team completed detailed screening and investigation of the existing forensic DNA data repositories and associated legislation in these countries. The scope also included relevant concurrent projects and a wide spectrum of different activities in relation to forensics DNA use. The state of forensic DNA analysis was also determined in the neighboring Slovenia and Croatia, which already have functional national DNA databases. There is a need for a 'regional supplement' to the current documentation and standards pertaining to forensic application of DNA databases, which should include regional-specific preliminary aims and recommendations.

Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication

PubMed Central

Seco, Elena M.

2017-01-01

Abstract Firmicutes have two distinct replicative DNA polymerases, the PolC leading strand polymerase, and PolC and DnaE synthesizing the lagging strand. We have reconstituted in vitro Bacillus subtilis bacteriophage SPP1 θ-type DNA replication, which initiates unidirectionally at oriL. With this system we show that DnaE is not only restricted to lagging strand synthesis as previously suggested. DnaG primase and DnaE polymerase are required for initiation of DNA replication on both strands. DnaE and DnaG synthesize in concert a hybrid RNA/DNA ‘initiation primer’ on both leading and lagging strands at the SPP1 oriL region, as it does the eukaryotic Pol α complex. DnaE, as a RNA-primed DNA polymerase, extends this initial primer in a reaction modulated by DnaG and one single-strand binding protein (SSB, SsbA or G36P), and hands off the initiation primer to PolC, a DNA-primed DNA polymerase. Then, PolC, stimulated by DnaG and the SSBs, performs the bulk of DNA chain elongation at both leading and lagging strands. Overall, these modulations by the SSBs and DnaG may contribute to the mechanism of polymerase switch at Firmicutes replisomes. PMID:28575448

Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli

PubMed Central

Kato, Jun-ichi; Katayama, Tsutomu

2001-01-01

The bacterial DnaA protein binds to the chromosomal origin of replication to trigger a series of initiation reactions, which leads to the loading of DNA polymerase III. In Escherichia coli, once this polymerase initiates DNA synthesis, ATP bound to DnaA is efficiently hydrolyzed to yield the ADP-bound inactivated form. This negative regulation of DnaA, which occurs through interaction with the β-subunit sliding clamp configuration of the polymerase, functions in the temporal blocking of re-initiation. Here we show that the novel DnaA-related protein, Hda, from E.coli is essential for this regulatory inactivation of DnaA in vitro and in vivo. Our results indicate that the hda gene is required to prevent over-initiation of chromosomal replication and for cell viability. Hda belongs to the chaperone-like ATPase family, AAA+, as do DnaA and certain eukaryotic proteins essential for the initiation of DNA replication. We propose that the once-per-cell-cycle rule of replication depends on the timely interaction of AAA+ proteins that comprise the apparatus regulating the activity of the initiator of replication. PMID:11483528

Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli.

PubMed

Kato , J; Katayama, T

2001-08-01

The bacterial DnaA protein binds to the chromosomal origin of replication to trigger a series of initiation reactions, which leads to the loading of DNA polymerase III. In Escherichia coli, once this polymerase initiates DNA synthesis, ATP bound to DnaA is efficiently hydrolyzed to yield the ADP-bound inactivated form. This negative regulation of DnaA, which occurs through interaction with the beta-subunit sliding clamp configuration of the polymerase, functions in the temporal blocking of re-initiation. Here we show that the novel DnaA-related protein, Hda, from E.coli is essential for this regulatory inactivation of DnaA in vitro and in vivo. Our results indicate that the hda gene is required to prevent over-initiation of chromosomal replication and for cell viability. Hda belongs to the chaperone-like ATPase family, AAA(+), as do DnaA and certain eukaryotic proteins essential for the initiation of DNA replication. We propose that the once-per-cell-cycle rule of replication depends on the timely interaction of AAA(+) proteins that comprise the apparatus regulating the activity of the initiator of replication.

Expression of the p12 subunit of human DNA polymerase δ (Pol δ), CDK inhibitor p21(WAF1), Cdt1, cyclin A, PCNA and Ki-67 in relation to DNA replication in individual cells.

PubMed

Zhao, Hong; Zhang, Sufang; Xu, Dazhong; Lee, Marietta Ywt; Zhang, Zhongtao; Lee, Ernest Yc; Darzynkiewicz, Zbigniew

2014-01-01

We recently reported that the p12 subunit of human DNA polymerase δ (Pol δ4) is degraded by CRL4(Cdt2) which regulates the licensing factor Cdt1 and p21(WAF1) during the G1 to S transition. Presently, we performed multiparameter laser scanning cytometric analyses of changes in levels of p12, Cdt1 and p21(WAF1), detected immunocytochemically in individual cells, vis-à-vis the initiation and completion of DNA replication. The latter was assessed by pulse-labeling A549 cells with the DNA precursor ethynyl-2'-deoxyribose (EdU). The loss of p12 preceded the initiation of DNA replication and essentially all cells incorporating EdU were p12 negative. Completion of DNA replication and transition to G2 phase coincided with the re-appearance and rapid rise of p12 levels. Similar to p12 a decline of p21(WAF1) and Cdt1 was seen at the end of G1 phase and all DNA replicating cells were p21(WAF1) and Cdt1 negative. The loss of p21(WAF1) preceded that of Cdt1 and p12 and the disappearance of the latter coincided with the onset of DNA replication. Loss of p12 leads to conversion of Pol δ4 to its trimeric form, Pol δ3, so that the results provide strong support to the notion that Pol δ3 is engaged in DNA replication during unperturbed progression through the S phase of cell cycle. Also assessed was a correlation between EdU incorporation, likely reflecting the rate of DNA replication in individual cells, and the level of expression of positive biomarkers of replication cyclin A, PCNA and Ki-67 in these cells. Of interest was the observation of stronger correlation between EdU incorporation and expression of PCNA (r = 0.73) than expression of cyclin A (r = 0.47) or Ki-67 (r = 0.47).

The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP–DnaA-specific initiation complexes

PubMed Central

Keyamura, Kenji; Fujikawa, Norie; Ishida, Takuma; Ozaki, Shogo; Su’etsugu, Masayuki; Fujimitsu, Kazuyuki; Kagawa, Wataru; Yokoyama, Shigeyuki; Kurumizaka, Hitoshi; Katayama, Tsutomu

2007-01-01

Escherichia coli DiaA is a DnaA-binding protein that is required for the timely initiation of chromosomal replication during the cell cycle. In this study, we determined the crystal structure of DiaA at 1.8 Å resolution. DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Mutational analysis revealed that the DnaA-binding activity and formation of homotetramers are required for the stimulation of initiation by DiaA. DiaA tetramers can bind multiple DnaA molecules simultaneously. DiaA stimulated the assembly of multiple DnaA molecules on oriC, conformational changes in ATP–DnaA-specific initiation complexes, and unwinding of oriC duplex DNA. The mutant DiaA proteins are defective in these stimulations. DiaA associated also with ADP–DnaA, and stimulated the assembly of inactive ADP–DnaA–oriC complexes. Specific residues in the putative phosphosugar-binding motif of DiaA were required for the stimulation of initiation and formation of ATP–DnaA-specific–oriC complexes. Our data indicate that DiaA regulates initiation by a novel mechanism, in which DiaA tetramers most likely bind to multiple DnaA molecules and stimulate the assembly of specific ATP–DnaA–oriC complexes. These results suggest an essential role for DiaA in the promotion of replication initiation in a cell cycle coordinated manner. PMID:17699754

The unholy trinity: taxonomy, species delimitation and DNA barcoding

PubMed Central

DeSalle, Rob; Egan, Mary G; Siddall, Mark

2005-01-01

Recent excitement over the development of an initiative to generate DNA sequences for all named species on the planet has in our opinion generated two major areas of contention as to how this ‘DNA barcoding’ initiative should proceed. It is critical that these two issues are clarified and resolved, before the use of DNA as a tool for taxonomy and species delimitation can be universalized. The first issue concerns how DNA data are to be used in the context of this initiative; this is the DNA barcode reader problem (or barcoder problem). Currently, many of the published studies under this initiative have used tree building methods and more precisely distance approaches to the construction of the trees that are used to place certain DNA sequences into a taxonomic context. The second problem involves the reaction of the taxonomic community to the directives of the ‘DNA barcoding’ initiative. This issue is extremely important in that the classical taxonomic approach and the DNA approach will need to be reconciled in order for the ‘DNA barcoding’ initiative to proceed with any kind of community acceptance. In fact, we feel that DNA barcoding is a misnomer. Our preference is for the title of the London meetings—Barcoding Life. In this paper we discuss these two concerns generated around the DNA barcoding initiative and attempt to present a phylogenetic systematic framework for an improved barcoder as well as a taxonomic framework for interweaving classical taxonomy with the goals of ‘DNA barcoding’. PMID:16214748

Detection of an ALK Fusion in Colorectal Carcinoma by Hybrid Capture-Based Assay of Circulating Tumor DNA.

PubMed

Lai, Andrea Z; Schrock, Alexa B; Erlich, Rachel L; Ross, Jeffrey S; Miller, Vincent A; Yakirevich, Evgeny; Ali, Siraj M; Braiteh, Fadi

2017-07-01

ALK rearrangements have been observed in 0.05%-2.5% of patients with colorectal cancers (CRCs) and are predicted to be oncogenic drivers largely mutually exclusive of KRAS, NRAS, or BRAF alterations. Here we present the case of a patient with metastatic CRC who was treatment naïve at the time of molecular testing. Initial ALK immunohistochemistry (IHC) staining was negative, but parallel genomic profiling of both circulating tumor DNA (ctDNA) and tissue using similar hybrid capture-based assays each identified an identical STRN-ALK fusion. Subsequent ALK IHC staining of the same specimens was positive, suggesting that the initial result was a false negative. This report is the first instance of an ALK fusion in CRC detected using a ctDNA assay. Current guidelines for colorectal cancer (CRC) only recommend genomic assessment of KRAS, NRAS, BRAF, and microsatellite instability (MSI) status. ALK rearrangements are rare in CRC, but patients with activating ALK fusions have responded to targeted therapies ALK rearrangements can be detected by genomic profiling of ctDNA from blood or tissue, and this methodology may be informative in cases where immunohistochemistry (IHC) or other standard testing is negative. © AlphaMed Press 2017.

Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication.

PubMed

Camara, Johanna E; Breier, Adam M; Brendler, Therese; Austin, Stuart; Cozzarelli, Nicholas R; Crooke, Elliott

2005-08-01

Initiation of DNA replication from the Escherichia coli chromosomal origin is highly regulated, assuring that replication occurs precisely once per cell cycle. Three mechanisms for regulation of replication initiation have been proposed: titration of free DnaA initiator protein by the datA locus, sequestration of newly replicated origins by SeqA protein and regulatory inactivation of DnaA (RIDA), in which active ATP-DnaA is converted to the inactive ADP-bound form. DNA microarray analyses showed that the level of initiation in rapidly growing cells that lack datA was indistinguishable from that in wild-type cells, and that the absence of SeqA protein caused only a modest increase in initiation, in agreement with flow-cytometry data. In contrast, cells lacking Hda overinitiated replication twofold, implicating RIDA as the predominant mechanism preventing extra initiation events in a cell cycle.

Radiation cataracts: mechanisms involved in their long delayed occurrence but then rapid progression.

PubMed

Wolf, Norman; Pendergrass, William; Singh, Narendra; Swisshelm, Karen; Schwartz, Jeffrey

2008-02-05

This study was directed to assess the DNA damage and DNA repair response to X-ray inflicted lens oxidative damage and to investigate the subsequent changes in lens epithelial cell (LEC) behavior in vivo that led to long delayed but then rapidly developing cataracts. Two-month-old C57Bl/6 female mice received 11 Grays (Gy) of soft x-irradiation to the head only. The animals' eyes were examined for cataract status in 30 day intervals by slit lamp over an 11 month period post-irradiation. LEC migration, DNA fragment, free DNA retention, and reactive oxygen species (ROS) presence were established in the living lenses with fluorescent dyes using laser scanning confocal microscopy (LSCM). The extent and removal of initial LEC DNA damage were determined by comet assay. Immunohistochemistry was used to determine the presence of oxidized DNA and the response of a DNA repair protein in the lenses. This treatment resulted in advanced cortical cataracts that developed 5-11 months post-irradiation but then appeared suddenly within a 30 day period. The initially incurred DNA strand breaks were repaired within 30 min, but DNA damage remained as shown 72 h post-irradiation by the presence of the DNA adduct, 8-hydroxyguanosine (8-OHG), and a DNA repair protein, XRCC1. This was followed months later by abnormal behavior by LEC descendant cells with abnormal differentiation and migration patterns as seen with LSCM and fluorescent dyes. The sudden development of cortical cataracts several months post-irradiation coupled with the above findings suggests an accumulation of damaged descendants from the initially x-irradiated LECs. As these cells migrate abnormally and leave acellular lens surface sites, eventually a crisis point may arrive for lens entry of environmental O(2) with resultant ROS formation that overwhelms protection by resident antioxidant enzymes and results in the coagulation of lens proteins. The events seen in this study indicate the retention and transmission of progenitor cell DNA damage in descendant LEC. The cellular and molecular events parallel those previously reported for LSCM observations in age-related cataracts.

Investigation of a regulatory agency enquiry into potential porcine circovirus type 1 contamination of the human rotavirus vaccine, Rotarix: approach and outcome.

PubMed

Dubin, Gary; Toussaint, Jean-François; Cassart, Jean-Pol; Howe, Barbara; Boyce, Donna; Friedland, Leonard; Abu-Elyazeed, Remon; Poncelet, Sylviane; Han, Htay Htay; Debrus, Serge

2013-11-01

In January 2010, porcine circovirus type 1 (PCV1) DNA was unexpectedly detected in the oral live-attenuated human rotavirus vaccine, Rotarix (GlaxoSmithKline [GSK] Vaccines) by an academic research team investigating a novel, highly sensitive analysis not routinely used for adventitious agent screening. GSK rapidly initiated an investigation to confirm the source, nature and amount of PCV1 in the vaccine manufacturing process and to assess potential clinical implications of this finding. The investigation also considered the manufacturer's inactivated poliovirus (IPV)-containing vaccines, since poliovirus vaccine strains are propagated using the same cell line as the rotavirus vaccine strain. Results confirmed the presence of PCV1 DNA and low levels of PCV1 viral particles at all stages of the Rotarix manufacturing process. PCV type 2 DNA was not detected at any stage. When tested in human cell lines, productive PCV1 infection was not observed. There was no immunological or clinical evidence of PCV1 infection in infants who had received Rotarix in clinical trials. PCV1 DNA was not detected in the IPV-containing vaccine manufacturing process beyond the purification stage. Retrospective testing confirmed the presence of PCV1 DNA in Rotarix since the initial stages of its development and in vaccine lots used in clinical studies conducted pre- and post-licensure. The acceptable safety profile observed in clinical trials of Rotarix therefore reflects exposure to PCV1 DNA. The investigation into the presence of PCV1 in Rotarix could serve as a model for risk assessment in the event of new technologies identifying adventitious agents in the manufacturing of other vaccines and biological products.

Investigation of a regulatory agency enquiry into potential porcine circovirus type 1 contamination of the human rotavirus vaccine, Rotarix™

PubMed Central

Dubin, Gary; Toussaint, Jean-François; Cassart, Jean-Pol; Howe, Barbara; Boyce, Donna; Friedland, Leonard; Abu-Elyazeed, Remon; Poncelet, Sylviane; Han, Htay Htay; Debrus, Serge

2013-01-01

In January 2010, porcine circovirus type 1 (PCV1) DNA was unexpectedly detected in the oral live-attenuated human rotavirus vaccine, Rotarix™ (GlaxoSmithKline [GSK] Vaccines) by an academic research team investigating a novel, highly sensitive analysis not routinely used for adventitious agent screening. GSK rapidly initiated an investigation to confirm the source, nature and amount of PCV1 in the vaccine manufacturing process and to assess potential clinical implications of this finding. The investigation also considered the manufacturer’s inactivated poliovirus (IPV)-containing vaccines, since poliovirus vaccine strains are propagated using the same cell line as the rotavirus vaccine strain. Results confirmed the presence of PCV1 DNA and low levels of PCV1 viral particles at all stages of the Rotarix™ manufacturing process. PCV type 2 DNA was not detected at any stage. When tested in human cell lines, productive PCV1 infection was not observed. There was no immunological or clinical evidence of PCV1 infection in infants who had received Rotarix™ in clinical trials. PCV1 DNA was not detected in the IPV-containing vaccine manufacturing process beyond the purification stage. Retrospective testing confirmed the presence of PCV1 DNA in Rotarix™ since the initial stages of its development and in vaccine lots used in clinical studies conducted pre- and post-licensure. The acceptable safety profile observed in clinical trials of Rotarix™ therefore reflects exposure to PCV1 DNA. The investigation into the presence of PCV1 in Rotarix™ could serve as a model for risk assessment in the event of new technologies identifying adventitious agents in the manufacturing of other vaccines and biological products. PMID:24056737

Impact of folic acid fortification on global DNA methylation and one-carbon biomarkers in the Women's Health Initiative Observational Study cohort.

PubMed

Bae, Sajin; Ulrich, Cornelia M; Bailey, Lynn B; Malysheva, Olga; Brown, Elissa C; Maneval, David R; Neuhouser, Marian L; Cheng, Ting-Yuan David; Miller, Joshua W; Zheng, Yingye; Xiao, Liren; Hou, Lifang; Song, Xiaoling; Buck, Katharina; Beresford, Shirley A A; Caudill, Marie A

2014-03-01

DNA methylation is an epigenetic mechanism that regulates gene expression and can be modified by one-carbon nutrients. The objective of this study was to investigate the impact of folic acid (FA) fortification of the US food supply on leukocyte global DNA methylation and the relationship between DNA methylation, red blood cell (RBC) folate, and other one-carbon biomarkers among postmenopausal women enrolled in the Women's Health Initiative Observational Study. We selected 408 women from the highest and lowest tertiles of RBC folate distribution matching on age and timing of the baseline blood draw, which spanned the pre- (1994-1995), peri- (1996-1997), or post-fortification (1998) periods. Global DNA methylation was assessed by liquid chromatography-tandem mass spectrometry and expressed as a percentage of total cytosine. We observed an interaction (P = 0.02) between fortification period and RBC folate in relation to DNA methylation. Women with higher (vs. lower) RBC folate had higher mean DNA methylation (5.12 vs. 4.99%; P = 0.05) in the pre-fortification period, but lower (4.95 vs. 5.16%; P = 0.03) DNA methylation in the post-fortification period. We also observed significant correlations between one-carbon biomarkers and DNA methylation in the pre-fortification period, but not in the peri- or post-fortification period. The correlation between plasma homocysteine and DNA methylation was reversed from an inverse relationship during the pre-fortification period to a positive relationship during the post-fortification period. Our data suggest that (1) during FA fortification, higher RBC folate status is associated with a reduction in leukocyte global DNA methylation among postmenopausal women and; (2) the relationship between one-carbon biomarkers and global DNA methylation is dependent on folate availability.

Monitoring Daily Dynamics of Early Tumor Response to Targeted Therapy by Detecting Circulating Tumor DNA in Urine

PubMed Central

Husain, Hatim; Melnikova, Vladislava O.; Kosco, Karena; Woodward, Brian; More, Soham; Pingle, Sandeep C.; Weihe, Elizabeth; Park, Ben Ho; Tewari, Muneesh; Erlander, Mark G.; Cohen, Ezra; Lippman, Scott M.; Kurzrock, Razelle

2017-01-01

Purpose Non-invasive drug biomarkers for the early assessment of tumor response can enable adaptive therapeutic decision-making and proof-of-concept studies for investigational drugs. Circulating tumor DNA (ctDNA) is released into the circulation by tumor cell turnover and has been shown to be detectable in urine. Experimental Design We tested the hypothesis that dynamic changes in epidermal growth factor receptor (EGFR) activating (exon 19del and L858R) and resistance (T790M) mutation levels detected in urine could inform tumor response within days of therapy for advanced non-small cell lung cancer (NSCLC) patients receiving osimertinib, a second line third generation anti-EGFR tyrosine kinase inhibitor. Results Eight of nine evaluable NSCLC patients had detectable T790M-mutant DNA fragments in pre-treatment baseline samples. Daily monitoring of mutations in urine indicated a pattern of intermittent spikes throughout week 1 suggesting apoptosis with an overall decrease in fragment numbers between baselines to day 7 preceding radiographic response assessed at 6-12 weeks. Conclusions These findings suggest drug-induced tumor apoptosis within days of initial dosing. Daily sampling of ctDNA may enable early assessment of patient response and proof-of-concept studies for drug development. PMID:28420725

Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication.

PubMed

Liberek, K; Osipiuk, J; Zylicz, M; Ang, D; Skorko, J; Georgopoulos, C

1990-02-25

The process of initiation of lambda DNA replication requires the assembly of the proper nucleoprotein complex at the origin of replication, ori lambda. The complex is composed of both phage and host-coded proteins. The lambda O initiator protein binds specifically to ori lambda. The lambda P initiator protein binds to both lambda O and the host-coded dnaB helicase, giving rise to an ori lambda DNA.lambda O.lambda P.dnaB structure. The dnaK and dnaJ heat shock proteins have been shown capable of dissociating this complex. The thus freed dnaB helicase unwinds the duplex DNA template at the replication fork. In this report, through cross-linking, size chromatography, and protein affinity chromatography, we document some of the protein-protein interactions occurring at ori lambda. Our results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that the dnaJ protein specifically interacts with the dnaB helicase.

Timely binding of IHF and Fis to DARS2 regulates ATP–DnaA production and replication initiation

PubMed Central

Kasho, Kazutoshi; Fujimitsu, Kazuyuki; Matoba, Toshihiro; Oshima, Taku; Katayama, Tsutomu

2014-01-01

In Escherichia coli, the ATP-bound form of DnaA (ATP–DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP–DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP–DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP–DnaA was fully active in replication initiation and underwent DnaA–ATP hydrolysis. ADP–DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP–DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP–DnaA production, thereby promoting timely initiation. Moreover, we show that IHF–DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP–DnaA and replication initiation in coordination with the cell cycle and growth phase. PMID:25378325

Analysis of Heat-Labile Sites Generated by Reactions of Depleted Uranium and Ascorbate in Plasmid DNA

PubMed Central

Wilson, Janice; Young, Ashley; Civitello, Edgar R.

2013-01-01

The goal of this study was to characterize how depleted uranium (DU) causes DNA damage. Procedures were developed to assess the ability of organic and inorganic DNA adducts to convert to single strand breaks (SSB) in pBR322 plasmid DNA in the presence of heat or piperidine. DNA adducts formed by methyl methanesulfonate (MMS), cis-platin (cis-Pt), and chromic chloride were compared to those formed by reaction of uranyl acetate (UA) and ascorbate (Asc). Uranyl ion in the presence of Asc produced U-DNA adducts that converted to SSB upon heating. Piperidine, which acted on DNA methylated by MMS to convert methyl-DNA adducts to SSB, served in the opposite fashion with U-DNA adducts by decreasing SSB. The observation that piperidine also decreased the gel shift for metal-DNA adducts formed by monofunctional cis-Pt and chromic chloride was interpreted to suggest that piperidine served to remove U-DNA adducts. Radical scavengers did not affect formation of U-induced SSB, suggesting that SSB arose from the presence of U-DNA adducts and not from free radicals. A model is proposed to predict how U-DNA adducts may serve as initial lesions that convert to SSB or AP sites. Results suggest that DU can act as a chemical genotoxin that does not require radiation for its mode of action. Characterizing the DNA lesions formed by DU is necessary to assess the relative importance of different DNA lesions in the formation of DU-induced mutations. Understanding mechanisms of formation of DU-induced mutations may contribute to identification of biomarkers of DU exposures in humans. PMID:24218036

Mechanism of chimera formation during the Multiple Displacement Amplification reaction.

PubMed

Lasken, Roger S; Stockwell, Timothy B

2007-04-12

Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. Here, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2-21 nucleotides (nts) in the new templates. Formation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications.

Mechanism of chimera formation during the Multiple Displacement Amplification reaction

PubMed Central

Lasken, Roger S; Stockwell, Timothy B

2007-01-01

Background Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. Results Here, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2–21 nucleotides (nts) in the new templates. Conclusion Formation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications. PMID:17430586

The molecular landscape of pediatric acute myeloid leukemia reveals recurrent structural alterations and age-specific mutational interactions | Office of Cancer Genomics

Cancer.gov

We present the molecular landscape of pediatric acute myeloid leukemia (AML) and characterize nearly 1,000 participants in Children’s Oncology Group (COG) AML trials. The COG–National Cancer Institute (NCI) TARGET AML initiative assessed cases by whole-genome, targeted DNA, mRNA and microRNA sequencing and CpG methylation profiling. Validated DNA variants corresponded to diverse, infrequent mutations, with fewer than 40 genes mutated in >2% of cases.

Healing assessment of tile sets for error tolerance in DNA self-assembly.

PubMed

Hashempour, M; Mashreghian Arani, Z; Lombardi, F

2008-12-01

An assessment of the effectiveness of healing for error tolerance in DNA self-assembly tile sets for algorithmic/nano-manufacturing applications is presented. Initially, the conditions for correct binding of a tile to an existing aggregate are analysed using a Markovian approach; based on this analysis, it is proved that correct aggregation (as identified with a so-called ideal tile set) is not always met for the existing tile sets for nano-manufacturing. A metric for assessing tile sets for healing by utilising punctures is proposed. Tile sets are investigated and assessed with respect to features such as error (mismatched tile) movement, punctured area and bond types. Subsequently, it is shown that the proposed metric can comprehensively assess the healing effectiveness of a puncture type for a tile set and its capability to attain error tolerance for the desired pattern. Extensive simulation results are provided.

Impact of Storage Time on Hepatitis B Virus DNA Stability in Clinical Specimens Determined by Quantitative Real-time PCR.

PubMed

Zhang, Xiaolian; Yang, Dongmei; Lu, Yu; Lao, Xianjun; Qin, Xue; Li, Shan

2016-01-01

Detecting blood levels of hepatitis B virus (HBV) DNA must be accurate and credible. Shipment and storage conditions of clinical samples affect the quality of nucleic acids and can interfere with HBV DNA analysis. The aim of our study was to compare HBV DNA stability in plasma specimens at 4 degrees C for different storage periods. Blood samples from 30 hepatitis B surface antigen (HBsAg) positive patients were collected in tubes containing EDTA-K2. Each sample was divided into eight aliquots, one of which was measured immediately for the initial viral load. The remaining aliquots were then stored at 4 degrees C and assessed after 1, 2, 3, 7, 14, 21, and 30 days of storage. Quantification of HBV DNA was performed by real-time polymerase chain reaction (RT-PCR), and the difference in HBV DNA concentrations between two different time points was analysed with a paired-samples t-test. HBV DNA was measured in a range of 2.00 - 8.00 IU/mL, with low within-run and between-run coefficients of variation (< 10%). Storing plasma for one month at 4 degrees C revealed no significant decrease in HBV DNA level (p = 0.231), and no trend was evident to indicate continued reduction over a 3-week storage period. Based on the results of this study, storing plasma for up to one month at 4 degrees C does not affect the stability of HBV DNA, regardless of the initial viral load.

Biological characterization of a novel in vitro cell irradiator

PubMed Central

Fowler, Tyler L.; Fisher, Michael M.; Bailey, Alison M.; Bednarz, Bryan P.

2017-01-01

To evaluate the overall robustness of a novel cellular irradiator we performed a series of well-characterized, dose-responsive assays to assess the consequences of DNA damage. We used a previously described novel irradiation system and a traditional 137Cs source to irradiate a cell line. The generation of reactive oxygen species was assessed using chloromethyl-H2DCFDA dye, the induction of DNA DSBs was observed using the comet assay, and the initiation of DNA break repair was assessed through γH2AX image cytometry. A high correlation between physical absorbed dose and biologic dose was seen for the production of intracellular reactive oxygen species, physical DNA double strand breaks, and modulation of the cellular double stand break pathway. The results compared favorably to irradiation with a traditional 137Cs source. The rapid, straightforward tests described form a reasonable approach for biologic characterization of novel irradiators. These additional testing metrics go beyond standard physics testing such as Monte Carlo simulation and thermo-luminescent dosimeter evaluation to confirm that a novel irradiator can produce the desired dose effects in vitro. Further, assessment of these biological metrics confirms that the physical handling of the cells during the irradiation process results in biologic effects that scale appropriately with dose. PMID:29232400

Timely binding of IHF and Fis to DARS2 regulates ATP-DnaA production and replication initiation.

PubMed

Kasho, Kazutoshi; Fujimitsu, Kazuyuki; Matoba, Toshihiro; Oshima, Taku; Katayama, Tsutomu

2014-12-01

In Escherichia coli, the ATP-bound form of DnaA (ATP-DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP-DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP-DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP-DnaA was fully active in replication initiation and underwent DnaA-ATP hydrolysis. ADP-DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP-DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP-DnaA production, thereby promoting timely initiation. Moreover, we show that IHF-DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP-DnaA and replication initiation in coordination with the cell cycle and growth phase. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

PubMed

Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

2016-12-13

Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

DEVELOPMENT OF DNA MICROARRAYS FOR ECOLOGICAL EXPOSURE ASSESSMENT

EPA Science Inventory

EPA/ORD is moving forward with a computational toxicology initiative in FY 04 which aims to integrate genomics and computational methods to provide a mechanistic basis for prediction of exposure and effects of chemical stressors in the environment.

The goal of the presen...

Strand invasion structures in the inverted repeat of Candida albicans mitochondrial DNA reveal a role for homologous recombination in replication.

PubMed

Gerhold, Joachim M; Aun, Anu; Sedman, Tiina; Jõers, Priit; Sedman, Juhan

2010-09-24

Molecular recombination and transcription are proposed mechanisms to initiate mitochondrial DNA (mtDNA) replication in yeast. We conducted a comprehensive analysis of mtDNA from the yeast Candida albicans. Two-dimensional agarose gel electrophoresis of mtDNA intermediates reveals no bubble structures diagnostic of specific replication origins, but rather supports recombination-driven replication initiation of mtDNA in yeast. Specific species of Y structures together with DNA copy number analyses of a C. albicans mutant strain provide evidence that a region in a mainly noncoding inverted repeat is predominantly involved in replication initiation via homologous recombination. Our further findings show that the C. albicans mtDNA forms a complex branched network that does not contain detectable amounts of circular molecules. We provide topological evidence for recombination-driven mtDNA replication initiation and introduce C. albicans as a suitable model organism to study wild-type mtDNA maintenance in yeast. Copyright © 2010 Elsevier Inc. All rights reserved.

Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex.

PubMed

Su'etsugu, Masayuki; Takata, Makoto; Kubota, Toshio; Matsuda, Yusaku; Katayama, Tsutomu

2004-06-01

In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding. Copyright Blackwell Publishing Limited

Hda-mediated inactivation of the DnaA protein and dnaA gene autoregulation act in concert to ensure homeostatic maintenance of the Escherichia coli chromosome

PubMed Central

Riber, Leise; Olsson, Jan A.; Jensen, Rasmus B.; Skovgaard, Ole; Dasgupta, Santanu; Marinus, Martin G.; Løbner-Olesen, Anders

2006-01-01

Initiation of DNA replication in Eschericia coli requires the ATP-bound form of the DnaA protein. The conversion of DnaA–ATP to DnaA–ADP is facilitated by a complex of DnaA, Hda (homologous to DnaA), and DNA-loaded β-clamp proteins in a process termed RIDA (regulatory inactivation of DnaA). Hda-deficient cells initiate replication at each origin mainly once per cell cycle, and the rare reinitiation events never coincide with the end of the origin sequestration period. Therefore, RIDA is not the predominant mechanism to prevent immediate reinitiation from oriC. The cellular level of Hda correlated directly with dnaA gene expression such that Hda deficiency led to reduced dnaA gene expression, and overproduction of Hda led to DnaA overproduction. Hda-deficient cells were very sensitive to variations in the cellular level of DnaA, and DnaA overproduction led to uncontrolled initiation of replication from oriC, causing severe growth retardation or cell death. Based on these observations, we propose that both RIDA and dnaA gene autoregulation are required as homeostatic mechanisms to ensure that initiation of replication occurs at the same time relative to cell mass in each cell cycle. PMID:16882985

Hda-mediated inactivation of the DnaA protein and dnaA gene autoregulation act in concert to ensure homeostatic maintenance of the Escherichia coli chromosome.

PubMed

Riber, Leise; Olsson, Jan A; Jensen, Rasmus B; Skovgaard, Ole; Dasgupta, Santanu; Marinus, Martin G; Løbner-Olesen, Anders

2006-08-01

Initiation of DNA replication in Eschericia coli requires the ATP-bound form of the DnaA protein. The conversion of DnaA-ATP to DnaA-ADP is facilitated by a complex of DnaA, Hda (homologous to DnaA), and DNA-loaded beta-clamp proteins in a process termed RIDA (regulatory inactivation of DnaA). Hda-deficient cells initiate replication at each origin mainly once per cell cycle, and the rare reinitiation events never coincide with the end of the origin sequestration period. Therefore, RIDA is not the predominant mechanism to prevent immediate reinitiation from oriC. The cellular level of Hda correlated directly with dnaA gene expression such that Hda deficiency led to reduced dnaA gene expression, and overproduction of Hda led to DnaA overproduction. Hda-deficient cells were very sensitive to variations in the cellular level of DnaA, and DnaA overproduction led to uncontrolled initiation of replication from oriC, causing severe growth retardation or cell death. Based on these observations, we propose that both RIDA and dnaA gene autoregulation are required as homeostatic mechanisms to ensure that initiation of replication occurs at the same time relative to cell mass in each cell cycle.

MTBP, the partner of Treslin, contains a novel DNA-binding domain that is essential for proper initiation of DNA replication.

PubMed

Kumagai, Akiko; Dunphy, William G

2017-11-01

Treslin, which is essential for incorporation of Cdc45 into the replicative helicase, possesses a partner called MTBP (Mdm2-binding protein). We have analyzed Xenopus and human MTBP to assess its role in DNA replication. Depletion of MTBP from Xenopus egg extracts, which also removes Treslin, abolishes DNA replication. These extracts be can rescued with recombinant Treslin-MTBP but not Treslin or MTBP alone. Thus, Treslin-MTBP is collectively necessary for replication. We have identified a C-terminal region of MTBP (the CTM domain) that binds efficiently to both double-stranded DNA and G-quadruplex (G4) DNA. This domain also exhibits homology with budding yeast Sld7. Mutants of MTBP without a functional CTM domain are defective for DNA replication in Xenopus egg extracts. These mutants display an impaired localization to chromatin and the inability to support loading of Cdc45. Human cells harboring such a mutant also display severe S-phase defects. Thus, the CTM domain of MTBP plays a critical role in localizing Treslin-MTBP to the replication apparatus for initiation. © 2017 Kumagai and Dunphy. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Evaluation of the metabolic fate of munitions material (TNT & RDX) in plant systems. Initial assessment of plant DNA mutation spectra as a biomarker

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

Leung, F.; Cataldo, D.A.; Fellows, R.J.

1995-09-01

Munitions material can enter the environment as a result of manufacturing activities and field usage. Predictor methodologies, or biomarkers would enhance evaluation of environmental impacts. The goal of this exploratory study deoxyribonucleic acid (DNA) mutation frequency as a biomarker for munitions exposure. The approach e resolution of an effective repetitive sequence probe for the identification of characteristic mutations, and (2) the development of a testing media [a clonal cell line of carrot (Daucus carota) spension cells]. Commercially available probes demonstrated marginal resolution therefore a low-C{sub o}t library was then constructed. Three colonies from the low-C{sub o}t DNA library were screenedmore » and the DNA isolates sequenced. A suspension culture of carrot (Daucus carota) was developed. A mutation spectra experiment was initiated at a 10-mg TNT/L exposure concentration with the attempt to clone over 1500 single TNT-exposed cells. Over the following six months greater than 98% of the initially isolated cells were unable to survive and produce micro calluses. The remaining calli were too few to be statistically significant and the experiment was terminated. The biomarker concept itself remains to be disproved, but the need for large numbers of uniform clones to differentiate true mutations suggest that more direct techniques using whole tissues need to be developed.« less

Localized DNA melting and structural pertubations in the origin of replication, oriC, of Escherichia coli in vitro and in vivo.

PubMed Central

Gille, H; Messer, W

1991-01-01

The leftmost region of the Escherichia coli origin of DNA replication (oriC) contains three tandemly repeated AT-rich 13mers which have been shown to become single-stranded during the early stages of initiation in vitro. Melting is induced by the ATP form of DnaA, the initiator protein of DNA replication. KMnO4 was used to probe for single-stranded regions and altered DNA conformation during the initiation of DNA replication at oriC in vitro and in vivo. Unpairing in the AT-rich 13mer region is thermodynamically stable even in the absence of DnaA protein, but only when divalent cations are omitted from the reaction. In the presence of Mg2+, oriC melting is strictly DnaA dependent. The sensitive region is distinct from that detected in the absence of DnaA as it is located further to the left within the minimal origin. In addition, the DNA is severely distorted between the three 13mers and the IHF binding site in oriC. A change of conformation can also be observed during the initiation of DNA replication in vivo. This is the first in vivo evidence for a structural change at the 13mers during initiation complex formation. Images PMID:2026151

Dynamic interaction of Y RNAs with chromatin and initiation proteins during human DNA replication

PubMed Central

Zhang, Alice Tianbu; Langley, Alexander R.; Christov, Christo P.; Kheir, Eyemen; Shafee, Thomas; Gardiner, Timothy J.; Krude, Torsten

2011-01-01

Non-coding Y RNAs are required for the initiation of chromosomal DNA replication in mammalian cells. It is unknown how they perform this function or if they associate with a nuclear structure during DNA replication. Here, we investigate the association of Y RNAs with chromatin and their interaction with replication proteins during DNA replication in a human cell-free system. Our results show that fluorescently labelled Y RNAs associate with unreplicated euchromatin in late G1 phase cell nuclei before the initiation of DNA replication. Following initiation, Y RNAs are displaced locally from nascent and replicated DNA present in replication foci. In intact human cells, a substantial fraction of endogenous Y RNAs are associated with G1 phase nuclei, but not with G2 phase nuclei. Y RNAs interact and colocalise with the origin recognition complex (ORC), the pre-replication complex (pre-RC) protein Cdt1, and other proteins implicated in the initiation of DNA replication. These data support a molecular ‘catch and release’ mechanism for Y RNA function during the initiation of chromosomal DNA replication, which is consistent with Y RNAs acting as replication licensing factors. PMID:21610089

Assessment of primer/template mismatch effects on real-time PCR amplification of target taxa for GMO quantification.

PubMed

Ghedira, Rim; Papazova, Nina; Vuylsteke, Marnik; Ruttink, Tom; Taverniers, Isabel; De Loose, Marc

2009-10-28

GMO quantification, based on real-time PCR, relies on the amplification of an event-specific transgene assay and a species-specific reference assay. The uniformity of the nucleotide sequences targeted by both assays across various transgenic varieties is an important prerequisite for correct quantification. Single nucleotide polymorphisms (SNPs) frequently occur in the maize genome and might lead to nucleotide variation in regions used to design primers and probes for reference assays. Further, they may affect the annealing of the primer to the template and reduce the efficiency of DNA amplification. We assessed the effect of a minor DNA template modification, such as a single base pair mismatch in the primer attachment site, on real-time PCR quantification. A model system was used based on the introduction of artificial mismatches between the forward primer and the DNA template in the reference assay targeting the maize starch synthase (SSIIb) gene. The results show that the presence of a mismatch between the primer and the DNA template causes partial to complete failure of the amplification of the initial DNA template depending on the type and location of the nucleotide mismatch. With this study, we show that the presence of a primer/template mismatch affects the estimated total DNA quantity to a varying degree.

Structural Confirmation of a Bent and Open Model for the Initiation Complex of T7 RNA Polymerase

PubMed Central

Turingan, Rosemary S.; Liu, Cuihua; Hawkins, Mary E.; Martin, Craig T.

2008-01-01

T7 RNA polymerase is known to induce bending of its promoter DNA upon binding, as evidenced by gel-shift assays and by recent end-to-end fluorescence energy transfer distance measurements. Crystal structures of promoter-bound and initially transcribing complexes, however, lack downstream DNA, providing no information on the overall path of the DNA through the protein. Crystal structures of the elongation complex do include downstream DNA and provide valuable guidance in the design of models for the complete melted bubble structure at initiation. In the current study, we test a specific structural model for the initiation complex, obtained by alignment of the C-terminal regions of the protein structures from both initiation and elongation and then simple transferal of the downstream DNA from the elongation complex onto the initiation complex. FRET measurement of distances from a point upstream on the promoter DNA to various points along the downstream helix reproduce the expected helical periodicity in the distances and support the model’s orientation and phasing of the downstream DNA. The model also makes predictions about the extent of melting downstream of the active site. By monitoring fluorescent base analogs incorporated at various positions in the DNA we have mapped the downstream edge of the bubble, confirming the model. The initially melted bubble, in the absence of substrate, encompasses 7–8 bases and is sufficient to allow synthesis of a 3 base transcript before further melting is required. The results demonstrate that despite massive changes in the N-terminal portion of the protein and in the DNA upstream of the active site, the DNA downstream of the active site is virtually identical in both initiation and elongation complexes. PMID:17253774

STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

PubMed Central

Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

2009-01-01

Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

The Escherichia coli Cryptic Prophage Protein YfdR Binds to DnaA and Initiation of Chromosomal Replication Is Inhibited by Overexpression of the Gene Cluster yfdQ-yfdR-yfdS-yfdT

PubMed Central

Noguchi, Yasunori; Katayama, Tsutomu

2016-01-01

The initiation of bacterial chromosomal replication is regulated by multiple pathways. To explore novel regulators, we isolated multicopy suppressors for the cold-sensitive hda-185 ΔsfiA(sulA) mutant. Hda is crucial for the negative regulation of the initiator DnaA and the hda-185 mutation causes severe replication overinitiation at the replication origin oriC. The SOS-associated division inhibitor SfiA inhibits FtsZ ring formation, an essential step for cell division regulation during the SOS response, and ΔsfiA enhances the cold sensitivity of hda-185 cells in colony formation. One of the suppressors comprised the yfdQ-yfdR-yfdS-yfdT gene cluster carried on a cryptic prophage. Increased copy numbers of yfdQRT or yfdQRS inhibited not only hda-185-dependent overinitiation, but also replication overinitiation in a hyperactive dnaA mutant, and in a mutant lacking an oriC-binding initiation-inhibitor SeqA. In addition, increasing the copy number of the gene set inhibited the growth of cells bearing specific, initiation-impairing dnaA mutations. In wild-type cells, multicopy supply of yfdQRT or yfdQRS also inhibited replication initiation and increased hydroxyurea (HU)-resistance, as seen in cells lacking DiaA, a stimulator of DnaA assembly on oriC. Deletion of the yfdQ-yfdR-yfdS-yfdT genes did not affect either HU resistance or initiation regulation. Furthermore, we found that DnaA bound specifically to YfdR in soluble protein extracts oversupplied with YfdQRST. Purified YfdR also bound to DnaA, and DnaA Phe46, an amino acid residue crucial for DnaA interactions with DiaA and DnaB replicative helicase was important for this interaction. Consistently, YfdR moderately inhibited DiaA-DnaA and DnaB-DnaA interactions. In addition, protein extracts oversupplied with YfdQRST inhibited replication initiation in vitro. Given the roles of yfdQ and yfdS in cell tolerance to specific environmental stresses, the yfdQ-yfdR-yfdS-yfdT genes might downregulate the initiator DnaA-oriC complex under specific growth conditions. PMID:26973617

The Escherichia coli Cryptic Prophage Protein YfdR Binds to DnaA and Initiation of Chromosomal Replication Is Inhibited by Overexpression of the Gene Cluster yfdQ-yfdR-yfdS-yfdT.

PubMed

Noguchi, Yasunori; Katayama, Tsutomu

2016-01-01

The initiation of bacterial chromosomal replication is regulated by multiple pathways. To explore novel regulators, we isolated multicopy suppressors for the cold-sensitive hda-185 ΔsfiA(sulA) mutant. Hda is crucial for the negative regulation of the initiator DnaA and the hda-185 mutation causes severe replication overinitiation at the replication origin oriC. The SOS-associated division inhibitor SfiA inhibits FtsZ ring formation, an essential step for cell division regulation during the SOS response, and ΔsfiA enhances the cold sensitivity of hda-185 cells in colony formation. One of the suppressors comprised the yfdQ-yfdR-yfdS-yfdT gene cluster carried on a cryptic prophage. Increased copy numbers of yfdQRT or yfdQRS inhibited not only hda-185-dependent overinitiation, but also replication overinitiation in a hyperactive dnaA mutant, and in a mutant lacking an oriC-binding initiation-inhibitor SeqA. In addition, increasing the copy number of the gene set inhibited the growth of cells bearing specific, initiation-impairing dnaA mutations. In wild-type cells, multicopy supply of yfdQRT or yfdQRS also inhibited replication initiation and increased hydroxyurea (HU)-resistance, as seen in cells lacking DiaA, a stimulator of DnaA assembly on oriC. Deletion of the yfdQ-yfdR-yfdS-yfdT genes did not affect either HU resistance or initiation regulation. Furthermore, we found that DnaA bound specifically to YfdR in soluble protein extracts oversupplied with YfdQRST. Purified YfdR also bound to DnaA, and DnaA Phe46, an amino acid residue crucial for DnaA interactions with DiaA and DnaB replicative helicase was important for this interaction. Consistently, YfdR moderately inhibited DiaA-DnaA and DnaB-DnaA interactions. In addition, protein extracts oversupplied with YfdQRST inhibited replication initiation in vitro. Given the roles of yfdQ and yfdS in cell tolerance to specific environmental stresses, the yfdQ-yfdR-yfdS-yfdT genes might downregulate the initiator DnaA-oriC complex under specific growth conditions.

Investigation of sodium arsenite, thioacetamide, and diethanolamine in the alkaline comet assay: Part of the JaCVAM comet validation exercise.

PubMed

Beevers, Carol; Henderson, Debbie; Lillford, Lucinda

2015-07-01

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined sodium arsenite, thioacetamide, and diethanolamine. Using the JaCVAM approved study protocol version 14.2, each chemical was tested in male rats up to maximum tolerated dose levels and DNA damage in the liver and stomach was assessed approximately 3h after the final administration by gavage. Histopathology assessments of liver and stomach sections from the same animals were also examined for evidence of cytotoxicity or necrosis. No evidence of DNA damage was observed in the stomach of animals treated with sodium arsenite at 7.5, 15, or 30 mg/kg/day. However, equivocal findings were found in the liver, where increases in DNA migration were observed in two independent experiments, but not in all treated animals and not at the same dose levels. Thioacetamide caused an increase in DNA migration in the stomach of rats treated at 19, 38, and 75 mg/kg/day, but not in the liver, despite evidence of marked hepatotoxicity following histopathology assessments. No evidence of DNA damage was observed in the stomach or liver of animals treated with diethanolamine at 175, 350, or 700 mg/kg/day. Copyright © 2015 Elsevier B.V. All rights reserved.

Structure of a bacterial RNA polymerase holoenzyme open promoter complex

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

Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka

2015-09-08

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstreammore » of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.« less

Structure of a bacterial RNA polymerase holoenzyme open promoter complex

DOE PAGES

Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka; ...

2015-09-08

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstreammore » of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Additionally a RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σ A dissociation.« less

Novel methylation panel for the early detection of colorectal tumors in stool DNA.

PubMed

Azuara, Daniel; Rodriguez-Moranta, Francisco; de Oca, Javier; Soriano-Izquierdo, Antonio; Mora, Josefina; Guardiola, Jordi; Biondo, Sebastiano; Blanco, Ignacio; Peinado, Miguel Angel; Moreno, Victor; Esteller, Manel; Capellá, Gabriel

2010-07-01

Previous studies showed that the assessment of promoter hypermethylation of a limited number of genes in tumor biopsies may identify the majority of colorectal tumors. This study aimed to assess the clinical usefulness of a panel of methylation biomarkers in stool DNA in the identification of colorectal tumors, using methylation-specific melting curve analysis (MS-MCA), a technique that simultaneously analyzes all cytosine-phosphate-guanine (CpG) residues within a promoter. The promoter methylation status of 4 tumor-related genes (RARB2, p16INK4a, MGMT, and APC) was analyzed in DNA stool samples and corresponding tissues in an initial set of 12 patients with newly diagnosed primary colorectal carcinomas and 20 patients with newly diagnosed colorectal adenomas, using methylation-specific polymerase chain reaction. Results were replicated in a set of 82 patients (20 healthy subjects, 16 patients with inflammatory bowel disease (IBD), 20 patients with adenomas, and 26 patients with carcinomas), using MS-MCA analyses. In the initial set, >or= 1 positive methylation marker was detected in the stools of 9 of 12 patients (75%) with carcinomas and 12 of 20 patients (60%) with adenomas, with no false-positive results. Stool analyses missed 7 methylated lesions (25%). In the replication set, stool DNA testing detected 16 of 26 carcinomas (62%) and 8 of 20 adenomas (40%). The MS-MCAs missed 14 methylated tumors (37%). No aberrant methylation was evident in healthy subjects, but the RARB2 marker was positive in 2 of 15 stool samples (13%) of patients with IBD. Analysis via MS-MCA of a panel of methylation markers in stool DNA may offer a good alternative in the early, noninvasive detection of colorectal tumors.

Controlled initiation of chromosomal replication in Escherichia coli requires functional Hda protein.

PubMed

Camara, Johanna Eltz; Skarstad, Kirsten; Crooke, Elliott

2003-05-01

Regulatory inactivation of DnaA helps ensure that the Escherichia coli chromosome is replicated only once per cell cycle, through accelerated hydrolysis of active replication initiator ATP-DnaA to inactive ADP-DnaA. Analysis of deltahda strains revealed that the regulatory inactivation of DnaA component Hda is necessary for maintaining controlled initiation but not for cell growth or viability.

Using dual-polarization interferometry to study surface-initiated DNA hybridization chain reactions in real time.

PubMed

Huang, Fujian; Xu, Pingping; Liang, Haojun

2014-01-15

In this study we used dual-polarization interferometry to investigate DNA hybridization chain reactions (HCRs) at solid-liquid interfaces. We monitored the effects of variations in mass, thickness, and density of the immobilized initiator on the subsequent HCRs at various salt concentrations. At low salt concentrations, the single-stranded DNA (ssDNA) initiator was attached uniformly to the chip surface. At high salt concentrations, it lay on the surface at the onset of the immobilization process, but the approaching ssDNA forced the pre-immobilized ssDNA strands to extend into solution as a result of increased electrostatic repulsion between the pre-adsorbed and approaching ssDNA chains. Injection of a mixture of H1 and H2 increased the mass and thickness of the films initially, but thereafter the thickness decreased. These changes indicate that the long double-stranded DNA that formed lay on the surface, rather than extended into the solution, thereby suppressing the subsequent initiation activity of the released single-strand parts of H1 and H2. Increasing the salt concentration increased the HCR efficiency and reaction rate. The HCR efficiency of the initiator ssDNA immobilized on its 5' end was higher than that immobilized on its 3' end, suggesting that the released single-strand parts of H1 and H2 close to the chip surface decreased the initiation activity relative to those of the ones extending into solution. © 2013 Elsevier B.V. All rights reserved.

The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2

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

Jha, Jyoti K.; Li, Mi; Ghirlando, Rodolfo

Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations inrctBthat reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in amore » dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding. IMPORTANCE The capacity of proteins to undergo remodeling provides opportunities to control their function. However, remodeling remains a poorly understood aspect of the structure-function paradigm due to its dynamic nature. Here we have studied remodeling of the initiator of replication ofVibrio choleraeChr2 by the molecular chaperone, DnaK. We show that DnaK binds to a site on the Chr2 initiator (RctB) that promotes initiation by reducing the initiator’s propensity to dimerize. Dimerization of the initiator of the putative plasmid progenitor of Chr2 is also reduced by DnaK, which promotes initiation. Paradoxically, the DnaK binding also promotes replication inhibition by reducing an autoinhibitory activity of RctB. In the plasmid-to-chromosome transition, it appears that the initiator has acquired an autoinhibitory activity and along with it a new chaperone activity that apparently helps to control replication inhibition independently of replication promotion.« less

Low-concentration exposure to BPA, BPF and BPAF induces oxidative DNA bases lesions in human peripheral blood mononuclear cells.

PubMed

Mokra, Katarzyna; Woźniak, Katarzyna; Bukowska, Bożena; Sicińska, Paulina; Michałowicz, Jaromir

2018-06-01

Because bisphenol A (BPA) and some of its analogs have been supposed to influence development of cancer, we have assessed the effect of BPA, bisphenol S (BPS), bisphenol F (BPF) and bisphenol AF (BPAF) on DNA bases oxidation, which is a key process in cancer initiation. The analysis was conducted on human peripheral blood mononuclear cells (PBMCs), which are very useful model to assess genotoxic potential of various toxicants in different cell types. In order to determine oxidative damage to DNA pyrimidines and purines, alkaline version of the comet assay with DNA glycosylases, i.e. endonuclease III (Nth) and human 8-oxoguanine DNA glycosylase (hOGG1) was used. PBMCs were exposed to BPA or its analogs in the concentrations of 0.01, 0.1 and 1 μg/mL for 4 h and 0.001, 0.01 and 0.1 μg/mL for 48 h. We have observed that BPA, BPS, BPF and particularly BPAF caused oxidative damage to DNA pyrimidines and more strongly to purines in human PBMCs. The results have also shown that BPS, which is the most commonly used as a substitute for BPA in the manufacture induced definitely the smallest oxidative DNA bases lesions in PBMCs. Moreover, we have noticed that BPA, BPF and BPAF caused DNA damage at very low concentration of 1 ng/mL. Copyright © 2018 Elsevier Ltd. All rights reserved.

A multigene molecular phylogenetic assessment of true morels (Morchella) in turkey

USDA-ARS?s Scientific Manuscript database

A collection of 247 true morels (Morchella spp.) primarily from the Mediterranean and Aegean Regions of Southern Turkey, were analyzed for species diversity using partial RNA polymerase I (RPB1) and nuclear ribosomal large subunit (LSU) rDNA gene sequences. Based on the result of this initial scree...

The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse.

PubMed Central

Urano, Y; Kominami, R; Mishima, Y; Muramatsu, M

1980-01-01

Approximately one kilobase pairs surrounding and upstream the transcription initiation site of a cloned ribosomal DNA (rDNA) of the mouse were sequenced. The putative transcription initiation site was determined by two independent methods: one nuclease S1 protection and the other reverse transcriptase elongation mapping using isolated 45S ribosomal RNA precursor (45S RNA) and appropriate restriction fragments of rDNA. Both methods gave an identical result; 45S RNA had a structure starting from ACTCTTAG---. Characteristically, mouse rDNA had many T clusters (greater than or equal to 5) upstream the initiation site, the longest being 21 consecutive T's. A pentadecanucleotide, TGCCTCCCGAGTGCA, appeared twice within 260 nucleotides upstream the putative initiation site. No such characteristic sequences were found downstream this site. Little similarity was found in the upstream of the transcription initiation site between the mouse, Xenopus laevis and Saccharomyces cerevisiae rDNA. Images PMID:6162156

DNA polymeric films as a support for cell growth as a new material for regenerative medicine: Compatibility and applicability.

PubMed

Jayme, Cristiano Ceron; de Paula, Leonardo Barcelos; Rezende, Nayara; Calori, Italo Rodrigo; Franchi, Leonardo Pereira; Tedesco, Antonio Claudio

2017-11-15

DNA polymeric films (DNA-PFs) are a promising drug delivery system (DDS) in modern medicine. In this study, we evaluated the growth behavior of oral squamous cell carcinoma (OSCC) cells on DNA-PFs. The morphological, biochemical, and cytometric features of OSCC cell adhesion on DNA-PFs were also assessed. An initial, temporary alteration in cell morphology was observed at early time points owing to the inhibition of cell attachment to the film, which then returned to a normal morphological state at later time points. MTT and resazurin assays showed a moderate reduction in cell viability related to increased DNA concentration in the DNA-PFs. Flow cytometry studies showed low cytotoxicity of DNA-PFs, with cell viabilities higher than 90% in all the DNA-PFs tested. Flow cytometric cell cycle analysis also showed average cell cycle phase distributions at later time points, indicating that OSCC cell growth is maintained in the presence of DNA-PFs. These results show high biocompatibility of DNA-PFs and suggest their use in designing "dressing material," where the DNA film acts as a support for cell growth, or with incorporation of active or photoactive compounds, which can induce tissue regeneration and are useful to treat many diseases, especially oral cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Transcription initiation complex structures elucidate DNA opening.

PubMed

Plaschka, C; Hantsche, M; Dienemann, C; Burzinski, C; Plitzko, J; Cramer, P

2016-05-19

Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts.

Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction.

PubMed Central

Dodson, M; Echols, H; Wickner, S; Alfano, C; Mensa-Wilmot, K; Gomes, B; LeBowitz, J; Roberts, J D; McMacken, R

1986-01-01

The O protein of bacteriophage lambda localizes the initiation of DNA replication to a unique site on the lambda genome, ori lambda. By means of electron microscopy, we infer that the binding of O to ori lambda initiates a series of protein addition and transfer reactions that culminate in localized unwinding of the origin DNA, generating a prepriming structure for the initiation of DNA replication. We can define three stages of this prepriming reaction, the first two of which we have characterized previously. First, dimeric O protein binds to multiple DNA binding sites and self-associates to form a nucleoprotein structure, the O-some. Second, lambda P and host DnaB proteins interact with the O-some to generate a larger complex that includes additional DNA from an A + T-rich region adjacent to the O binding sites. Third, the addition of the DnaJ, DnaK, and Ssb proteins and ATP results in an origin-specific unwinding reaction, probably catalyzed by the helicase activity of DnaB. The unwinding reaction is unidirectional, proceeding "rightward" from the origin. The minimal DNA sequence competent for unwinding consists of two O binding sites and the adjacent A + T-rich region to the right of the binding sites. We conclude that the lambda O protein localizes and initiates a six-protein sequential reaction responsible for but preceding the precise initiation of DNA replication. Specialized nucleoprotein structures similar to the O-some may be a general feature of DNA transactions requiring extraordinary precision in localization and control. Images PMID:3020552

Cost assessment of the automated VERSANT 440 Molecular System versus the semi-automated System 340 bDNA Analyzer platforms.

PubMed

Elbeik, Tarek; Loftus, Richard A; Beringer, Scott

2007-11-01

Labor, supply and waste were evaluated for HIV-1 and HCV bDNA on the semi-automated System 340 bDNA Analyzer and the automated VERSANT 440 Molecular System (V440). HIV-1 sample processing was evaluated using a 24- and 48-position centrifuge rotor. Vigilance time (hands-on manipulations plus incubation time except initial target hybridization) and disposables were approximately 37 and 12% lower for HIV-1, and 64 and 31% lower for HCV bDNA, respectively, with V440. Biohazardous solid waste was approximately twofold lower for both assays and other waste types were the same for either assay on both platforms. HIV-1 sample processing vigilance time for the 48-position rotor was reduced by 2 h. V440 provides cost savings and improved workflow.

Systemic Lupus Erythematosus with and without Anti-dsDNA Antibodies: Analysis from a Large Monocentric Cohort.

PubMed

Conti, Fabrizio; Ceccarelli, Fulvia; Perricone, Carlo; Massaro, Laura; Marocchi, Elisa; Miranda, Francesca; Spinelli, Francesca Romana; Truglia, Simona; Alessandri, Cristiano; Valesini, Guido

2015-01-01

The anti-dsDNA antibodies are a marker for Systemic Lupus Erythematosus (SLE) and 70-98% of patients test positive. We evaluated the demographic, clinical, laboratory, and therapeutical features of a monocentric SLE cohort according to the anti-dsDNA status. We identified three groups: anti-dsDNA + (persistent positivity); anti-dsDNA ± (initial positivity and subsequent negativity during disease course); anti-dsDNA - (persistent negativity). Disease activity was assessed by the European Consensus Lupus Activity Measurement (ECLAM). We evaluated 393 patients (anti-dsDNA +: 62.3%; anti-dsDNA ±: 13.3%; anti-dsDNA -: 24.4%). The renal involvement was significantly more frequent in anti-dsDNA + (30.2%), compared with anti-dsDNA ± and anti-dsDNA - (21.1% and 18.7%, resp.; P = 0.001). Serositis resulted significantly more frequent in anti-dsDNA - (82.3%) compared to anti-dsDNA + and anti-dsDNA ± (20.8% and 13.4%, resp.; P < 0.0001). The reduction of C4 serum levels was identified significantly more frequently in anti-dsDNA + and anti-dsDNA ± (40.0% and 44.2%, resp.) compared with anti-dsDNA - (21.8%, P = 0.005). We did not identify significant differences in the mean ECLAM values before and after modification of anti-dsDNA status (P = 0.7). Anti-dsDNA status influences the clinical and immunological features of SLE patients. Nonetheless, it does not appear to affect disease activity.

Herpes simplex virus type 2 (HSV-2) genital shedding in HSV-2-/HIV-1-co-infected women receiving effective combination antiretroviral therapy.

PubMed

Péré, Héléne; Rascanu, Aida; LeGoff, Jérome; Matta, Mathieu; Bois, Frédéric; Lortholary, Olivier; Leroy, Valériane; Launay, Odile; Bélec, Laurent

2016-03-01

The dynamics of genital shedding of HSV-2 DNA was assessed in HIV-1-infected women taking combination antiretroviral therapy (cART). HIV-1 RNA, HIV-1 DNA and HSV DNA loads were measured during 12-18 months using frozen plasma, PBMC and cervicovaginal lavage samples from 22 HIV-1-infected women, including 17 women naive for antiretroviral therapy initiating cART and 5 women with virological failure switching to a new regimen. Nineteen (86%) women were HSV-2-seropositive. Among HSV-2-/HIV-1-co-infected women, HIV-1 RNA loads showed a rapid fall from baseline after one month of cART, in parallel in paired plasma and cervicovaginal secretions. In contrast, HIV-1 DNA loads did not show significant variations from baseline up to 18 months of treatment in both systemic and genital compartments. HSV DNA was detected at least once in 12 (63%) of 19 women during follow up: HSV-2 shedding in the genital compartment was observed in 11% of cervicovaginal samples at baseline and in 16% after initiating or switching cART. Cervicovaginal HIV-1 RNA loads were strongly associated with plasma HIV-1 RNA loads over time, but not with cervicovaginal HSV DNA loads. Reactivation of genital HSV-2 replication frequently occurred despite effective cART in HSV-2-/HIV-1-co-infected women. Genital HSV-2 replication under cART does not influence cervicovaginal HIV-1 RNA or DNA shedding. © The Author(s) 2015.

Amplification of bovine papillomavirus DNA by N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet irradiation, or infection with herpes simplex virus

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

Schmitt, J.; Schlehofer, J.R.; Mergener, K.

1989-09-01

Treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or irradiation with ultraviolet light (uv254 nm) induces amplification of integrated as well as episomal sequences of bovine papillomavirus (BPV) type 1 DNA in BPV-1-transformed mouse C127 cells (i.e., ID13 cells). This is shown by filter in situ hybridization and Southern blot analysis of cellular DNA. Similarly, infection of ID13 cells with herpes simplex virus (HSV) type 1 which has been shown to be mutagenic for host cell DNA leads to amplification of BPV DNA sequences. In contrast to this induction of DNA amplification by initiators, treatment of ID13 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)more » does not result in increased synthesis of BPV DNA nor does TPA treatment modulate the initiator-induced DNA amplification. Similar to other cell systems infection with adeno-associated virus (AAV) type 2 inhibits BPV-1 DNA amplification irrespective of the inducing agent. In contrast to initiator-induced DNA amplification, treatment with carcinogen (MNNG) or tumor promoters or combination of MNNG and promoter of C127 cells prior to transformation by BPV-1 does not lead to an increase in the number of transformed foci. The induction of amplification of papillomavirus DNA by initiating agents possibly represents one of the mechanisms by which the observed synergism between papillomavirus infection and initiators in tumorigenesis might occur.« less

Unrepaired DNA damage in macrophages causes elevation of particulate matter- induced airway inflammatory response.

PubMed

Luo, Man; Bao, Zhengqiang; Xu, Feng; Wang, Xiaohui; Li, Fei; Li, Wen; Chen, Zhihua; Ying, Songmin; Shen, Huahao

2018-04-14

The inflammatory cascade can be initiated with the recognition of damaged DNA. Macrophages play an essential role in particulate matter (PM)-induced airway inflammation. In this study, we aim to explore the PM induced DNA damage response of macrophages and its function in airway inflammation. The DNA damage response and inflammatory response were assessed using bone marrow-derived macrophages following PM treatment and mouse model instilled intratracheally with PM. We found that PM induced significant DNA damage both in vitro and in vivo and simultaneously triggered a rapid DNA damage response, represented by nuclear RPA, 53BP1 and γH2AX foci formation. Genetic ablation or chemical inhibition of the DNA damage response sensor amplified the production of cytokines including Cxcl1, Cxcl2 and Ifn-γ after PM stimulation in bone marrow-derived macrophages. Similar to that seen in vitro , mice with myeloid-specific deletion of RAD50 showed higher levels of airway inflammation in response to the PM challenge, suggesting a protective role of DNA damage sensor during inflammation. These data demonstrate that PM exposure induces DNA damage and activation of DNA damage response sensor MRN complex in macrophages. Disruption of MRN complex lead to persistent, unrepaired DNA damage that causes elevated inflammatory response.

DNA Dosimetry Assessment for Sunscreen Genotoxic Photoprotection

PubMed Central

Schuch, André Passaglia; Lago, Juliana Carvalhães; Yagura, Teiti; Menck, Carlos Frederico Martins

2012-01-01

Background Due to the increase of solar ultraviolet radiation (UV) incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter. Methodology/Principal Findings The Sun Protection Factor for DNA (DNA-SPF) is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD) and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF). Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations. Conclusions/Significance The application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage. PMID:22768281

Properties of internalization factors contributing to the uptake of extracellular DNA into tumor-initiating stem cells of mouse Krebs-2 cell line.

PubMed

Dolgova, Evgeniya V; Potter, Ekaterina A; Proskurina, Anastasiya S; Minkevich, Alexandra M; Chernych, Elena R; Ostanin, Alexandr A; Efremov, Yaroslav R; Bayborodin, Sergey I; Nikolin, Valeriy P; Popova, Nelly A; Kolchanov, Nikolay A; Bogachev, Sergey S

2016-05-25

Previously, we demonstrated that poorly differentiated cells of various origins, including tumor-initiating stem cells present in the ascites form of mouse cancer cell line Krebs-2, are capable of naturally internalizing both linear double-stranded DNA and circular plasmid DNA. The method of co-incubating Krebs-2 cells with extracellular plasmid DNA (pUC19) or TAMRA-5'-dUTP-labeled polymerase chain reaction (PCR) product was used. It was found that internalized plasmid DNA isolated from Krebs-2 can be transformed into competent Escherichia coli cells. Thus, the internalization processes taking place in the Krebs-2 cell subpopulation have been analyzed and compared, as assayed by E. coli colony formation assay (plasmid DNA) and cytofluorescence (TAMRA-DNA). We showed that extracellular DNA both in the form of plasmid DNA and a PCR product is internalized by the same subpopulation of Krebs-2 cells. We found that the saturation threshold for Krebs-2 ascites cells is 0.5 μg DNA/10(6) cells. Supercoiled plasmid DNA, human high-molecular weight DNA, and 500 bp PCR fragments are internalized into the Krebs-2 tumor-initiating stem cells via distinct, non-competing internalization pathways. Under our experimental conditions, each cell may harbor 340-2600 copies of intact plasmid material, or up to 3.097 ± 0.044×10(6) plasmid copies (intact or not), as detected by quantitative PCR. The internalization dynamics of extracellular DNA, copy number of the plasmids taken up by the cells, and competition between different types of double-stranded DNA upon internalization into tumor-initiating stem cells of mouse ascites Krebs-2 have been comprehensively analyzed. Investigation of the extracellular DNA internalization into tumor-initiating stem cells is an important part of understanding their properties and possible destruction mechanisms. For example, a TAMRA-labeled DNA probe may serve as an instrument to develop a target for the therapy of cancer, aiming at elimination of tumor stem cells, as well as developing a straightforward test system for the quantification of poorly differentiated cells, including tumor-initiating stem cells, in the bulk tumor sample (biopsy or surgery specimen).

Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA

PubMed Central

Jameson, Katie H; Rostami, Nadia; Fogg, Mark J; Turkenburg, Johan P; Grahl, Anne; Murray, Heath; Wilkinson, Anthony J

2014-01-01

Chromosome copy number in cells is controlled so that the frequency of initiation of DNA replication matches that of cell division. In bacteria, this is achieved through regulation of the interaction between the initiator protein DnaA and specific DNA elements arrayed at the origin of replication. DnaA assembles at the origin and promotes DNA unwinding and the assembly of a replication initiation complex. SirA is a DnaA-interacting protein that inhibits initiation of replication in diploid Bacillus subtilis cells committed to the developmental pathway leading to formation of a dormant spore. Here we present the crystal structure of SirA in complex with the N-terminal domain of DnaA revealing a heterodimeric complex. The interacting surfaces of both proteins are α-helical with predominantly apolar side-chains packing in a hydrophobic interface. Site-directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo. Localization of GFP–SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA. The SirA interacting surface of DnaA corresponds closely to the HobA-interacting surface of DnaA from Helicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor. PMID:25041308

Assessment of DNA damage and repair efficiency in drug naïve schizophrenia using comet assay.

PubMed

Muraleedharan, Aparna; Menon, Vikas; Rajkumar, Ravi Philip; Chand, Parkash

2015-09-01

The etiology of schizophrenia continues to be confounding and elusive. Some knowledge gaps exist in the neurodegenerative theory of schizophrenia. Oxidative DNA damage and repair deficits are relevant to the mechanisms of neurodegeneration but have not been studied in drug naïve schizophrenia. The present study used the comet assay technique to study the extent of DNA damage in circulating peripheral lymphocytes of patients with drug naïve schizophrenia (n = 40) along with an age and gender matched control group (n = 40). We also assessed the DNA repair efficiency in cases following incubation in a nutrient medium. All the assayed comet parameters demonstrated significantly greater baseline DNA damage in cases in comparison to the controls except for head diameter (p < 0.001 for all significant results, p = 0.32 for head diameter). Gender, age and duration of illness (p = 0.21, 0.69 and 0.12 respectively for tail length) did not influence any of the parameters significantly. Significant decrease was noted in the comet tail length and percentage of DNA in comet tail (p < 0.001 for both) in cases following incubation suggesting that the DNA repair machinery was preserved. No difference in DNA repair efficiency was noted between the genders (p = 0.23 for tail length). Our findings confirm the presence of significant baseline DNA damage in schizophrenia even prior to the initiation of anti-psychotic treatment. Additionally, intact genomic repair efficiency was noted in this group as a whole. These results provide some evidence for oxidative DNA damage as molecular link underpinning neurodegeneration in drug naïve schizophrenia. Copyright © 2015 Elsevier Ltd. All rights reserved.

DNA replication depends on photosynthetic electron transport in cyanobacteria.

PubMed

Ohbayashi, Ryudo; Watanabe, Satoru; Kanesaki, Yu; Narikawa, Rei; Chibazakura, Taku; Ikeuchi, Masahiko; Yoshikawa, Hirofumi

2013-07-01

The freshwater cyanobacterium Synechococcus elongatus PCC 7942 exhibits light-dependent growth. Although it has been reported that DNA replication also depends on light irradiation in S. elongatus 7942, the involvement of the light in the regulation of DNA replication remains unclear. To elucidate the regulatory pathway of DNA replication by light, we studied the effect of several inhibitors, including two electron transport inhibitors, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), on DNA replication in S. elongatus 7942. DCMU inhibited only DNA replication initiation, whereas DBMIB blocked both the initiation and progression of DNA replication. These results suggest that DNA replication depends on the photosynthetic electron transport activity and initiation and progression of DNA replication are regulated in different ways. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Forensic individual age estimation with DNA: From initial approaches to methylation tests.

PubMed

Freire-Aradas, A; Phillips, C; Lareu, M V

2017-07-01

Individual age estimation is a key factor in forensic science analysis that can provide very useful information applicable to criminal, legal, and anthropological investigations. Forensic age inference was initially based on morphological inspection or radiography and only later began to adopt molecular approaches. However, a lack of accuracy or technical problems hampered the introduction of these DNA-based methodologies in casework analysis. A turning point occurred when the epigenetic signature of DNA methylation was observed to gradually change during an individual´s lifespan. In the last four years, the number of publications reporting DNA methylation age-correlated changes has gradually risen and the forensic community now has a range of age methylation tests applicable to forensic casework. Most forensic age predictor models have been developed based on blood DNA samples, but additional tissues are now also being explored. This review assesses the most widely adopted genes harboring methylation sites, detection technologies, statistical age-predictive analyses, and potential causes of variation in age estimates. Despite the need for further work to improve predictive accuracy and establishing a broader range of tissues for which tests can analyze the most appropriate methylation sites, several forensic age predictors have now been reported that provide consistency in their prediction accuracies (predictive error of ±4 years); this makes them compelling tools with the potential to contribute key information to help guide criminal investigations. Copyright © 2017 Central Police University.

DNA methylation in lung tissues of mouse offspring exposed in utero to polycyclic aromatic hydrocarbons.

PubMed

Fish, Trevor J; Benninghoff, Abby D

2017-11-01

Polycyclic aromatic hydrocarbons (PAHs) comprise an important class of environmental pollutants that are known to cause lung cancer in animals and are suspected lung carcinogens in humans. Moreover, evidence from cell-based studies points to PAHs as modulators of the epigenome. The objective of this work was to assess patterns of genome-wide DNA methylation in lung tissues of adult offspring initiated in utero with the transplacental PAH carcinogens dibenzo [def,p]chrysene (DBC) or benzo [a]pyrene (BaP). Genome-wide methylation patterns for normal (not exposed), normal adjacent and lung tumor tissues obtained from adult offspring were determined using methylated DNA immunoprecipitation (MeDIP) with the NimbleGen mouse DNA methylation CpG island array. Lung tumor incidence in 45-week old mice initiated with BaP was 32%, much lower than that of the DBC-exposed offspring at 96%. Also, male offspring appeared more susceptible to BaP as compared to females. Distinct patterns of DNA methylation were associated with non-exposed, normal adjacent and adenocarcinoma lung tissues, as determined by principal components, hierarchical clustering and gene ontology analyses. From these methylation profiles, a set of genes of interest was identified that includes potential important targets for epigenetic modification during the process of lung tumorigenesis in animals exposed to environmental PAHs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Prediction of cellular radiosensitivity from DNA damage induced by gamma-rays and carbon ion irradiation in canine tumor cells.

PubMed

Wada, Seiichi; Van Khoa, Tran; Kobayashi, Yasuhiko; Funayama, Tomoo; Ogihara, Kikumi; Ueno, Shunji; Ito, Nobuhiko

2005-11-01

Diseases of companion animals are shifting from infectious diseases to neoplasms (cancer), and since radiation therapy is one of the effective choices available for cancer treatment, the application of radiotherapy in veterinary medicine is likely to increase. However tumor tissues have different radiosensitivities, and therefore it is important to determine the intrinsic radiosensitivity of tumors in individual patients in advance of radiotherapy. We have studied the relationship between the surviving cell fraction measured by a clonogenic assay and DNA double strand breaks detected by a comet assay under neutral conditions in three canine tumor cell lines, after gamma-ray and carbon ion irradiation. In all the cell lines, cell death assessed by the clonogenic assay was much higher following irradiation with carbon ions than with gamma-rays. The initial and residual (4 hr) DNA damage due to gamma-ray and carbon ion irradiation were higher in a radiosensitive cell line than in a radioresistant cell line. The surviving cell fraction at 2 Gy (SF2) showed a tendency for correlation with both the initial and residual DNA damage. In particular, the residual damage per Gy was significantly correlated with SF2, regardless of the type of radiation. This indicates that cellular radiosensitivity can be predicted by detection of radiation-induced residual DNA damage.

In vivo gamma-rays induced initial DNA damage and the effect of famotidine in mouse leukocytes as assayed by the alkaline comet assay.

PubMed

Mozdarani, Hossein; Nasirian, Borzo; Haeri, S Abolghasem

2007-03-01

Ionizing radiation induces a variety of lesions in DNA, each of which can be used as a bio-indicator for biological dosimetry or the study of the radioprotective effects of substances. To assess gamma ray-induced DNA damage in vivo in mouse leukocytes at various doses and the effect of famotidine, blood was collected from Balb/c male mice after irradiation with 4 Gy gamma-rays at different time intervals post-irradiation. To assess the response, mice were irradiated with doses of gamma-rays at 1 to 4 Grays. Famotidine was injected intra-peritoneally (i.p) at a dose of 5 mg/kg at various time intervals before irradiation. Four slides were prepared from each sample and alkaline comet assay was performed using standard protocols. Results obtained show that radiation significantly increases DNA damage in leukocytes in a dose dependent manner (p < 0.01) when using appropriate sampling time after irradiation, because increasing sampling time after irradiation resulted in a time dependent disappearance of DNA damage. Treatment with only 5 mg/kg famotidine before 4 Gy irradiation led to almost 50% reduction in DNA damage when compared with those animals which received radiation alone. The radioprotective capability of famotidine might be attributed to radical scavenging properties and an anti-oxidation mechanism.

Chromosome damage evolution after low and high LET irradiation

NASA Astrophysics Data System (ADS)

Andreev, Sergey; Eidelman, Yuri

Ionizing radiation induces DNA and chromatin lesions which are converted to chromosome lesions detected in the first post-irradiation mitosis by classic cytogenetic techniques as chromosomal aberrations (CAs). These techniques allow to monitor also delayed aberrations observed after many cell generations post-irradiation - the manifestation of chromosomal instability phenotype (CIN). The problem discussed is how to predict time evolution from initial to delayed DNA/chromosome damage. To address this question, in the present work a mechanistic model of CIN is elaborated which integrates pathways of (*) DNA damage induction and its conversion to chromosome lesions (aberrations), (**) lesion transmission and generation through cell cycles. Delayed aberrations in subsequent cycles are formed in the model owing to two pathways, DNA damage generation de novo as well as CA transmission from previous cycles. DNA damage generation rate is assumed to consist of bystander and non-bystander components. Bystander signals impact all cells roughly equally, whereas non-bystander DSB generation rate differs for the descendants of unirradiated and irradiated cells. Monte Carlo simulation of processes underlying CIN allows to predict the time evolution of initial radiation-induced damage - kinetics curve for delayed unstable aberrations (dicentrics) together with dose response and RBE as a function of time after high vs low LET irradiation. The experimental data for radiation-induced CIN in TK6 lymphoblastoid cells and human lymphocytes irradiated with low (gamma) and high (Fe, C) LET radiation are analyzed on the basis of the proposed model. One of the conclusions is that without bystander signaling, just taking into account the initial DNA damage and non-bystander DSB generation, it is impossible to describe the available experimental data for high-LET-induced CIN. The exact contribution of bystander effects for high vs low LET remains unknown, but the relative contribution may be assessed at large times after initial acute irradiation. RBE for delayed aberrations depends on LET, time and cell line, which probably reflects a genetic background for bystander component. The proposed modeling approach creates a basis for integration of complex network of bystander/inflammatory signaling in systems-level platform for quantification of radiation induced CIN.

Crystal Structures of the E. coli Transcription Initiation Complexes with a Complete Bubble

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

Zuo, Yuhong; Steitz, Thomas A.

2015-05-01

During transcription initiation, RNA polymerase binds to promoter DNA to form an initiation complex containing a DNA bubble and enters into abortive cycles of RNA synthesis before escaping the promoter to transit into the elongation phase for processive RNA synthesis. Here we present the crystal structures of E. coli transcription initiation complexes containing a complete transcription bubble and de novo synthesized RNA oligonucleotides at about 6-Å resolution. The structures show how RNA polymerase recognizes DNA promoters that contain spacers of different lengths and reveal a bridging interaction between the 5'-triphosphate of the nascent RNA and the σ factor that maymore » function to stabilize the short RNA-DNA hybrids during the early stage of transcription initiation. The conformation of the RNA oligonucleotides and the paths of the DNA strands in the complete initiation complexes provide insights into the mechanism that controls both the abortive and productive RNA synthesis.« less

Mechanisms and regulation of DNA replication initiation in eukaryotes

PubMed Central

Parker, Matthew W.; Botchan, Michael R.; Berger, James M.

2017-01-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a given cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the Origin Recognition Complex (ORC), and subsequent activation of the helicase by incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms. PMID:28094588

Mechanisms and regulation of DNA replication initiation in eukaryotes.

PubMed

Parker, Matthew W; Botchan, Michael R; Berger, James M

2017-04-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis.

PubMed

Washington, Tracy A; Smith, Janet L; Grossman, Alan D

2017-10-01

DnaA is the widely conserved bacterial AAA+ ATPase that functions as both the replication initiator and a transcription factor. In many organisms, DnaA controls expression of its own gene and likely several others during growth and in response to replication stress. To evaluate the effects of DnaA on gene expression, separate from its role in replication initiation, we analyzed changes in mRNA levels in Bacillus subtilis cells with and without dnaA, using engineered strains in which dnaA is not essential. We found that dnaA was required for many of the changes in gene expression in response to replication stress. We also found that dnaA indirectly affected expression of several regulons during growth, including those controlled by the transcription factors Spo0A, AbrB, PhoP, SinR, RemA, Rok and YvrH. These effects were largely mediated by the effects of DnaA on expression of sda. DnaA activates transcription of sda, and Sda inhibits histidine protein kinases required for activation of the transcription factor Spo0A. We also found that loss of dnaA caused a decrease in the development of genetic competence. Together, our results indicate that DnaA plays an important role in modulating cell physiology, separate from its role in replication initiation. © 2017 John Wiley & Sons Ltd.

Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription.

PubMed

Kogoma, T

1997-06-01

Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored.

Cypermethrin and lambda-cyhalothrin induced in vivo alterations in nucleic acids and protein contents in a freshwater catfish, Clarias batrachus (Linnaeus; Family-Clariidae).

PubMed

Kumar, Amit; Sharma, Bechan; Pandey, Ravi S

2009-08-01

The fresh water fish, Clarias batrachus, were exposed to sub-acute concentrations of cypermethrin and lambda-cyhalothrin for 96 h to assess their impact on the levels of nucleic acids and protein in different organs of fish. DNA content was found almost unchanged with a single exception of liver, which showed significant increment in the levels of DNA in response to the separate treatments of both compounds. Both RNA and protein contents declined in brain, liver, and muscle while sharp increase was observed in gills. However, in kidney, RNA contents depicted significant enhancement only at higher concentrations, with initial decline at lower concentrations. The trends of alterations in RNA/DNA and protein/DNA ratios were quite similar to the corresponding results explained above for RNA and protein. The results clearly indicated that both of these pyrethroids exerted their effects at transcriptional and translational levels while DNA synthesis was found to be unaffected by these compounds with an exception of liver.

Application of virtual screening and molecular dynamics for the analysis of selectivity of inhibitors of HU proteins targeted to the DNA-recognition site

NASA Astrophysics Data System (ADS)

Talyzina, A. A.; Agapova, Yu. K.; Podshivalov, D. D.; Timofeev, V. I.; Sidorov-Biryukov, D. D.; Rakitina, T. V.

2017-11-01

DNA-Binding HU proteins are essential for the maintenance of genomic DNA supercoiling and compaction in prokaryotic cells and are promising pharmacological targets for the design of new antibacterial agents. The virtual screening for low-molecular-weight compounds capable of specifically interacting with the DNA-recognition loop of the HU protein from the mycoplasma Spiroplasma melliferum was performed. The ability of the initially selected ligands to form stable complexes with the protein target was assessed by molecular dynamics simulation. One compound, which forms an unstable complex, was eliminated by means of a combination of computational methods, resulting in a decrease in the number of compounds that will pass to the experimental test phase. This approach can be used to solve a wide range of problems related to the search for and validation of low-molecular-weight inhibitors specific for a particular protein target.

Generation and Repair of AID-initiated DNA Lesions in B Lymphocytes

PubMed Central

Chen, Zhangguo; Wang, Jing H.

2014-01-01

Activation-induced deaminase (AID) initiates the secondary antibody diversification process in B lymphocytes. In mammalian B cells, this process includes somatic hypermutation (SHM) and class switch recombination (CSR), both of which require AID. AID induces U:G mismatch lesions in DNA that are subsequently converted into point mutations or DNA double stranded breaks during SHM/CSR. In a physiological context, AID targets immunoglobulin (Ig) loci to mediate SHM/CSR. However, recent studies reveal genome-wide access of AID to numerous non-Ig loci. Thus, AID poses a threat to the genome of B cells if AID-initiated DNA lesions cannot be properly repaired. In this review, we focus on the molecular mechanisms that regulate the specificity of AID targeting and the repair pathways responsible for processing AID-initiated DNA lesions. PMID:24748462

Ultrasensitive and selective signal-on electrochemical DNA detection via exonuclease III catalysis and hybridization chain reaction amplification.

PubMed

Ren, Wang; Gao, Zhong Feng; Li, Nian Bing; Luo, Hong Qun

2015-01-15

This work reported a novel, ultrasensitive, and selective platform for electrochemical detection of DNA, employing an integration of exonuclease III (Exo-III) assisted target recycling and hybridization chain reaction (HCR) for the dual signal amplification strategy. The hairpin capture probe DNA (C-DNA) with an Exo-III 3' overhang end was self-assembled on a gold electrode. In the presence of target DNA (T-DNA), C-DNA hybridized with the T-DNA to form a duplex region, exposing its 5' complementary sequence (initiator). Exo-III was applied to selectively digest duplex region from its 3-hydroxyl termini until the duplex was fully consumed, leaving the remnant initiator. The intact T-DNA spontaneously dissociated from the structure and then initiated the next hybridization process as a result of catalysis of the Exo-III. HCR event was triggered by the initiator and two hairpin helper signal probes labeled with methylene blue, facilitating the polymerization of oligonucleotides into a long nicked dsDNA molecule. The numerous exposed remnant initiators can trigger more HCR events. Because of integration of dual signal amplification and the specific HCR process reaction, the resultant sensor showed a high sensitivity for the detection of the target DNA in a linear range from 1.0 fM to 1.0 nM, and a detection limit as low as 0.2 fM. The proposed dual signal amplification strategy provides a powerful tool for detecting different sequences of target DNA by changing the sequence of capture probe and signal probes, holding a great potential for early diagnosis in gene-related diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Base Excision Repair of Tandem Modifications in a Methylated CpG Dinucleotide*

PubMed Central

Sassa, Akira; Çağlayan, Melike; Dyrkheeva, Nadezhda S.; Beard, William A.; Wilson, Samuel H.

2014-01-01

Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of altered in-tandem CpG dinucleotides; e.g. Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3′-flanking 8-oxoG. In contrast, the OGG1-initiated BER pathway is blocked due to the 5′-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase β activities. Furthermore, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide. PMID:24695738

Assessment of sperm DNA in patients submitted the assisted reproduction technology procedures.

PubMed

Tsuribe, Patrícia Miyuki; Lima Neto, João Ferreira; Golim, Marjorie de Assis; Dell'Aqua, Camila de Paula Freitas; Issa, João Paulo; Gobbo, Carlos Alberto Monte

2016-03-01

This study aimed to produce data on sperm quality while maintaining the integrity of sperm DNA samples taken from patients submitted to in vitro fertilization (IVF) procedures at our center, and determine whether increased levels of histones were associated with sperm DNA damage and decreased fertilization, cleavage, and pregnancy rates. Such findings might shed light on the physiology and outcomes of pregnancy. Semen samples from 27 patients divided into two groups were analyzed. The case group included individuals offered IVF; the control group had subjects with normal spermograms. Sperm DNA structure was assessed through phosphorylated histone H2AX analysis by flow cytometry. The patients with altered sperm parameters had more histones in sperm chromatin than the individuals with normal sperm parameters. Results indicated that increased levels of histone in sperm chromatin do not affect embryo production, but affect the cleavage rate, embryo quality, and might thus reduce pregnancy rates. The integrity of the paternal genome is of paramount importance in the initiation and maintenance of a viable pregnancy in patients treated with assisted reproduction technology procedures. Further studies on sperm diagnostic tests at a nuclear level might improve the treatment offered to infertile couples.

HumanMethylation450K Array–Identified Biomarkers Predict Tumour Recurrence/Progression at Initial Diagnosis of High-risk Non-muscle Invasive Bladder Cancer

PubMed Central

Kitchen, Mark O; Bryan, Richard T; Emes, Richard D; Luscombe, Christopher J; Cheng, KK; Zeegers, Maurice P; James, Nicholas D; Gommersall, Lyndon M; Fryer, Anthony A

2018-01-01

Background: High-risk non-muscle invasive bladder cancer (HR-NMIBC) is a clinically unpredictable disease. Despite clinical risk estimation tools, many patients are undertreated with intra-vesical therapies alone, whereas others may be over-treated with early radical surgery. Molecular biomarkers, particularly DNA methylation, have been reported as predictive of tumour/patient outcomes in numerous solid organ and haematologic malignancies; however, there are few reports in HR-NMIBC and none using genome-wide array assessment. We therefore sought to identify novel DNA methylation markers of HR-NMIBC clinical outcomes that might predict tumour behaviour at initial diagnosis and help guide patient management. Patients and methods: A total of 21 primary initial diagnosis HR-NMIBC tumours were analysed by Illumina HumanMethylation450 BeadChip arrays and subsequently bisulphite Pyrosequencing. In all, 7 had not recurred at 1 year after resection and 14 had recurred and/or progressed despite intra-vesical BCG. A further independent cohort of 32 HR-NMIBC tumours (17 no recurrence and 15 recurrence and/or progression despite BCG) were also assessed by bisulphite Pyrosequencing. Results: Array analyses identified 206 CpG loci that segregated non-recurrent HR-NMIBC tumours from clinically more aggressive recurrence/progression tumours. Hypermethylation of CpG cg11850659 and hypomethylation of CpG cg01149192 in combination predicted HR-NMIBC recurrence and/or progression within 1 year of diagnosis with 83% sensitivity, 79% specificity, and 83% positive and 79% negative predictive values. Conclusions: This is the first genome-wide DNA methylation analysis of a unique HR-NMIBC tumour cohort encompassing known 1-year clinical outcomes. Our analyses identified potential novel epigenetic markers that could help guide individual patient management in this clinically unpredictable disease. PMID:29343995

Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer.

PubMed

Diede, Scott J; Yao, Zizhen; Keyes, C Chip; Tyler, Ashlee E; Dey, Joyoti; Hackett, Christopher S; Elsaesser, Katrina; Kemp, Christopher J; Neiman, Paul E; Weiss, William A; Olson, James M; Tapscott, Stephen J

2013-12-01

Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development.

Monte Carlo approach in assessing damage in higher order structures of DNA

NASA Technical Reports Server (NTRS)

Chatterjee, A.; Schmidt, J. B.; Holley, W. R.

1994-01-01

We have developed a computer monitor of nuclear DNA in the form of chromatin fibre. The fibres are modeled as a ideal solenoid consisting of twenty helical turns with six nucleosomes per turn. The chromatin model, in combination with are Monte Carlo theory of radiation damage induces by charged particles, based on general features of tack structure and stopping power theory, has been used to evaluate the influence of DNA structure on initial damage. An interesting has emerged from our calculations. Our calculated results predict the existence of strong spatial correlations in damage sites associated with the symmetries in the solenoidal model. We have calculated spectra of short fragments of double stranded DNA produced by multiple double strand breaks induced by both high and low LET radiation. The spectra exhibit peaks at multiples of approximately 85 base pairs (the nucleosome periodicity), and approximately 1000 base pairs (solenoid periodicity). Preliminary experiments to investigate the fragment distributions from irradiated DNA, made by B. Rydberg at Lawrence Berkeley Laboratory, confirm the existence of short DNA fragments and are in substantial agreement with the predictions of our theory.

Chapter 23: International Standard reagents for harmonization of HPV serology and DNA assays--an update.

PubMed

Pagliusi, Sonia R; Dillner, Joakim; Pawlita, Michael; Quint, Wim G V; Wheeler, Cosette M; Ferguson, M

2006-08-31

International reference materials such as International Standard reagents facilitate quality assurance of essential biopharmaceutical products and related in vitro diagnostic tests. Standardization of antibody and DNA measurements and harmonization of laboratory procedures are key to the success of cancer prevention strategies through screening methods as well as for development and implementation of vaccination against the human papillomavirus (HPV). The WHO supported the preparation and initial analysis of a panel of candidate serological and DNA reference reagents aimed at facilitating inter-laboratory comparisons and detection of HPV worldwide. Two international collaborative studies assessed the performance of various HPV antibody and HPV-DNA detection assays and examined the feasibility of generating HPV antibody and DNA standard reagents. These studies showed that improvement in performance and comparability of assays is urgently needed and that the use of the same International Standard reference reagent could significantly improve performance and comparability. It is hoped that the establishment of International Units and International Standards for HPV antibody and DNA analysis will be pursued with high priority.

DNA reactivity as a mode of action and its relevance to cancer risk assessment.

PubMed

Preston, R Julian

2013-02-01

The ability of a chemical to induce mutations has long been a driver in the cancer risk assessment process. The default strategy has been that mutagenic chemicals demonstrate linear cancer dose responses, especially at low exposure levels. In the absence of additional confounding information, this is a reasonable approach, because risk assessment is appropriately considered as being protective of human health. The concept of mode of action has allowed for an opportunity to move off this default position; mutagenicity is now not considered as the driver but rather the mode of action is. In a more precise way, it is the set of key events that define a mode of action that is fundamental in defining the shape of a cancer dose response. A key event is an informative bioindicator of the cancer response and as such should be predictive of the tumor response, at least in a qualitative way. A clear example of the use of key events in cancer risk assessment is for DNA reactive chemicals. A series of such key events is initiated by the production of DNA damage in target cells from direct interaction of the chemical with DNA leading to the production of mutations by misreplication that results in enhanced cell replication. This enhanced cell replication eventually leads to the development of preneoplastic cells and ultimately overt neoplasms. The response of each of these key events to dose of the chemical can inform the cancer dose-response curve shape. Thus, the dose-response curve for any DNA-reactive chemical can be predicted from knowledge of its mode of action and the behavior of the induced key events.

Re-wiring of energy metabolism promotes viability during hyperreplication stress in E. coli

PubMed Central

Campion, Christopher; Weimann, Allan

2017-01-01

Chromosome replication in Escherichia coli is initiated by DnaA. DnaA binds ATP which is essential for formation of a DnaA-oriC nucleoprotein complex that promotes strand opening, helicase loading and replisome assembly. Following initiation, DnaAATP is converted to DnaAADP primarily by the Regulatory Inactivation of DnaA process (RIDA). In RIDA deficient cells, DnaAATP accumulates leading to uncontrolled initiation of replication and cell death by accumulation of DNA strand breaks. Mutations that suppress RIDA deficiency either dampen overinitiation or permit growth despite overinitiation. We characterize mutations of the last group that have in common that distinct metabolic routes are rewired resulting in the redirection of electron flow towards the cytochrome bd-1. We propose a model where cytochrome bd-1 lowers the formation of reactive oxygen species and hence oxidative damage to the DNA in general. This increases the processivity of replication forks generated by overinitiation to a level that sustains viability. PMID:28129339

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

PubMed

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

2002-03-01

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

Promoter binding, initiation, and elongation by bacteriophage T7 RNA polymerase. A single-molecule view of the transcription cycle.

PubMed

Skinner, Gary M; Baumann, Christoph G; Quinn, Diana M; Molloy, Justin E; Hoggett, James G

2004-01-30

A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k(off)= 2.9 s(-1)) of T7 RNAP and promoter DNA, the transition to the elongation mode (k(for) = 0.36 s(-1)), and the processive synthesis (k(pol) = 43 nt s(-1)) and release of a gene-length RNA transcript ( approximately 1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k(off) > k(for)), identifying a rate-limiting step between promoter DNA binding and promoter escape.

Methanol exposure does not produce oxidatively damaged DNA in lung, liver or kidney of adult mice, rabbits or primates

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

McCallum, Gordon P.; Siu, Michelle; Sweeting, J. Nicole

2011-01-15

In vitro and in vivo genotoxicity tests indicate methanol (MeOH) is not mutagenic, but carcinogenic potential has been claimed in one controversial long-term rodent cancer bioassay that has not been replicated. To determine whether MeOH could indirectly damage DNA via reactive oxygen species (ROS)-mediated mechanisms, we treated male CD-1 mice, New Zealand white rabbits and cynomolgus monkeys with MeOH (2.0 g/kg ip) and 6 h later assessed oxidative damage to DNA, measured as 8-oxo-2'-deoxyguanosine (8-oxodG) by HPLC with electrochemical detection. We found no MeOH-dependent increases in 8-oxodG in lung, liver or kidney of any species. Chronic treatment of CD-1 micemore » with MeOH (2.0 g/kg ip) daily for 15 days also did not increase 8-oxodG levels in these organs. These results were corroborated in DNA repair-deficient oxoguanine glycosylase 1 (Ogg1) knockout (KO) mice, which accumulated 8-oxodG in lung, kidney and liver with age, but exhibited no increase following MeOH, despite a 2-fold increase in renal 8-oxodG in Ogg1 KO mice following treatment with a ROS-initiating positive control, the renal carcinogen potassium bromate (KBrO{sub 3}; 100 mg/kg ip). These observations suggest that MeOH exposure does not promote the accumulation of oxidatively damaged DNA in lung, kidney or liver, and that environmental exposure to MeOH is unlikely to initiate carcinogenesis in these organs by DNA oxidation.« less

Genotoxicity Assessment of Drinking Water Disinfection Byproducts by DNA Damage and Repair Pathway Profiling Analysis.

PubMed

Lan, Jiaqi; Rahman, Sheikh Mokhlesur; Gou, Na; Jiang, Tao; Plewa, Micheal J; Alshawabkeh, Akram; Gu, April Z

2018-06-05

Genotoxicity is considered a major concern for drinking water disinfection byproducts (DBPs). Of over 700 DBPs identified to date, only a small number has been assessed with limited information for DBP genotoxicity mechanism(s). In this study, we evaluated genotoxicity of 20 regulated and unregulated DBPs applying a quantitative toxicogenomics approach. We used GFP-fused yeast strains that examine protein expression profiling of 38 proteins indicative of all known DNA damage and repair pathways. The toxicogenomics assay detected genotoxicity potential of these DBPs that is consistent with conventional genotoxicity assays end points. Furthermore, the high-resolution, real-time pathway activation and protein expression profiling, in combination with clustering analysis, revealed molecular level details in the genotoxicity mechanisms among different DBPs and enabled classification of DBPs based on their distinct DNA damage effects and repair mechanisms. Oxidative DNA damage and base alkylation were confirmed to be the main molecular mechanisms of DBP genotoxicity. Initial exploration of QSAR modeling using moleular genotoxicity end points (PELI) suggested that genotoxicity of DBPs in this study was correlated with topological and quantum chemical descriptors. This study presents a toxicogenomics-based assay for fast and efficient mechanistic genotoxicity screening and assessment of a large number of DBPs. The results help to fill in the knowledge gap in the understanding of the molecular mechanisms of DBP genotoxicity.

Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis*S⃞

PubMed Central

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-01-01

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only ∼100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain. PMID:18977760

Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis.

PubMed

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-12-26

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.

Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase.

PubMed

Lõoke, Marko; Maloney, Michael F; Bell, Stephen P

2017-02-01

Activation of the Mcm2-7 replicative DNA helicase is the committed step in eukaryotic DNA replication initiation. Although Mcm2-7 activation requires binding of the helicase-activating proteins Cdc45 and GINS (forming the CMG complex), an additional protein, Mcm10, drives initial origin DNA unwinding by an unknown mechanism. We show that Mcm10 binds a conserved motif located between the oligonucleotide/oligosaccharide fold (OB-fold) and A subdomain of Mcm2. Although buried in the interface between these domains in Mcm2-7 structures, mutations predicted to separate the domains and expose this motif restore growth to conditional-lethal MCM10 mutant cells. We found that, in addition to stimulating initial DNA unwinding, Mcm10 stabilizes Cdc45 and GINS association with Mcm2-7 and stimulates replication elongation in vivo and in vitro. Furthermore, we identified a lethal allele of MCM10 that stimulates initial DNA unwinding but is defective in replication elongation and CMG binding. Our findings expand the roles of Mcm10 during DNA replication and suggest a new model for Mcm10 function as an activator of the CMG complex throughout DNA replication. © 2017 Lõoke et al.; Published by Cold Spring Harbor Laboratory Press.

Changes in DnaA-dependent gene expression contribute to the transcriptional and developmental response of Bacillus subtilis to manganese limitation in Luria-Bertani medium.

PubMed

Hoover, Sharon E; Xu, Weihong; Xiao, Wenzhong; Burkholder, William F

2010-08-01

The SOS response to DNA damage in bacteria is a well-known component of the complex transcriptional responses to genotoxic environmental stresses such as exposure to reactive oxygen species, alkylating agents, and many of the antibiotics targeting DNA replication. However, bacteria such as Bacillus subtilis also respond to conditions that perturb DNA replication via a transcriptional response mediated by the replication initiation protein DnaA. In addition to regulating the initiation of DNA replication, DnaA directly regulates the transcription of specific genes. Conditions that perturb DNA replication can trigger the accumulation of active DnaA, activating or repressing the transcription of genes in the DnaA regulon. We report here that simply growing B. subtilis in LB medium altered DnaA-dependent gene expression in a manner consistent with the accumulation of active DnaA and that this was part of a general transcriptional response to manganese limitation. The SOS response to DNA damage was not induced under these conditions. One of the genes positively regulated by DnaA in Bacillus subtilis encodes a protein that inhibits the initiation of sporulation, Sda. Sda expression was induced as cells entered stationary phase in LB medium but not in LB medium supplemented with manganese, and the induction of Sda inhibited sporulation-specific gene expression and the onset of spore morphogenesis. In the absence of Sda, manganese-limited cells initiated spore development but failed to form mature spores. These data highlight that DnaA-dependent gene expression may influence the response of bacteria to a range of environmental conditions, including conditions that are not obviously associated with genotoxic stress.

Changes in DnaA-Dependent Gene Expression Contribute to the Transcriptional and Developmental Response of Bacillus subtilis to Manganese Limitation in Luria-Bertani Medium▿ †

PubMed Central

Hoover, Sharon E.; Xu, Weihong; Xiao, Wenzhong; Burkholder, William F.

2010-01-01

The SOS response to DNA damage in bacteria is a well-known component of the complex transcriptional responses to genotoxic environmental stresses such as exposure to reactive oxygen species, alkylating agents, and many of the antibiotics targeting DNA replication. However, bacteria such as Bacillus subtilis also respond to conditions that perturb DNA replication via a transcriptional response mediated by the replication initiation protein DnaA. In addition to regulating the initiation of DNA replication, DnaA directly regulates the transcription of specific genes. Conditions that perturb DNA replication can trigger the accumulation of active DnaA, activating or repressing the transcription of genes in the DnaA regulon. We report here that simply growing B. subtilis in LB medium altered DnaA-dependent gene expression in a manner consistent with the accumulation of active DnaA and that this was part of a general transcriptional response to manganese limitation. The SOS response to DNA damage was not induced under these conditions. One of the genes positively regulated by DnaA in Bacillus subtilis encodes a protein that inhibits the initiation of sporulation, Sda. Sda expression was induced as cells entered stationary phase in LB medium but not in LB medium supplemented with manganese, and the induction of Sda inhibited sporulation-specific gene expression and the onset of spore morphogenesis. In the absence of Sda, manganese-limited cells initiated spore development but failed to form mature spores. These data highlight that DnaA-dependent gene expression may influence the response of bacteria to a range of environmental conditions, including conditions that are not obviously associated with genotoxic stress. PMID:20511500

Deciphering DNA replication dynamics in eukaryotic cell populations in relation with their averaged chromatin conformations

NASA Astrophysics Data System (ADS)

Goldar, A.; Arneodo, A.; Audit, B.; Argoul, F.; Rappailles, A.; Guilbaud, G.; Petryk, N.; Kahli, M.; Hyrien, O.

2016-03-01

We propose a non-local model of DNA replication that takes into account the observed uncertainty on the position and time of replication initiation in eukaryote cell populations. By picturing replication initiation as a two-state system and considering all possible transition configurations, and by taking into account the chromatin’s fractal dimension, we derive an analytical expression for the rate of replication initiation. This model predicts with no free parameter the temporal profiles of initiation rate, replication fork density and fraction of replicated DNA, in quantitative agreement with corresponding experimental data from both S. cerevisiae and human cells and provides a quantitative estimate of initiation site redundancy. This study shows that, to a large extent, the program that regulates the dynamics of eukaryotic DNA replication is a collective phenomenon that emerges from the stochastic nature of replication origins initiation.

Long-lasting alterations to DNA methylation and ncRNAs could underlie the effects of fetal alcohol exposure in mice

PubMed Central

Laufer, Benjamin I.; Mantha, Katarzyna; Kleiber, Morgan L.; Diehl, Eric J.; Addison, Sean M. F.; Singh, Shiva M.

2013-01-01

SUMMARY Fetal alcohol spectrum disorders (FASDs) are characterized by life-long changes in gene expression, neurodevelopment and behavior. What mechanisms initiate and maintain these changes are not known, but current research suggests a role for alcohol-induced epigenetic changes. In this study we assessed alterations to adult mouse brain tissue by assaying DNA cytosine methylation and small noncoding RNA (ncRNA) expression, specifically the microRNA (miRNA) and small nucleolar RNA (snoRNA) subtypes. We found long-lasting alterations in DNA methylation as a result of fetal alcohol exposure, specifically in the imprinted regions of the genome harboring ncRNAs and sequences interacting with regulatory proteins. A large number of major nodes from the identified networks, such as Pten signaling, contained transcriptional repressor CTCF-binding sites in their promoters, illustrating the functional consequences of alcohol-induced changes to DNA methylation. Next, we assessed ncRNA expression using two independent array platforms and quantitative PCR. The results identified 34 genes that are targeted by the deregulated miRNAs. Of these, four (Pten, Nmnat1, Slitrk2 and Otx2) were viewed as being crucial in the context of FASDs given their roles in the brain. Furthermore, ∼20% of the altered ncRNAs mapped to three imprinted regions (Snrpn-Ube3a, Dlk1-Dio3 and Sfmbt2) that showed differential methylation and have been previously implicated in neurodevelopmental disorders. The findings of this study help to expand on the mechanisms behind the long-lasting changes in the brain transcriptome of FASD individuals. The observed changes could contribute to the initiation and maintenance of the long-lasting effect of alcohol. PMID:23580197

The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage

PubMed Central

Fu, Haiqing; Martin, Melvenia M.; Regairaz, Marie; Huang, Liang; You, Yang; Lin, Chi-Mei; Ryan, Michael; Kim, RyangGuk; Shimura, Tsutomu; Pommier, Yves; Aladjem, Mirit I.

2015-01-01

The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81 deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81 deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins. PMID:25879486

Fluorescence detection of thrombin using autocatalytic strand displacement cycle reaction and a dual-aptamer DNA sandwich assay.

PubMed

Niu, Shuyan; Qu, Lijing; Zhang, Qing; Lin, Jiehua

2012-02-15

A sensitive and specific sandwich assay for the detection of thrombin is described. Two affiliative aptamers were used to increase the assay specificity through sandwich recognition. Recognition DNA loaded on gold nanoparticles (AuNPs) partially hybridized with the initiator DNA, which was displaced by surviving DNA. After the initiator DNA was released into the solution, one hairpin structure was opened, which in turn opened another hairpin structure. The initiator DNA was displaced and released into the solution again by another hairpin structure because of the hybridized reaction. Then the released initiator DNA initiated another autocatalytic strand displacement reaction. A sophisticated network of three such duplex formation cycles was designed to amplify the fluorescence signal. Other proteins, such as bovine serum albumin and lysozyme, did not interfere with the detection of thrombin. This approach enables rapid and specific thrombin detection with reduced costs and minimized material consumption compared with traditional assay processes. The detection limit of thrombin was as low as 4.3 × 10⁻¹³ M based on the AuNP amplification and the autocatalytic strand displacement cycle reaction. This method could be used in biological samples with excellent selectivity. Copyright © 2011 Elsevier Inc. All rights reserved.

[The effects of TorR protein on initiation of DNA replication in Escherichia coli].

PubMed

Yuan, Yao; Jiaxin, Qiao; Jing, Li; Hui, Li; Morigen, Morigen

2015-03-01

The two-component systems, which could sense and respond to environmental changes, widely exist in bacteria as a signal transduction pathway. The bacterial CckA/CtrA, ArcA/ArcB and PhoP/PhoQ two-component systems are associated with initiation of DNA replication and cell division, however, the effects of the TorS/TorR system on cell cycle and DNA replication remains unknown. The TorS/TorR system in Escherichia coli can sense changes in trimethylamine oxide (TMAO) concentration around the cells. However, it is unknown if it also affects initiation of DNA replication. We detected DNA replication patterns in ΔtorS and ΔtorR mutant strains by flow cytometry. We found that the average number of replication origins (oriCs) per cell and doubling time in ΔtorS mutants were the same while the average number of oriCs in ΔtorR mutants was increased compared with that in wild-type cells. These results indicated that absence of TorR led to an earlier initiation of DNA replication than that in wild-type cells. Strangely, neither overexpression of TorR nor co-expression of TorR and TorS could restore ΔtorR mutant phenotype to the wild type. However, overexpression of SufD in both wild type and ΔtorR mutants promoted initiation of DNA replication, while mutation of SufD delayed it in ΔtorR mutants. Thus, TorR may affect initiation of DNA replication indirectly through regulating gene expression of sufD.

Conserved Sequences at the Origin of Adenovirus DNA Replication

PubMed Central

Stillman, Bruce W.; Topp, William C.; Engler, Jeffrey A.

1982-01-01

The origin of adenovirus DNA replication lies within an inverted sequence repetition at either end of the linear, double-stranded viral DNA. Initiation of DNA replication is primed by a deoxynucleoside that is covalently linked to a protein, which remains bound to the newly synthesized DNA. We demonstrate that virion-derived DNA-protein complexes from five human adenovirus serological subgroups (A to E) can act as a template for both the initiation and the elongation of DNA replication in vitro, using nuclear extracts from adenovirus type 2 (Ad2)-infected HeLa cells. The heterologous template DNA-protein complexes were not as active as the homologous Ad2 DNA, most probably due to inefficient initiation by Ad2 replication factors. In an attempt to identify common features which may permit this replication, we have also sequenced the inverted terminal repeated DNA from human adenovirus serotypes Ad4 (group E), Ad9 and Ad10 (group D), and Ad31 (group A), and we have compared these to previously determined sequences from Ad2 and Ad5 (group C), Ad7 (group B), and Ad12 and Ad18 (group A) DNA. In all cases, the sequence around the origin of DNA replication can be divided into two structural domains: a proximal A · T-rich region which is partially conserved among these serotypes, and a distal G · C-rich region which is less well conserved. The G · C-rich region contains sequences similar to sequences present in papovavirus replication origins. The two domains may reflect a dual mechanism for initiation of DNA replication: adenovirus-specific protein priming of replication, and subsequent utilization of this primer by host replication factors for completion of DNA synthesis. Images PMID:7143575

Localization of DNA methyltransferase-1 during oocyte differentiation, in vitro maturation and early embryonic development in cow

PubMed Central

Lodde, V.; Modina, S.C.; Franciosi, F.; Zuccari, E.; Tessaro, I.; Luciano, A.M.

2009-01-01

DNA methyltransferase-1 (Dnmt1) is involved in the maintenance of DNA methylation patterns and is crucial for normal mammalian development. The aim of the present study was to assess the localization of Dnmt1 in cow, during the latest phases of oocyte differentiation and during the early stages of segmentation. Dnmt1 expression and localization were assessed in oocytes according to the chromatin configuration, which in turn provides an important epigenetic mechanism for the control of global gene expression and represents a morphological marker of oocyte differentiation. We found that the initial chromatin condensation was accompanied by a slight increase in the level of global DNA methylation, as assessed by 5-methyl-cytosine immunostaining followed by laser scanning confocal microscopy analysis (LSCM). RT-PCR confirmed the presence of Dnmt1 transcripts throughout this phase of oocyte differentiation. Analogously, Dnmt1 immunodetection and LSCM indicated that the protein was always present and localized in the cytoplasm, regardless the chromatin configuration and the level of global DNA methylation. Moreover, our data indicate that while Dnmt1 is retained in the cytoplasm in metaphase II stage oocytes and zygotes, it enters the nuclei of 8–16 cell stage embryos. As suggested in mouse, the functional meaning of the presence of Dnmt1 in the bovine embryo nuclei could be the maintainement of the methylation pattern of imprinted genes. In conclusion, the present work provides useful elements for the study of Dnmt1 function during the late stage of oocyte differentiation, maturation and early embryonic development in mammals. PMID:22073356

Mutant DnaAs of Escherichia coli that are refractory to negative control

PubMed Central

Chodavarapu, Sundari; Felczak, Magdalena M.; Simmons, Lyle A.; Murillo, Alec; Kaguni, Jon M.

2013-01-01

DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli. PMID:23990329

Mutant DnaAs of Escherichia coli that are refractory to negative control.

PubMed

Chodavarapu, Sundari; Felczak, Magdalena M; Simmons, Lyle A; Murillo, Alec; Kaguni, Jon M

2013-12-01

DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli.

Elucidating polyploidization of bermudagrasses as assessed by organelle and nuclear DNA markers.

PubMed

Gulsen, Osman; Ceylan, Ahmet

2011-12-01

Clarification of relationships among ploidy series of Cynodon accessions could be beneficial to bermudagrass breeding programs, and would enhance our understanding of the evolutionary biology of this warm season grass species. This study was initiated to elucidate polyploidization among Cynodon accessions with different ploidy series collected from Turkey based on chloroplast and nuclear DNA. Forty Cynodon accessions including 7 diploids, 3 triploids, 10 tetraploids, 11 pentaploids, and 9 hexaploids were analyzed using chloroplast DNA restriction fragment-length polymorphism (cpDNA RFLP), chloroplast DNA simple sequence repeat (cpDNA SSR), and nuclear DNA markers based on neighbor-joining (NJ) and principle component analyses (PCA). All three-marker systems with two statistical algorithms clustered the diploids apart from the other ploidy levels. Assuming autopolyploidy, spontaneous polyploidization followed by rapid diversification among the higher ploidy levels than the diploids is likely in Cynodon's evolution. Few tetraploid and hexaploid accessions were clustered with or closely to the group of diploids, supporting the hypothesis above. Eleven haplotypes as estimated by cpDNA RFLP and SSR markers were detected. This study indicated that the diploids had different organelle genome from the rest of the ploidy series and provided valuable insight into relationships among ploidy series of Cynodon accessions based on cp and nuclear DNAs.

Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers.

PubMed

Zhu, Bin; Wang, Longfei; Mitsunobu, Hitoshi; Lu, Xueling; Hernandez, Alfredo J; Yoshida-Takashima, Yukari; Nunoura, Takuro; Tabor, Stanley; Richardson, Charles C

2017-03-21

A DNA polymerase is encoded by the deep-sea vent phage NrS-1. NrS-1 has a unique genome organization containing genes that are predicted to encode a helicase and a single-stranded DNA (ssDNA)-binding protein. The gene for an unknown protein shares weak homology with the bifunctional primase-polymerases (prim-pols) from archaeal plasmids but is missing the zinc-binding domain typically found in primases. We show that this gene product has efficient DNA polymerase activity and is processive in DNA synthesis in the presence of the NrS-1 helicase and ssDNA-binding protein. Remarkably, this NrS-1 DNA polymerase initiates DNA synthesis from a specific template DNA sequence in the absence of any primer. The de novo DNA polymerase activity resides in the N-terminal domain of the protein, whereas the C-terminal domain enhances DNA binding.

Dpb11 may function with RPA and DNA to initiate DNA replication.

PubMed

Bruck, Irina; Dhingra, Nalini; Martinez, Matthew P; Kaplan, Daniel L

2017-01-01

Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA. A Dpb11 mutant specifically defective for DNA binding did not exhibit tight binding to RPA in the presence of DNA, suggesting that Dpb11-interaction with DNA may promote the recruitment of RPA to melted DNA. We then characterized a mutant of Dpb11 that is specifically defective in DNA binding in budding yeast cells. Expression of dpb11-m1,2,3,5,ΔC results in a substantial decrease in RPA recruitment to origins, suggesting that Dpb11 interaction with DNA may be required for RPA recruitment to origins. Expression of dpb11-m1,2,3,5,ΔC also results in diminished GINS interaction with Mcm2-7 during S phase, while Cdc45 interaction with Mcm2-7 is like wild-type. The reduced GINS interaction with Mcm2-7 may be an indirect consequence of diminished origin melting. We propose that the tight interaction between Dpb11, CDK-phosphorylated RPA, and branched-DNA may be required for the essential function of stabilizing melted origin DNA in vivo. We also propose an alternative model, wherein Dpb11-DNA interaction is required for some other function in DNA replication initiation, such as helicase activation.

Sensitivity to methylmercury toxicity is enhanced in oxoguanine glycosylase 1 knockout murine embryonic fibroblasts and is dependent on cellular proliferation capacity

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

Ondovcik, Stephanie L.; Tamblyn, Laura; McPherson, John Peter

2013-07-01

Methylmercury (MeHg) is a persistent environmental contaminant with potent neurotoxic action for which the underlying molecular mechanisms remain to be conclusively delineated. Our objectives herein were twofold: first, to corroborate our previous findings of an increased sensitivity of spontaneously-immortalized oxoguanine glycosylase 1-null (Ogg1{sup −/−}) murine embryonic fibroblasts (MEFs) to MeHg through generation of Simian virus 40 (SV40) large T antigen-immortalized wild-type and Ogg1{sup −/−} MEFs; and second, to determine whether MeHg toxicity is proliferation-dependent. As with the spontaneously-immortalized cells used previously, the SV40 large T antigen-immortalized cells exhibited similar tendencies to undergo MeHg-initiated cell cycle arrest, with increased sensitivity inmore » the Ogg1{sup −/−} MEFs as measured by clonogenic survival and DNA damage. Compared to exponentially growing cells, those seeded at a higher density exhibited compromised proliferation, which proved protective against MeHg-mediated cell cycle arrest and induction of DNA double strand breaks (DSBs), measured by phosphorylation of the core histone H2A variant (H2AX) on serine 139 (γH2AX), and by its functional confirmation by micronucleus assessment. This enhanced sensitivity of Ogg1{sup −/−} MEFs to MeHg toxicity using discrete SV40 immortalization corroborates our previous studies, and suggests a novel role for OGG1 in minimizing MeHg-initiated DNA lesions that trigger replication-associated DSBs. Furthermore, proliferative capacity may determine MeHg toxicity in vivo and in utero. Accordingly, variations in cellular proliferative capacity and interindividual variability in repair activity may modulate the risk of toxicological consequences following MeHg exposure. - Highlights: • SV40 large T antigen-immortalized Ogg1{sup −/−} cells are more sensitive to MeHg. • Sensitivity to MeHg is dependent on cellular proliferation capacity. • OGG1 maintains genomic integrity following MeHg-initiated DNA damage. • OGG1 may limit MeHg-initiated DNA lesions that trigger replication-associated DSBs. • Variations in proliferation and repair activity may modulate toxicological risk.« less

Nucleic acid and protein synthesis during lateral root initiation in Marsilea quadrifolia (Marsileaceae)

NASA Technical Reports Server (NTRS)

Lin, B. L.; Raghavan, V.

1991-01-01

The pattern of DNA, RNA, and protein synthesis during lateral root initiation in Marsilea quadrifolia L. was monitored by autoradiography of incorporated of 3H-thymidine, 3H-uridine, and 3H-leucine, respectively. DNA synthesis was associated with the enlargement of the lateral root initial prior to its division. Consistent with histological studies, derivatives of the lateral root initial as well as the cells of the adjacent inner cortex and pericycle of the parent root also continued to synthesize DNA. RNA and protein synthetic activities were found to be higher in the lateral root initials than in the endodermal initials of the same longitudinal layer. The data suggest a role for nucleic acid and protein synthesis during cytodifferentiation of a potential endodermal cell into a lateral root initial.

The Yeast INO80 Complex Operates as a Tunable DNA Length-Sensitive Switch to Regulate Nucleosome Sliding.

PubMed

Zhou, Coral Y; Johnson, Stephanie L; Lee, Laura J; Longhurst, Adam D; Beckwith, Sean L; Johnson, Matthew J; Morrison, Ashby J; Narlikar, Geeta J

2018-02-15

The yeast INO80 chromatin remodeling complex plays essential roles in regulating DNA damage repair, replication, and promoter architecture. INO80's role in these processes is likely related to its ability to slide nucleosomes, but the underlying mechanism is poorly understood. Here we use ensemble and single-molecule enzymology to study INO80-catalyzed nucleosome sliding. We find that the rate of nucleosome sliding by INO80 increases ∼100-fold when the flanking DNA length is increased from 40 to 60 bp. Furthermore, once sliding is initiated, INO80 moves the nucleosome rapidly at least 20 bp without pausing to re-assess flanking DNA length, and it can change the direction of nucleosome sliding without dissociation. Finally, we show that the Nhp10 module of INO80 plays an auto-inhibitory role, tuning INO80's switch-like response to flanking DNA. Our results indicate that INO80 is a highly processive remodeling motor that is tightly regulated by both substrate cues and non-catalytic subunits. Copyright © 2018 Elsevier Inc. All rights reserved.

DNA methylation assessment from human slow- and fast-twitch skeletal muscle fibers

PubMed Central

Begue, Gwénaëlle; Raue, Ulrika; Jemiolo, Bozena

2017-01-01

A new application of the reduced representation bisulfite sequencing method was developed using low-DNA input to investigate the epigenetic profile of human slow- and fast-twitch skeletal muscle fibers. Successful library construction was completed with as little as 15 ng of DNA, and high-quality sequencing data were obtained with 32 ng of DNA. Analysis identified 143,160 differentially methylated CpG sites across 14,046 genes. In both fiber types, selected genes predominantly expressed in slow or fast fibers were hypomethylated, which was supported by the RNA-sequencing analysis. These are the first fiber type-specific methylation data from human skeletal muscle and provide a unique platform for future research. NEW & NOTEWORTHY This study validates a low-DNA input reduced representation bisulfite sequencing method for human muscle biopsy samples to investigate the methylation patterns at a fiber type-specific level. These are the first fiber type-specific methylation data reported from human skeletal muscle and thus provide initial insight into basal state differences in myosin heavy chain I and IIa muscle fibers among young, healthy men. PMID:28057818

Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts

PubMed Central

1993-01-01

Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase- like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M- phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine- treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation. PMID:8253833

A universal colorimetry for nucleic acids and aptamer-specific ligands detection based on DNA hybridization amplification.

PubMed

Li, Shuang; Shang, Xinxin; Liu, Jia; Wang, Yujie; Guo, Yingshu; You, Jinmao

2017-07-01

We present a universal amplified-colorimetric for detecting nucleic acid targets or aptamer-specific ligand targets based on gold nanoparticle-DNA (GNP-DNA) hybridization chain reaction (HCR). The universal arrays consisted of capture probe and hairpin DNA-GNP. First, capture probe recognized target specificity and released the initiator sequence. Then dispersed hairpin DNA modified GNPs were cross-linked to form aggregates through HCR events triggered by initiator sequence. As the aggregates accumulate, a significant red-to purple color change can be easily visualized by the naked eye. We used miRNA target sequence (miRNA-203) and aptamer-specific ligand (ATP) as target molecules for this proof-of-concept experiment. Initiator sequence (DNA2) was released from the capture probe (MNP/DNA1/2 conjugates) under the strong competitiveness of miRNA-203. Hairpin DNA (H1 and H2) can be complementary with the help of initiator DNA2 to form GNP-H1/GNP-H2 aggregates. The absorption ratio (A 620 /A 520 ) values of solutions were a sensitive function of miRNA-203 concentration covering from 1.0 × 10 -11  M to 9.0 × 10 -10  M, and as low as 1.0 × 10 -11  M could be detected. At the same time, the color changed from light wine red to purple and then to light blue have occurred in the solution. For ATP, initiator sequence (5'-end of DNA3) was released from the capture probe (DNA3) under the strong combination of aptamer-ATP. The present colorimetric for specific detection of ATP exhibited good sensitivity and 1.0 × 10 -8  M ATP could be detected. The proposed strategy also showed good performances for qualitative analysis and quantitative analysis of intracellular nucleic acids and aptamer-specific ligands. Copyright © 2017 Elsevier Inc. All rights reserved.

Epigenetic approaches for the detection of fetal DNA in maternal plasma

PubMed Central

Tsui, Dana WY; Chiu, Rossa WK

2010-01-01

The presence of fetal DNA in the plasma of pregnant women has opened up new possibilities for noninvasive prenatal diagnosis. Over the past decades, different types of fetal markers have been developed, initially based on discriminative genetic markers such as male-specific signals or paternally-inherited polymorphisms, and gradually evolved to the detection of fetal-specific transcripts or epigenetic signatures. This development has extended the coverage of the application of cell-free fetal DNA to essentially all pregnancies, regardless of the gender of the fetus or its polymorphic status. In this review, we present an overview of the development of noninvasive prenatal diagnosis through epigenetics. We introduce the basis of how fetal DNA could be detected from a large background of maternal DNA in maternal plasma based on fetal-specific DNA methylation patterns. We evaluate the methodologies involved and discuss the factors that affect the robustness of the detection. We review the progress in adopting fetal epigenetic markers for noninvasive prenatal assessment of fetal chromosomal aneuploidies and pregnancy-associated disorders. We conclude with comments on the future directions regarding the search for new fetal epigenetic markers and the clinical implementation of epigenetic approaches for noninvasive prenatal diagnosis. PMID:21327153

Linking JNK Activity to the DNA Damage Response

PubMed Central

Picco, Vincent

2013-01-01

The activity of c-Jun N-terminal kinase (JNK) was initially described as ultraviolet- and oncogene-induced kinase activity on c-Jun. Shortly after this initial discovery, JNK activation was reported for a wider variety of DNA-damaging agents, including γ-irradiation and chemotherapeutic compounds. As the DNA damage response mechanisms were progressively uncovered, the mechanisms governing the activation of JNK upon genotoxic stresses became better understood. In particular, a recent set of papers links the physical breakage in DNA, the activation of the transcription factor NF-κB, the secretion of TNF-α, and an autocrine activation of the JNK pathway. In this review, we will focus on the pathway that is initiated by a physical break in the DNA helix, leading to JNK activation and the resultant cellular consequences. The implications of these findings will be discussed in the context of cancer therapy with DNA-damaging agents. PMID:24349633

Insights into the Initiation of Eukaryotic DNA Replication.

PubMed

Bruck, Irina; Perez-Arnaiz, Patricia; Colbert, Max K; Kaplan, Daniel L

2015-01-01

The initiation of DNA replication is a highly regulated event in eukaryotic cells to ensure that the entire genome is copied once and only once during S phase. The primary target of cellular regulation of eukaryotic DNA replication initiation is the assembly and activation of the replication fork helicase, the 11-subunit assembly that unwinds DNA at a replication fork. The replication fork helicase, called CMG for Cdc45-Mcm2-7, and GINS, assembles in S phase from the constituent Cdc45, Mcm2-7, and GINS proteins. The assembly and activation of the CMG replication fork helicase during S phase is governed by 2 S-phase specific kinases, CDK and DDK. CDK stimulates the interaction between Sld2, Sld3, and Dpb11, 3 initiation factors that are each required for the initiation of DNA replication. DDK, on the other hand, phosphorylates the Mcm2, Mcm4, and Mcm6 subunits of the Mcm2-7 complex. Sld3 recruits Cdc45 to Mcm2-7 in a manner that depends on DDK, and recent work suggests that Sld3 binds directly to Mcm2-7 and also to single-stranded DNA. Furthermore, recent work demonstrates that Sld3 and its human homolog Treslin substantially stimulate DDK phosphorylation of Mcm2. These data suggest that the initiation factor Sld3/Treslin coordinates the assembly and activation of the eukaryotic replication fork helicase by recruiting Cdc45 to Mcm2-7, stimulating DDK phosphorylation of Mcm2, and binding directly to single-stranded DNA as the origin is melted.

Role of the Escherichia coli grpE heat shock protein in the initiation of bacteriophage lambda DNA replication.

PubMed

Osipiuk, J; Zylicz, M

1991-01-01

Initiation of replication of lambda DNA requires assembly of the proper nucleoprotein complex consisting of the lambda origin of replication-lambda O-lambda P-dnaB proteins. The dnaJ, dnaK and grpE heat shock proteins destabilize the lambda P-dnaB interaction in this complex permitting dnaB helicase to unwind lambda DNA near ori lambda sequence. First step of this disassembling reaction is the binding of dnaK protein to lambda P protein. In this report we examined the influence of dnaJ and grpE proteins on stability of the lambda P-dnaK complex. Our results show that grpE alone dissociates this complex, but both grpE and dnaJ together do not. These results suggest that, in the presence of grpE protein, dnaK protein has a higher affinity for lambda P protein complexed with dnaJ protein than in the situation where grpE protein is not used.

The role of DNA repair in herpesvirus pathogenesis.

PubMed

Brown, Jay C

2014-10-01

In cells latently infected with a herpesvirus, the viral DNA is present in the cell nucleus, but it is not extensively replicated or transcribed. In this suppressed state the virus DNA is vulnerable to mutagenic events that affect the host cell and have the potential to destroy the virus' genetic integrity. Despite the potential for genetic damage, however, herpesvirus sequences are well conserved after reactivation from latency. To account for this apparent paradox, I have tested the idea that host cell-encoded mechanisms of DNA repair are able to control genetic damage to latent herpesviruses. Studies were focused on homologous recombination-dependent DNA repair (HR). Methods of DNA sequence analysis were employed to scan herpesvirus genomes for DNA features able to activate HR. Analyses were carried out with a total of 39 herpesvirus DNA sequences, a group that included viruses from the alpha-, beta- and gamma-subfamilies. The results showed that all 39 genome sequences were enriched in two or more of the eight recombination-initiating features examined. The results were interpreted to indicate that HR can stabilize latent herpesvirus genomes. The results also showed, unexpectedly, that repair-initiating DNA features differed in alpha- compared to gamma-herpesviruses. Whereas inverted and tandem repeats predominated in alpha-herpesviruses, gamma-herpesviruses were enriched in short, GC-rich initiation sequences such as CCCAG and depleted in repeats. In alpha-herpesviruses, repair-initiating repeat sequences were found to be concentrated in a specific region (the S segment) of the genome while repair-initiating short sequences were distributed more uniformly in gamma-herpesviruses. The results suggest that repair pathways are activated differently in alpha- compared to gamma-herpesviruses. Copyright © 2014. Published by Elsevier Inc.

Amino acid-dependent signaling via S6K1 and MYC is essential for regulation of rDNA transcription

PubMed Central

Kang, Jian; Kusnadi, Eric P.; Ogden, Allison J.; Hicks, Rodney J.; Bammert, Lukas; Kutay, Ulrike; Hung, Sandy; Sanij, Elaine; Hannan, Ross D.; Hannan, Katherine M.; Pearson, Richard B.

2016-01-01

Dysregulation of RNA polymerase I (Pol I)-dependent ribosomal DNA (rDNA) transcription is a consistent feature of malignant transformation that can be targeted to treat cancer. Understanding how rDNA transcription is coupled to the availability of growth factors and nutrients will provide insight into how ribosome biogenesis is maintained in a tumour environment characterised by limiting nutrients. We demonstrate that modulation of rDNA transcription initiation, elongation and rRNA processing is an immediate, co-regulated response to altered amino acid abundance, dependent on both mTORC1 activation of S6K1 and MYC activity. Growth factors regulate rDNA transcription initiation while amino acids modulate growth factor-dependent rDNA transcription by primarily regulating S6K1-dependent rDNA transcription elongation and processing. Thus, we show for the first time amino acids regulate rRNA synthesis by a distinct, post-initiation mechanism, providing a novel model for integrated control of ribosome biogenesis that has implications for understanding how this process is dysregulated in cancer. PMID:27385002

Track structure based modelling of light ion radiation effects on nuclear and mitochondrial DNA

NASA Astrophysics Data System (ADS)

Schmitt, Elke; Ottolenghi, Andrea; Dingfelder, Michael; Friedland, Werner; Kundrat, Pavel; Baiocco, Giorgio

2016-07-01

Space radiation risk assessment is of great importance for manned spaceflights in order to estimate risks and to develop counter-measures to reduce them. Biophysical simulations with PARTRAC can help greatly to improve the understanding of initial biological response to ionizing radiation. Results from modelling radiation quality dependent DNA damage and repair mechanisms up to chromosomal aberrations (e.g. dicentrics) can be used to predict radiation effects depending on the kind of mixed radiation field exposure. Especially dicentric yields can serve as a biomarker for an increased risk due to radiation and hence as an indicator for the effectiveness of the used shielding. PARTRAC [1] is a multi-scale biophysical research MC code for track structure based initial DNA damage and damage response modelling. It integrates physics, radiochemistry, detailed nuclear DNA structure and molecular biology of DNA repair by NHEJ-pathway to assess radiation effects on cellular level [2]. Ongoing experiments with quasi-homogeneously distributed compared to sub-micrometre focused bunches of protons, lithium and carbon ions allow a separation of effects due to DNA damage complexity on nanometre scale from damage clustering on (sub-) micrometre scale [3, 4]. These data provide an unprecedented benchmark for the DNA damage response model in PARTRAC and help understand the mechanisms leading to cell killing and chromosomal aberrations (e.g. dicentrics) induction. A large part of space radiation is due to a mixed ion field of high energy protons and few heavier ions that can be only partly absorbed by the shielding. Radiation damage induced by low-energy ions significantly contributes to the high relative biological efficiency (RBE) of ion beams around Bragg peak regions. For slow light ions the physical cross section data basis in PARTRAC has been extended to investigate radiation quality effects in the Bragg peak region [5]. The resulting range and LET values agree with ICRU data and SRIM calculations. Preliminary studies regarding the biological endpoints DSB (cluster) and chromosomal aberrations have been performed for selected light ions up to neon. Validation with experimental data as well as further calculations are underway and final results will be presented at the meeting. Mitochondrial alterations have been implicated in radiation-induced cardiovascular effects. To extend the applicability of PARTRAC biophysical tool towards effects on mitochondria, the nuclear DNA and chromatin as the primary target of radiation has been complemented by a model of mitochondrial DNA (mtDNA) to mimic a coronary cell with thousand mitochondria contained in the cytoplasm. Induced mtDNA damage (SSB, DSB) has been scored for 60Co photons and 5 MeV alpha-particle irradiation, assuming alternative radical scavenging capacities within the mitochondria. While direct radiation effects in mtDNA are identical to nuclear DNA, indirect effects in mtDNA are in general larger due to lower scavenging and the lack of DNA-protecting histones. These simulations complement the scarce experimental data on radiation-induced mtDNA damage and help elucidate the relative roles of initial mtDNA versus nuclear DNA damage and of pathways that amplify their respective effects. Ongoing and planned developments of PARTRAC include coupling with a radiation transport code and track-structure based calculations of cell killing for RBE studies on macroscopic scales within a mixed ion field. [1] Friedland, Dingfelder et al. (2011): "Track structures, DNA targets and radiation effects in the biophysical Monte Carlo simulation code PARTRAC", Mutat. Res. 711, 28-40 [2] Friedland et al. (2013): "Track structure based modelling of chromosome aberrations after photon and alpha-particle irradiation", Mutat. Res. 756, 213-223 [3] Schmid, Friedland et al. (2015): "Sub-micrometer 20 MeV protons or 45 MeV lithium spot irradiation enhances yields of dicentric chromosomes due to clustering of DNA double-strand breaks", Mutat. Res. 793, 30-40 [4] Friedland, Schmitt, Kundrat (2015): "Modelling Proton bunches focussed to submicrometre scales: Low-LET Radiation damage in high-LET-like spatial structure", Radiat. Prot. Dosim. 166, 34-37 [5] Schmitt, Friedland, Kundrat, Dingfelder, Ottolenghi (2015): "Cross section scaling for track structure simulations of low-energy ions in liquid water", Radiat. Prot. Dosim. 166, 15-18} Supported by the European Atomic Energy Community's Seventh Framework Programme (FP7/2007-2011) under grant agreement no 249689 "DoReMi" and the German Federal Ministry on Education and Research (KVSF-Projekt "LET-Verbund").

Convergence and reproducibility in molecular dynamics simulations of the DNA duplex d(GCACGAACGAACGAACGC).

PubMed

Galindo-Murillo, Rodrigo; Roe, Daniel R; Cheatham, Thomas E

2015-05-01

The structure and dynamics of DNA are critically related to its function. Molecular dynamics simulations augment experiment by providing detailed information about the atomic motions. However, to date the simulations have not been long enough for convergence of the dynamics and structural properties of DNA. Molecular dynamics simulations performed with AMBER using the ff99SB force field with the parmbsc0 modifications, including ensembles of independent simulations, were compared to long timescale molecular dynamics performed with the specialized Anton MD engine on the B-DNA structure d(GCACGAACGAACGAACGC). To assess convergence, the decay of the average RMSD values over longer and longer time intervals was evaluated in addition to assessing convergence of the dynamics via the Kullback-Leibler divergence of principal component projection histograms. These molecular dynamics simulations-including one of the longest simulations of DNA published to date at ~44μs-surprisingly suggest that the structure and dynamics of the DNA helix, neglecting the terminal base pairs, are essentially fully converged on the ~1-5μs timescale. We can now reproducibly converge the structure and dynamics of B-DNA helices, omitting the terminal base pairs, on the μs time scale with both the AMBER and CHARMM C36 nucleic acid force fields. Results from independent ensembles of simulations starting from different initial conditions, when aggregated, match the results from long timescale simulations on the specialized Anton MD engine. With access to large-scale GPU resources or the specialized MD engine "Anton" it is possible for a variety of molecular systems to reproducibly and reliably converge the conformational ensemble of sampled structures. This article is part of a Special Issue entitled: Recent developments of molecular dynamics. Copyright © 2014. Published by Elsevier B.V.

Convergence and reproducibility in molecular dynamics simulations of the DNA duplex d(GCACGAACGAACGAACGC)

PubMed Central

Galindo-Murillo, Rodrigo; Roe, Daniel R.; Cheatham, Thomas E.

2014-01-01

Background The structure and dynamics of DNA are critically related to its function. Molecular dynamics (MD) simulations augment experiment by providing detailed information about the atomic motions. However, to date the simulations have not been long enough for convergence of the dynamics and structural properties of DNA. Methods MD simulations performed with AMBER using the ff99SB force field with the parmbsc0 modifications, including ensembles of independent simulations, were compared to long timescale MD performed with the specialized Anton MD engine on the B-DNA structure d(GCACGAACGAACGAACGC). To assess convergence, the decay of the average RMSD values over longer and longer time intervals was evaluated in addition to assessing convergence of the dynamics via the Kullback-Leibler divergence of principal component projection histograms. Results These MD simulations —including one of the longest simulations of DNA published to date at ~44 μs—surprisingly suggest that the structure and dynamics of the DNA helix, neglecting the terminal base pairs, are essentially fully converged on the ~1–5 μs timescale. Conclusions We can now reproducibly converge the structure and dynamics of B-DNA helices, omitting the terminal base pairs, on the μs time scale with both the AMBER and CHARMM C36 nucleic acid force fields. Results from independent ensembles of simulations starting from different initial conditions, when aggregated, match the results from long timescale simulations on the specialized Anton MD engine. General Significance With access to large-scale GPU resources or the specialized MD engine “Anton” it is possibly for a variety of molecular systems to reproducibly and reliably converge the conformational ensemble of sampled structures. PMID:25219455

Dpb11 may function with RPA and DNA to initiate DNA replication

PubMed Central

Bruck, Irina; Dhingra, Nalini; Martinez, Matthew P.

2017-01-01

Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA. A Dpb11 mutant specifically defective for DNA binding did not exhibit tight binding to RPA in the presence of DNA, suggesting that Dpb11-interaction with DNA may promote the recruitment of RPA to melted DNA. We then characterized a mutant of Dpb11 that is specifically defective in DNA binding in budding yeast cells. Expression of dpb11-m1,2,3,5,ΔC results in a substantial decrease in RPA recruitment to origins, suggesting that Dpb11 interaction with DNA may be required for RPA recruitment to origins. Expression of dpb11-m1,2,3,5,ΔC also results in diminished GINS interaction with Mcm2-7 during S phase, while Cdc45 interaction with Mcm2-7 is like wild-type. The reduced GINS interaction with Mcm2-7 may be an indirect consequence of diminished origin melting. We propose that the tight interaction between Dpb11, CDK-phosphorylated RPA, and branched-DNA may be required for the essential function of stabilizing melted origin DNA in vivo. We also propose an alternative model, wherein Dpb11-DNA interaction is required for some other function in DNA replication initiation, such as helicase activation. PMID:28467467

Sensitive detection of multiple pathogens using a single DNA probe.

PubMed

Nordin, Noordiana; Yusof, Nor Azah; Abdullah, Jaafar; Radu, Son; Hushiarian, Roozbeh

2016-12-15

A simple but promising electrochemical DNA nanosensor was designed, constructed and applied to differentiate a few food-borne pathogens. The DNA probe was initially designed to have a complementary region in Vibrio parahaemolyticus (VP) genome and to make different hybridization patterns with other selected pathogens. The sensor was based on a screen printed carbon electrode (SPCE) modified with polylactide-stabilized gold nanoparticles (PLA-AuNPs) and methylene blue (MB) was employed as the redox indicator binding better to single-stranded DNA. The immobilization and hybridization events were assessed using differential pulse voltammetry (DPV). The fabricated biosensor was able to specifically distinguish complementary, non-complementary and mismatched oligonucleotides. DNA was measured in the range of 2.0×10(-9)-2.0×10(-13)M with a detection limit of 5.3×10(-12)M. The relative standard deviation for 6 replications of DPV measurement of 0.2µM complementary DNA was 4.88%. The fabricated DNA biosensor was considered stable and portable as indicated by a recovery of more than 80% after a storage period of 6 months at 4-45°C. Cross-reactivity studies against various food-borne pathogens showed a reliably sensitive detection of VP. Copyright © 2016 Elsevier B.V. All rights reserved.

Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners

PubMed Central

Felicori, Liza; Jameson, Katie H.; Roblin, Pierre; Fogg, Mark J.; Garcia-Garcia, Transito; Ventroux, Magali; Cherrier, Mickaël V.; Bazin, Alexandre; Noirot, Philippe; Wilkinson, Anthony J.; Molina, Franck; Terradot, Laurent; Noirot-Gros, Marie-Françoise

2016-01-01

YabA negatively regulates initiation of DNA replication in low-GC Gram-positive bacteria. The protein exerts its control through interactions with the initiator protein DnaA and the sliding clamp DnaN. Here, we combined X-ray crystallography, X-ray scattering (SAXS), modeling and biophysical approaches, with in vivo experimental data to gain insight into YabA function. The crystal structure of the N-terminal domain (NTD) of YabA solved at 2.7 Å resolution reveals an extended α-helix that contributes to an intermolecular four-helix bundle. Homology modeling and biochemical analysis indicates that the C-terminal domain (CTD) of YabA is a small Zn-binding domain. Multi-angle light scattering and SAXS demonstrate that YabA is a tetramer in which the CTDs are independent and connected to the N-terminal four-helix bundle via flexible linkers. While YabA can simultaneously interact with both DnaA and DnaN, we found that an isolated CTD can bind to either DnaA or DnaN, individually. Site-directed mutagenesis and yeast-two hybrid assays identified DnaA and DnaN binding sites on the YabA CTD that partially overlap and point to a mutually exclusive mode of interaction. Our study defines YabA as a novel structural hub and explains how the protein tetramer uses independent CTDs to bind multiple partners to orchestrate replication initiation in the bacterial cell. PMID:26615189

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

PubMed Central

2018-01-01

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

Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation.

PubMed

Zhu, Jing; Ding, Yongshun; Liu, Xingti; Wang, Lei; Jiang, Wei

2014-09-15

Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

PubMed

NandyMazumdar, Monali; Nedialkov, Yuri; Svetlov, Dmitri; Sevostyanova, Anastasia; Belogurov, Georgiy A; Artsimovitch, Irina

2016-12-27

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

454 Pyrosequencing to Describe Microbial Eukaryotic Community Composition, Diversity and Relative Abundance: A Test for Marine Haptophytes

PubMed Central

Egge, Elianne; Bittner, Lucie; Andersen, Tom; Audic, Stéphane; de Vargas, Colomban; Edvardsen, Bente

2013-01-01

Next generation sequencing of ribosomal DNA is increasingly used to assess the diversity and structure of microbial communities. Here we test the ability of 454 pyrosequencing to detect the number of species present, and assess the relative abundance in terms of cell numbers and biomass of protists in the phylum Haptophyta. We used a mock community consisting of equal number of cells of 11 haptophyte species and compared targeting DNA and RNA/cDNA, and two different V4 SSU rDNA haptophyte-biased primer pairs. Further, we tested four different bioinformatic filtering methods to reduce errors in the resulting sequence dataset. With sequencing depth of 11000–20000 reads and targeting cDNA with Haptophyta specific primers Hap454 we detected all 11 species. A rarefaction analysis of expected number of species recovered as a function of sampling depth suggested that minimum 1400 reads were required here to recover all species in the mock community. Relative read abundance did not correlate to relative cell numbers. Although the species represented with the largest biomass was also proportionally most abundant among the reads, there was generally a weak correlation between proportional read abundance and proportional biomass of the different species, both with DNA and cDNA as template. The 454 sequencing generated considerable spurious diversity, and more with cDNA than DNA as template. With initial filtering based only on match with barcode and primer we observed 100-fold more operational taxonomic units (OTUs) at 99% similarity than the number of species present in the mock community. Filtering based on quality scores, or denoising with PyroNoise resulted in ten times more OTU99% than the number of species. Denoising with AmpliconNoise reduced the number of OTU99% to match the number of species present in the mock community. Based on our analyses, we propose a strategy to more accurately depict haptophyte diversity using 454 pyrosequencing. PMID:24069303

Impact of DNA repair on the dose-response of colorectal cancer formation induced by dietary carcinogens.

PubMed

Fahrer, Jörg; Kaina, Bernd

2017-08-01

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers, which is causally linked to dietary habits, notably the intake of processed and red meat. Processed and red meat contain dietary carcinogens, including heterocyclic aromatic amines (HCAs) and N-nitroso compounds (NOC). NOC are agents that induce various N-methylated DNA adducts and O 6 -methylguanine (O 6 -MeG), which are removed by base excision repair (BER) and O 6 -methylguanine-DNA methyltransferase (MGMT), respectively. HCAs such as the highly mutagenic 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) cause bulky DNA adducts, which are removed from DNA by nucleotide excision repair (NER). Both O 6 -MeG and HCA-induced DNA adducts are linked to the occurrence of KRAS and APC mutations in colorectal tumors of rodents and humans, thereby driving CRC initiation and progression. In this review, we focus on DNA repair pathways removing DNA lesions induced by NOC and HCA and assess their role in protecting against mutagenicity and carcinogenicity in the large intestine. We further discuss the impact of DNA repair on the dose-response relationship in colorectal carcinogenesis in view of recent studies, demonstrating the existence of 'no effect' point of departures (PoDs), i.e. thresholds for genotoxicity and carcinogenicity. The available data support the threshold concept for NOC with DNA repair being causally involved. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantitation of DNA adducts by stable isotope dilution mass spectrometry

PubMed Central

Tretyakova, Natalia; Goggin, Melissa; Janis, Gregory

2012-01-01

Exposure to endogenous and exogenous chemicals can lead to the formation of structurally modified DNA bases (DNA adducts). If not repaired, these nucleobase lesions can cause polymerase errors during DNA replication, leading to heritable mutations potentially contributing to the development of cancer. Due to their critical role in cancer initiation, DNA adducts represent mechanism-based biomarkers of carcinogen exposure, and their quantitation is particularly useful for cancer risk assessment. DNA adducts are also valuable in mechanistic studies linking tumorigenic effects of environmental and industrial carcinogens to specific electrophilic species generated from their metabolism. While multiple experimental methodologies have been developed for DNA adduct analysis in biological samples – including immunoassay, HPLC, and 32P-postlabeling – isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) generally has superior selectivity, sensitivity, accuracy, and reproducibility. As typical DNA adducts concentrations in biological samples are between 0.01 – 10 adducts per 108 normal nucleotides, ultrasensitive HPLC-ESI-MS/MS methodologies are required for their analysis. Recent developments in analytical separations and biological mass spectrometry – especially nanoflow HPLC, nanospray ionization MS, chip-MS, and high resolution MS – have pushed the limits of analytical HPLC-ESI-MS/MS methodologies for DNA adducts, allowing researchers to accurately measure their concentrations in biological samples from patients treated with DNA alkylating drugs and in populations exposed to carcinogens from urban air, drinking water, cooked food, alcohol, and cigarette smoke. PMID:22827593

Cost effectiveness of fecal DNA screening for colorectal cancer: a systematic review and quality appraisal of the literature.

PubMed

Skally, Mairead; Hanly, Paul; Sharp, Linda

2013-06-01

Fecal DNA (fDNA) testing is a noninvasive potential alternative to current colorectal cancer screening tests. We conducted a systematic review and quality assessment of studies of cost-effectiveness of fDNA as a colorectal cancer screening tool (compared with no screening and other screening modalities), and identified key variables that impinged on cost-effectiveness. We searched MEDLINE, Embase, and the Centre for Reviews and Dissemination for cost-effectiveness studies of fDNA-based screening, published in English by September 2011. Studies that undertook an economic evaluation of fDNA, using either a cost-effectiveness or cost-utility analysis, compared with other relevant screening modalities and/or no screening were included. Additional inclusion criteria related to the presentation of data pertaining to model variables including time horizon, costs, fDNA performance characteristics, screening uptake, and comparators. A total of 369 articles were initially identified for review. After removing duplicates and applying inclusion and exclusion criteria, seven articles were included in the final review. Data was abstracted on key descriptor variables including screening scenarios, time horizon, costs, test performance characteristics, screening uptake, comparators, and incremental cost-effectiveness ratios. Quality assessment was undertaken using a standard checklist for economic evaluations. Studies cited by cost-effectiveness articles as the source of data on fDNA test performance characteristics were also reviewed. Seven cost-effectiveness studies were included, from the USA (4), Canada (1), Israel (1), and Taiwan (1). Markov models (5), a partially observable Markov decision process model (1) and MISCAN and SimCRC (1) microsimulation models were used. All studies took a third-party payer perspective and one included, in addition, a societal perspective. Comparator screening tests, screening intervals, and specific fDNA tests varied between studies. fDNA sensitivity and specificity parameters were derived from 12 research studies and one meta-analysis. Outcomes assessed were life-years gained and quality-adjusted life-years gained. fDNA was cost-effective when compared with no screening in six studies. Compared with other screening modalities, fDNA was not considered cost-effective in any of the base-case analyses: in five studies it was dominated by all alternatives considered. Sensitivity analyses identified cost, compliance, and test parameters as key influential parameters. In general, poor presentation of "study design" and "data collection" details lowered the quality of included articles. Although the literature searches were designed for high sensitivity, the possibility cannot be excluded that some eligible studies may have been missed. Reports (such as Health Technology Assessments produced by government agencies) and other forms of grey literature were excluded because they are difficult to identify systematically and/or may not report methods and results in sufficient detail for assessment. On the basis of the available (albeit limited) evidence, while fDNA is cost-effective when compared with no screening, it is currently dominated by most of the other available screening options. Cost and test performance appear to be the main influences on cost-effectiveness.

Investigation of the Causes of Breast Cancer at the Cellular Level: Isolation of In Vivo Binding Sites of the Human Origin Recognition Complex

DTIC Science & Technology

2002-08-01

We study the process of DNA replication in proliferating human cells. Our efforts are directed to the identification and characterization of proteins...that promote DNA replication (initiators) as well as the DNA sequences recognized by them (replicators) . We have focused in a group of initiator...to be a critical factor for the coordination of DNA replication with the cell division cycle. hOrclp levels are higher between the exit of mitosis and

Control of Initiation of DNA Replication in Bacillus subtilis and Escherichia coli

PubMed Central

Jameson, Katie H.; Wilkinson, Anthony J.

2017-01-01

Initiation of DNA Replication is tightly regulated in all cells since imbalances in chromosomal copy number are deleterious and often lethal. In bacteria such as Bacillus subtilis and Escherichia coli, at the point of cytokinesis, there must be two complete copies of the chromosome to partition into the daughter cells following division at mid-cell during vegetative growth. Under conditions of rapid growth, when the time taken to replicate the chromosome exceeds the doubling time of the cells, there will be multiple initiations per cell cycle and daughter cells will inherit chromosomes that are already undergoing replication. In contrast, cells entering the sporulation pathway in B. subtilis can do so only during a short interval in the cell cycle when there are two, and only two, chromosomes per cell, one destined for the spore and one for the mother cell. Here, we briefly describe the overall process of DNA replication in bacteria before reviewing initiation of DNA replication in detail. The review covers DnaA-directed assembly of the replisome at oriC and the multitude of mechanisms of regulation of initiation, with a focus on the similarities and differences between E. coli and B. subtilis. PMID:28075389

Initiator-catalyzed self-assembly of duplex-looped DNA hairpin motif based on strand displacement reaction for logic operations and amplified biosensing.

PubMed

Bi, Sai; Yue, Shuzhen; Wu, Qiang; Ye, Jiayan

2016-09-15

Here we program an initiator-catalyzed self-assembly of duplex-looped DNA hairpin motif based on strand displacement reaction. Due to the recycling of initiator and performance in a cascade manner, this system is versatilely extended to logic operations, including the construction of concatenated logic circuits with a feedback function and a biocomputing keypad-lock security system. Compared with previously reported molecular security systems, the prominent feature of our keypad lock is that it can be spontaneously reset and recycled with no need of any external stimulus and human intervention. Moreover, through integrating with an isothermal amplification technique of rolling circle amplification (RCA), this programming catalytic DNA self-assembly strategy readily achieves sensitive and selective biosensing of initiator. Importantly, a magnetic graphene oxide (MGO) is introduced to remarkably reduced background, which plays an important role in enhancing the signal-to-noise ratio and improving the detection sensitivity. Therefore, the proposed sophisticated DNA strand displacement-based methodology with engineering dynamic functions may find broad applications in the construction of programming DNA nanostructures, amplification biosensing platform, and large-scale DNA circuits. Copyright © 2016 Elsevier B.V. All rights reserved.

Clinical response to chemotherapy in locally advanced breast cancer was not associated with several polymorphisms in detoxification enzymes and DNA repair genes.

PubMed

Saadat, Mostafa; Khalili, Maryam; Nasiri, Meysam; Rajaei, Mehrdad; Omidvari, Shahpour; Saadat, Iraj

2012-03-02

The main aim of the present study was to investigate the association between several genetic polymorphisms (in glutathione S-transferase members and DNA repair genes) and clinical response to chemotherapy in locally advanced breast cancer. A sequential series of 101 patients were prospectively included in this study. Clinical assessment of treatment was accomplished by comparing initial tumor size with preoperative tumor size using revised RECIST guideline (version 1.1). Clinical response was regarded as a response or no response. There was no difference between non-responders and responders for the prevalence of genotypes of the study polymorphisms. Copyright © 2012 Elsevier Inc. All rights reserved.

Biomolecule Sequencer: Next-Generation DNA Sequencing Technology for In-Flight Environmental Monitoring, Research, and Beyond

NASA Technical Reports Server (NTRS)

Smith, David J.; Burton, Aaron; Castro-Wallace, Sarah; John, Kristen; Stahl, Sarah E.; Dworkin, Jason Peter; Lupisella, Mark L.

2016-01-01

On the International Space Station (ISS), technologies capable of rapid microbial identification and disease diagnostics are not currently available. NASA still relies upon sample return for comprehensive, molecular-based sample characterization. Next-generation DNA sequencing is a powerful approach for identifying microorganisms in air, water, and surfaces onboard spacecraft. The Biomolecule Sequencer payload, manifested to SpaceX-9 and scheduled on the Increment 4748 research plan (June 2016), will assess the functionality of a commercially-available next-generation DNA sequencer in the microgravity environment of ISS. The MinION device from Oxford Nanopore Technologies (Oxford, UK) measures picoamp changes in electrical current dependent on nucleotide sequences of the DNA strand migrating through nanopores in the system. The hardware is exceptionally small (9.5 x 3.2 x 1.6 cm), lightweight (120 grams), and powered only by a USB connection. For the ISS technology demonstration, the Biomolecule Sequencer will be powered by a Microsoft Surface Pro3. Ground-prepared samples containing lambda bacteriophage, Escherichia coli, and mouse genomic DNA, will be launched and stored frozen on the ISS until experiment initiation. Immediately prior to sequencing, a crew member will collect and thaw frozen DNA samples, connect the sequencer to the Surface Pro3, inject thawed samples into a MinION flow cell, and initiate sequencing. At the completion of the sequencing run, data will be downlinked for ground analysis. Identical, synchronous ground controls will be used for data comparisons to determine sequencer functionality, run-time sequence, current dynamics, and overall accuracy. We will present our latest results from the ISS flight experiment the first time DNA has ever been sequenced in space and discuss the many potential applications of the Biomolecule Sequencer for environmental monitoring, medical diagnostics, higher fidelity and more adaptable Space Biology Human Research Program investigations, and even life detection experiments for astrobiology missions.

Traces of archaic mitochondrial lineages persist in Austronesian-speaking Formosan populations.

PubMed

Trejaut, Jean A; Kivisild, Toomas; Loo, Jun Hun; Lee, Chien Liang; He, Chun Lin; Hsu, Chia Jung; Lee, Zheng Yan; Li, Zheng Yuan; Lin, Marie

2005-08-01

Genetic affinities between aboriginal Taiwanese and populations from Oceania and Southeast Asia have previously been explored through analyses of mitochondrial DNA (mtDNA), Y chromosomal DNA, and human leukocyte antigen loci. Recent genetic studies have supported the "slow boat" and "entangled bank" models according to which the Polynesian migration can be seen as an expansion from Melanesia without any major direct genetic thread leading back to its initiation from Taiwan. We assessed mtDNA variation in 640 individuals from nine tribes of the central mountain ranges and east coast regions of Taiwan. In contrast to the Han populations, the tribes showed a low frequency of haplogroups D4 and G, and an absence of haplogroups A, C, Z, M9, and M10. Also, more than 85% of the maternal lineages were nested within haplogroups B4, B5a, F1a, F3b, E, and M7. Although indicating a common origin of the populations of insular Southeast Asia and Oceania, most mtDNA lineages in Taiwanese aboriginal populations are grouped separately from those found in China and the Taiwan general (Han) population, suggesting a prevalence in the Taiwanese aboriginal gene pool of its initial late Pleistocene settlers. Interestingly, from complete mtDNA sequencing information, most B4a lineages were associated with three coding region substitutions, defining a new subclade, B4a1a, that endorses the origin of Polynesian migration from Taiwan. Coalescence times of B4a1a were 13.2 +/- 3.8 thousand years (or 9.3 +/- 2.5 thousand years in Papuans and Polynesians). Considering the lack of a common specific Y chromosomal element shared by the Taiwanese aboriginals and Polynesians, the mtDNA evidence provided here is also consistent with the suggestion that the proto-Oceanic societies would have been mainly matrilocal.

Validation of simple and cost-effective stains to assess acrosomal status, DNA damage and mitochondrial activity in rooster spermatozoa.

PubMed

Rui, Bruno R; Angrimani, Daniel S R; Losano, João Diego A; Bicudo, Luana de Cássia; Nichi, Marcílio; Pereira, Ricardo J G

2017-12-01

Several methods have been developed to evaluate spermatozoa function in birds but many of these are sometimes complicated, costly and not applicable to field studies (i.e., performed within poultry breeding facilities). The objective was, therefore, to validate efficient, practical and inexpensive procedures to determine DNA fragmentation, acrosomal integrity, and mitochondrial activity in poultry spermatozoa. Initially, ejaculates were individually diluted and divided into control (4°C, 4h) and UV-irradiated aliquots (room temperature, 4h), and then samples containing different percentages of DNA-damaged spermatozoa (0%, 25%, 50%, 75% and 100%) were subjected to Toluidine Blue (TB) and Sperm Chromatin Dispersion assessments (SCD). Fast Green-Rose Bengal (FG-RB) and FITC-PSA staining protocols were subsequently used to assess acrosome status in aliquots comprising assorted amounts of acrosome-reacted spermatozoa. Furthermore, to validate 3,3'-diaminobenzidine (DAB) assay, ejaculates containing different gradients of spermatozoa with great amounts of mitochondrial activity were concurrently evaluated using DAB and JC-1 stains. The proportion of spermatozoa with abnormal DNA integrity when evaluated using the TB assessment correlated significantly with the expected percentages of UV-irradiated spermatozoa and with SCD results. A significant linear regression coefficient was also observed between expected amounts of acrosome-intact spermatozoa and FG-RB readings, and there was a significant correlation of the data when FG-RB and FITC-PSA were used. Likewise, the use of the DAB assay enabled for accurately ascertaining percentages of rooster spermatozoa with greater and lesser mitochondrial function, and results were highly correlated to results with staining with JC-1. Altogether, findings of the present study indicate acrosomal status, DNA integrity and mitochondrial activity in rooster spermatozoa can be easily and reliably determined using FG-RB, TB and DAB stains. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and validation of a modified comet assay to phenotypically assess nucleotide excision repair.

PubMed

Langie, Sabine A S; Knaapen, Ad M; Brauers, Karen J J; van Berlo, Damien; van Schooten, Frederik-Jan; Godschalk, Roger W L

2006-03-01

There is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacities. Therefore, a modification of the alkaline comet assay was developed to determine the ability of human lymphocyte extracts to perform the initial steps of the nucleotide excision repair (NER) process, i.e. damage recognition and incision. Gel-embedded nucleoids from A549 cells, pre-exposed to 1 microM benzo[a]pyrene-diol-epoxide, were incubated with cell extracts from frozen or freshly isolated lymphocytes. The rate at which incisions are introduced and the subsequent increase in tail moment is indicative for the repair capacity of the extracts. Freshly prepared extracts from lymphocytes of human volunteers (n = 8) showed significant inter-individual variations in their DNA repair capacity, which correlated with the removal of bulky DNA lesions over a period of 48 h determined by (32)P-post-labelling (R(2) = 0.76, P = 0.005). Repeated measurements revealed a low inter-assay variation (11%). Storage of cell extracts for more than 3 weeks significantly reduced (up to 80%) the capacity to incise the damaged DNA as compared to freshly isolated extracts. This reduction was completely restored by addition of ATP to the extracts before use, as it is required for the incision step of NER. In contrast, extracts freshly prepared from frozen lymphocyte pellets can be used without loss of repair activity. DNA repair deficient XPA-/- and XPC-/- fibroblasts were used to further validate the assay. Although some residual capacity to incise the DNA was observed in these cells, the repair activity was restored to normal wild-type levels when a complementary mixture of both extracts (thereby restoring XPA and XPC deficiency) was used. These results demonstrate that this repair assay can be applied in molecular epidemiological studies to assess inter-individual differences in NER.

Safety assessment and public concerns for genetically modified food products: the Japanese experience.

PubMed

Hino, Akihiro

2002-01-01

The recombinant DNA (rDNA) technique is expected to bring about great progress in the improvement of breeding technology and the development of new plant varieties showing high quality and high yield, such as those with excellent pest and disease resistance, those with environmental stress tolerance, and so forth. In the United States and Canada, many genetically modified (GM) crop plants were commercialized as early as 1994. In Japan, 35 transgenic crop plants, such as herbicide tolerant soybean, cotton, and canola, and insect-resistant corn, cotton, and potatos, were authorized and considered marketable until April 2001. The general public, however, is not familiar with rDNA technology, and some people seem to feel uncomfortable with biotechnology, frequently because of the difficulty of the technology and lacking of sufficient information. New labeling systems were initiated in April 2001 in Japan to provide information regarding the use of GM crops as raw material.

Structures of RNA Polymerase Closed and Intermediate Complexes Reveal Mechanisms of DNA Opening and Transcription Initiation.

PubMed

Glyde, Robert; Ye, Fuzhou; Darbari, Vidya Chandran; Zhang, Nan; Buck, Martin; Zhang, Xiaodong

2017-07-06

Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ 54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ 54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ 54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Diversification of DnaA dependency for DNA replication in cyanobacterial evolution.

PubMed

Ohbayashi, Ryudo; Watanabe, Satoru; Ehira, Shigeki; Kanesaki, Yu; Chibazakura, Taku; Yoshikawa, Hirofumi

2016-05-01

Regulating DNA replication is essential for all living cells. The DNA replication initiation factor DnaA is highly conserved in prokaryotes and is required for accurate initiation of chromosomal replication at oriC. DnaA-independent free-living bacteria have not been identified. The dnaA gene is absent in plastids and some symbiotic bacteria, although it is not known when or how DnaA-independent mechanisms were acquired. Here, we show that the degree of dependency of DNA replication on DnaA varies among cyanobacterial species. Deletion of the dnaA gene in Synechococcus elongatus PCC 7942 shifted DNA replication from oriC to a different site as a result of the integration of an episomal plasmid. Moreover, viability during the stationary phase was higher in dnaA disruptants than in wild-type cells. Deletion of dnaA did not affect DNA replication or cell growth in Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120, indicating that functional dependency on DnaA was already lost in some nonsymbiotic cyanobacterial lineages during diversification. Therefore, we proposed that cyanobacteria acquired DnaA-independent replication mechanisms before symbiosis and such an ancestral cyanobacterium was the sole primary endosymbiont to form a plastid precursor.

Downstream DNA Tension Regulates the Stability of the T7 RNA Polymerase Initiation Complex

PubMed Central

Skinner, Gary M.; Kalafut, Bennett S.; Visscher, Koen

2011-01-01

Gene transcription by the enzyme RNA polymerase is tightly regulated. In many cases, such as in the lac operon in Escherichia coli, this regulation is achieved through the action of protein factors on DNA. Because DNA is an elastic polymer, its response to enzymatic processing can lead to mechanical perturbations (e.g., linear stretching and supercoiling) that can affect the operation of other DNA processing complexes acting elsewhere on the same substrate molecule. Using an optical-tweezers assay, we measured the binding kinetics between single molecules of bacteriophage T7 RNA polymerase and DNA, as a function of tension. We found that increasing DNA tension under conditions that favor formation of the open complex results in destabilization of the preinitiation complex. Furthermore, with zero ribonucleotides present, when the closed complex is favored, we find reduced tension sensitivity, implying that it is predominantly the open complex that is sensitive. This result strongly supports the “scrunching” model for T7 transcription initiation, as the applied tension acts against the movement of the DNA into the scrunched state, and introduces linear DNA tension as a potential regulatory quantity for transcription initiation. PMID:21320448

Effects of activated aflatoxin B/sub 1/ and caffeine on DNA replicon initiation in HeLa cells

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

Cramer, P.; Painter, R.B.

1981-01-01

Afatoxin B/sub 1/ (AFB/sub 1/) is activated by a rat microsomal extract (S-9) to form a product that inhibits DNA synthesis in HeLa cells. At 10/sup -7/ M, AFB/sub 1/ inhibited initiation of replicons, as shown in alkaline sucrose gradient profiles 30 min after incubation with the drug. Ninety minutes later, the profile of treated cells was similar to that of control, but 4 h later there was another effect on replicon initiation. At 10/sup -6/ M, the inhibition of initiation was greater than at 10/sup -7/ M and increased progressively. Four hours after removal of the drug, the gradientmore » profile showed low amounts of radioactivity in all size classes of DNA. When cells were incubated in medium containing caffeine (2 mM) even as late as 60 min after incubation with AFB/sub 1/, the inhibition of replicon initiation was prevented. If caffeine was later removed from the medium, replicon initiation was then inhibited. At 10/sup -7/ M or 10/sup -6/ M, AFB/sub 1/ had little immediate effect on chain elongation, but at 10/sup -5/ M, the gradient profiles showed an accumulation of low molecular weight DNA molecules, with no radioactivity in the region of high molecular weight DNA, owing to a block to chain elongation; this was not affected by caffeine. These results suggest that AFB/sub 1/ induces damage that changes the fonformation of chromatin so that initiation of new replicons cannot occur; in the presence of caffeine this change does not occur and DNA replication is not inhibited.« less

Regulated Eukaryotic DNA Replication Origin Firing with Purified Proteins

PubMed Central

Yeeles, Joseph T.P.; Deegan, Tom D.; Janska, Agnieszka; Early, Anne; Diffley, John F. X.

2016-01-01

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric MCM complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45, MCM, GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4 dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication. PMID:25739503

Regulated eukaryotic DNA replication origin firing with purified proteins.

PubMed

Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

2015-03-26

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication

PubMed Central

Spiering, Michelle M.; Hanoian, Philip; Gannavaram, Swathi; Benkovic, Stephen J.

2017-01-01

The opposite strand polarity of duplex DNA necessitates that the leading strand is replicated continuously whereas the lagging strand is replicated in discrete segments known as Okazaki fragments. The lagging-strand polymerase sometimes recycles to begin the synthesis of a new Okazaki fragment before finishing the previous fragment, creating a gap between the Okazaki fragments. The mechanism and signal that initiate this behavior—that is, the signaling mechanism—have not been definitively identified. We examined the role of RNA primer–primase complexes left on the lagging ssDNA from primer synthesis in initiating early lagging-strand polymerase recycling. We show for the T4 bacteriophage DNA replication system that primer–primase complexes have a residence time similar to the timescale of Okazaki fragment synthesis and the ability to block a holoenzyme synthesizing DNA and stimulate the dissociation of the holoenzyme to trigger polymerase recycling. The collision with primer–primase complexes triggering the early termination of Okazaki fragment synthesis has distinct advantages over those previously proposed because this signal requires no transmission to the lagging-strand polymerase through protein or DNA interactions, the mechanism for rapid dissociation of the holoenzyme is always collision, and no unique characteristics need to be assigned to either identical polymerase in the replisome. We have modeled repeated cycles of Okazaki fragment initiation using a collision with a completed Okazaki fragment or primer–primase complexes as the recycling mechanism. The results reproduce experimental data, providing insights into events related to Okazaki fragment initiation and the overall functioning of DNA replisomes. PMID:28507156

RNA primer-primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication.

PubMed

Spiering, Michelle M; Hanoian, Philip; Gannavaram, Swathi; Benkovic, Stephen J

2017-05-30

The opposite strand polarity of duplex DNA necessitates that the leading strand is replicated continuously whereas the lagging strand is replicated in discrete segments known as Okazaki fragments. The lagging-strand polymerase sometimes recycles to begin the synthesis of a new Okazaki fragment before finishing the previous fragment, creating a gap between the Okazaki fragments. The mechanism and signal that initiate this behavior-that is, the signaling mechanism-have not been definitively identified. We examined the role of RNA primer-primase complexes left on the lagging ssDNA from primer synthesis in initiating early lagging-strand polymerase recycling. We show for the T4 bacteriophage DNA replication system that primer-primase complexes have a residence time similar to the timescale of Okazaki fragment synthesis and the ability to block a holoenzyme synthesizing DNA and stimulate the dissociation of the holoenzyme to trigger polymerase recycling. The collision with primer-primase complexes triggering the early termination of Okazaki fragment synthesis has distinct advantages over those previously proposed because this signal requires no transmission to the lagging-strand polymerase through protein or DNA interactions, the mechanism for rapid dissociation of the holoenzyme is always collision, and no unique characteristics need to be assigned to either identical polymerase in the replisome. We have modeled repeated cycles of Okazaki fragment initiation using a collision with a completed Okazaki fragment or primer-primase complexes as the recycling mechanism. The results reproduce experimental data, providing insights into events related to Okazaki fragment initiation and the overall functioning of DNA replisomes.

Bacterial DNA induces pulmonary damage via TLR-9 through cross-talk with neutrophils.

PubMed

Itagaki, Kiyoshi; Adibnia, Yasaman; Sun, Shiqin; Zhao, Cong; Sursal, Tolga; Chen, Yu; Junger, Wolfgang; Hauser, Carl J

2011-12-01

Bacterial DNA (bDNA) contains hypomethylated "CpG" repeats that can be recognized by Toll-like receptor 9 (TLR-9) as a pathogen-associated molecular pattern. The ability of bDNA to initiate lung injury via TLR-9 has been inferred on the basis of studies using artificial CpG DNA. But the role of authentic bDNA in lung injury is still unknown. Moreover, the mechanisms by which CpG DNA species can lead to pulmonary injury are unknown, although neutrophils (PMNs) are thought to play a key role in the genesis of septic acute lung injury. We evaluated the effects of bDNA on PMN-endothelial cell (EC) interactions thought critical for initiation of acute lung injury. Using a biocapacitance system to monitor real-time changes in endothelial permeability, we demonstrate here that bDNA causes EC permeability in a dose-dependent manner uniquely in the presence of PMNs. These permeability changes are inhibited by chloroquine, suggesting TLR-9 dependency. When PMNs were preincubated with bDNA and applied to ECs or when bDNA was applied to ECs without PMNs, no permeability changes were detected. To study the underlying mechanisms, we evaluated the effects of bDNA on PMN-EC adherence. Bacterial DNA significantly increased PMN adherence to ECs in association with upregulated adhesion molecules in both cell types. Taken together, our results strongly support the conclusion that bDNA can initiate lung injury by stimulating PMN-EC adhesive interactions predisposing to endothelial permeability. Bacterial DNA stimulation of TLR-9 appears to promote enhanced gene expression of adhesion molecules in both cell types. This leads to PMN-EC cross-talk, which is required for injury to occur.

Helicase promotes replication re-initiation from an RNA transcript.

PubMed

Sun, Bo; Singh, Anupam; Sultana, Shemaila; Inman, James T; Patel, Smita S; Wang, Michelle D

2018-06-13

To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind. However, little is known about how DNA synthesis is resumed downstream of an obstacle. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances, the associated DNAP also moves forward. The presence of the DNAP increases both helicase's processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicase's novel role in replication re-initiation.

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

PubMed

Löb, D; Lengert, N; Chagin, V O; Reinhart, M; Casas-Delucchi, C S; Cardoso, M C; Drossel, B

2016-04-07

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase.

Initiation of DNA replication requires actin dynamics and formin activity.

PubMed

Parisis, Nikolaos; Krasinska, Liliana; Harker, Bethany; Urbach, Serge; Rossignol, Michel; Camasses, Alain; Dewar, James; Morin, Nathalie; Fisher, Daniel

2017-11-02

Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP-importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms. © 2017 The Authors.

The Neandertal genome and ancient DNA authenticity

PubMed Central

Green, Richard E; Briggs, Adrian W; Krause, Johannes; Prüfer, Kay; Burbano, Hernán A; Siebauer, Michael; Lachmann, Michael; Pääbo, Svante

2009-01-01

Recent advances in high-thoughput DNA sequencing have made genome-scale analyses of genomes of extinct organisms possible. With these new opportunities come new difficulties in assessing the authenticity of the DNA sequences retrieved. We discuss how these difficulties can be addressed, particularly with regard to analyses of the Neandertal genome. We argue that only direct assays of DNA sequence positions in which Neandertals differ from all contemporary humans can serve as a reliable means to estimate human contamination. Indirect measures, such as the extent of DNA fragmentation, nucleotide misincorporations, or comparison of derived allele frequencies in different fragment size classes, are unreliable. Fortunately, interim approaches based on mtDNA differences between Neandertals and current humans, detection of male contamination through Y chromosomal sequences, and repeated sequencing from the same fossil to detect autosomal contamination allow initial large-scale sequencing of Neandertal genomes. This will result in the discovery of fixed differences in the nuclear genome between Neandertals and current humans that can serve as future direct assays for contamination. For analyses of other fossil hominins, which may become possible in the future, we suggest a similar ‘boot-strap' approach in which interim approaches are applied until sufficient data for more definitive direct assays are acquired. PMID:19661919

Towards a comprehensive barcode library for arctic life - Ephemeroptera, Plecoptera, and Trichoptera of Churchill, Manitoba, Canada

PubMed Central

2009-01-01

Background This study reports progress in assembling a DNA barcode reference library for Ephemeroptera, Plecoptera, and Trichoptera ("EPTs") from a Canadian subarctic site, which is the focus of a comprehensive biodiversity inventory using DNA barcoding. These three groups of aquatic insects exhibit a moderate level of species diversity, making them ideal for testing the feasibility of DNA barcoding for routine biotic surveys. We explore the correlation between the morphological species delineations, DNA barcode-based haplotype clusters delimited by a sequence threshold (2%), and a threshold-free approach to biodiversity quantification--phylogenetic diversity. Results A DNA barcode reference library is built for 112 EPT species for the focal region, consisting of 2277 COI sequences. Close correspondence was found between EPT morphospecies and haplotype clusters as designated using a standard threshold value. Similarly, the shapes of taxon accumulation curves based upon haplotype clusters were very similar to those generated using phylogenetic diversity accumulation curves, but were much more computationally efficient. Conclusion The results of this study will facilitate other lines of research on northern EPTs and also bode well for rapidly conducting initial biodiversity assessments in unknown EPT faunas. PMID:20003245

Quantitative Profiling of DNA Damage and Apoptotic Pathways in UV Damaged Cells Using PTMScan Direct

PubMed Central

Stokes, Matthew P.; Silva, Jeffrey C.; Jia, Xiaoying; Lee, Kimberly A.; Polakiewicz, Roberto D.; Comb, Michael J.

2013-01-01

Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA. PMID:23344034

Molecular assessment of heterotrophy and prey digestion in zooxanthellate cnidarians.

PubMed

Leal, M C; Nejstgaard, J C; Calado, R; Thompson, M E; Frischer, M E

2014-08-01

Zooxanthellate cnidarians are trophically complex, relying on both autotrophy and heterotrophy. Although several aspects of heterotrophy have been studied in these organisms, information linking prey capture with digestion is still missing. We used prey-specific PCR-based tools to assess feeding and prey digestion of two zooxanthellate cnidarians - the tropical sea anemone Aiptasia sp. and the scleractinian coral Oculina arbuscula. Prey DNA disappeared rapidly for the initial 1-3 days, whereas complete digestion of prey DNA required up to 10 days in O. arbuscula and 5 or 6 days in Aiptasia sp. depending on prey species. These digestion times are considerably longer than previously reported from microscopy-based examination of zooxanthellate cnidarians and prey DNA breakdown in other marine invertebrates, but similar to prey DNA breakdown reported from terrestrial invertebrates such as heteroptera and spiders. Deprivation of external prey induced increased digestion rates during the first days after feeding in O. arbuscula, but after 6 days of digestion, there were no differences in the remaining prey levels in fed and unfed corals. This study indicates that prey digestion by symbiotic corals may be slower than previously reported and varies with the type of prey, the cnidarian species and its feeding history. These observations have important implications for bioenergetic and trophodynamic studies on zooxanthellate cnidarians. © 2013 John Wiley & Sons Ltd.

Repetitive transpositions of mitochondrial DNA sequences to the nucleus during the radiation of horseshoe bats (Rhinolophus, Chiroptera).

PubMed

Shi, Huizhen; Dong, Ji; Irwin, David M; Zhang, Shuyi; Mao, Xiuguang

2016-05-01

Transposition of mitochondrial DNA into the nucleus, which gives rise to nuclear mitochondrial DNAs (NUMTs), has been well documented in eukaryotes. However, very few studies have assessed the frequency of these transpositions during the evolutionary history of a specific taxonomic group. Here we used the horseshoe bats (Rhinolophus) as a case study to determine the frequency and relative timing of nuclear transfers of mitochondrial control region sequences. For this, phylogenetic and coalescent analyzes were performed on NUMTs and authentic mtDNA sequences generated from eight horseshoe bat species. Our results suggest at least three independent transpositions, including two ancient and one more recent, during the evolutionary history of Rhinolophus. The two ancient transpositions are represented by the NUMT-1 and -2 clades, with each clade consisting of NUMTs from almost all studied species but originating from different portions of the mtDNA genome. Furthermore, estimates of the most recent common ancestor for each clade corresponded to the time of the initial diversification of this genus. The recent transposition is represented by NUMT-3, which was discovered only in a specific subgroup of Rhinolophus and exhibited a close relationship to its mitochondrial counterpart. Our similarity searches of mtDNA in the R. ferrumequinum genome confirmed the presence of NUMT-1 and NUMT-2 clade sequences and, for the first time, assessed the extent of NUMTs in a bat genome. To our knowledge, this is the first study to report on the frequency of transpositions of mtDNA occurring before the common ancestry of a genus. Copyright © 2016 Elsevier B.V. All rights reserved.

Low concentration of arsenite exacerbates UVR-induced DNA strand breaks by inhibiting PARP-1 activity

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

Qin Xujun; Department of Toxicology, Fourth Military Medical University, Xi'an, Shaanxi, 710032; Hudson, Laurie G.

2008-10-01

Epidemiological studies have associated arsenic exposure with many types of human cancers. Arsenic has also been shown to act as a co-carcinogen even at low concentrations. However, the precise mechanism of its co-carcinogenic action is unknown. Recent studies indicate that arsenic can interfere with DNA-repair processes. Poly(ADP-ribose) polymerase (PARP)-1 is a zinc-finger DNA-repair protein, which can promptly sense DNA strand breaks and initiate DNA-repair pathways. In the present study, we tested the hypothesis that low concentrations of arsenic could inhibit PAPR-1 activity and so exacerbate levels of ultraviolet radiation (UVR)-induced DNA strand breaks. HaCat cells were treated with arsenite and/ormore » UVR, and then DNA strand breaks were assessed by comet assay. Low concentrations of arsenite ({<=} 2 {mu}M) alone did not induce significant DNA strand breaks, but greatly enhanced the DNA strand breaks induced by UVR. Further studies showed that 2 {mu}M arsenite effectively inhibited PARP-1 activity. Zinc supplementation of arsenite-treated cells restored PARP-1 activity and significantly diminished the exacerbating effect of arsenite on UVR-induced DNA strand breaks. Importantly, neither arsenite treatment, nor zinc supplementation changed UVR-triggered reactive oxygen species (ROS) formation, suggesting that their effects upon UVR-induced DNA strand breaks are not through a direct free radical mechanism. Combination treatments of arsenite with PARP-1 inhibitor 3-aminobenzamide or PARP-1 siRNA demonstrate that PARP-1 is the target of arsenite. Together, these findings show that arsenite at low concentration exacerbates UVR-induced DNA strand breaks by inhibiting PARP-1 activity, which may represent an important mechanism underlying the co-carcinogenicity of arsenic.« less

Is the detection of aquatic environmental DNA influenced by substrate type?

PubMed

Buxton, Andrew S; Groombridge, Jim J; Griffiths, Richard A

2017-01-01

The use of environmental DNA (eDNA) to assess the presence-absence of rare, cryptic or invasive species is hindered by a poor understanding of the factors that can remove DNA from the system. In aquatic systems, eDNA can be transported out either horizontally in water flows or vertically by incorporation into the sediment. Equally, eDNA may be broken down by various biotic and abiotic processes if the target organism leaves the system. We use occupancy modelling and a replicated mesocosm experiment to examine how detection probability of eDNA changes once the target species is no longer present. We hypothesise that detection probability falls faster with a sediment which has a large number of DNA binding sites such as topsoil or clay, over lower DNA binding capacity substrates such as sand. Water removed from ponds containing the target species (the great crested newt) initially showed high detection probabilities, but these fell to between 40% and 60% over the first 10 days and to between 10% and 22% by day 15: eDNA remained detectable at very low levels until day 22. Very little difference in detection was observed between the control group (no substrate) and the sand substrate. A small reduction in detection probability was observed between the control and clay substrates, but this was not significant. However, a highly significant reduction in detection probability was observed with a topsoil substrate. This result is likely to have stemmed from increased levels of PCR inhibition, suggesting that incorporation of DNA into the sentiment is of only limited importance. Surveys of aquatic species using eDNA clearly need to take account of substrate type as well as other environmental factors when collecting samples, analysing data and interpreting the results.

Regulation and function of DNA methylation in plants and animals

PubMed Central

He, Xin-Jian; Chen, Taiping; Zhu, Jian-Kang

2011-01-01

DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. PMID:21321601

How MCM loading and spreading specify eukaryotic DNA replication initiation sites.

PubMed

Hyrien, Olivier

2016-01-01

DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

Detection of Circulating Mucorales DNA in Critically Ill Burn Patients: Preliminary Report of a Screening Strategy for Early Diagnosis and Treatment.

PubMed

Legrand, Matthieu; Gits-Muselli, Maud; Boutin, Louis; Garcia-Hermoso, Dea; Maurel, Véronique; Soussi, Sabri; Benyamina, Mourad; Ferry, Axelle; Chaussard, Maïté; Hamane, Samia; Denis, Blandine; Touratier, Sophie; Guigue, Nicolas; Fréalle, Emilie; Jeanne, Mathieu; Shaal, Jean-Vivien; Soler, Charles; Mimoun, Maurice; Chaouat, Marc; Lafaurie, Matthieu; Mebazaa, Alexandre; Bretagne, Stéphane; Alanio, Alexandre

2016-11-15

 Invasive wound mucormycosis (IWM) is associated with an extremely poor outcome among critically ill burn patients. We describe the detection of circulating Mucorales DNA (cmDNA) for the early diagnosis of IWM in those patients and report the potential value of detecting cmDNA for treatment guidance.  Severely ill burn patients admitted to our tertiary referral center between October 2013 and February 2016 were included. Retrospective plasma samples were tested for the presence of cmDNA by quantitative real-time polymerase chain reaction (qPCR). Patients were then prospectively screened twice a week, and liposomal amphotericin-B therapy initiated based on a positive qPCR. The primary endpoint was the time between cmDNA detection and standard diagnosis. Secondary endpoints were the time from cmDNA detection and treatment initiation and mortality.  Seventy-seven patients (418 samples) were included. The average age was 46 (28-60) years, abbreviated burn severity index was 8 (7-10), and simplified acute physiology score was 33 (23-46). The total body surface area was 33% (22%-52%). cmDNA was detected 11 (4.5-15) days before standard diagnosis. The in-hospital mortality was 62% for patients with IWM and 24% for those without (P = .03). The mortality due to IWM was 80% during period A and 33% during period B (P = .46).  This study suggests that the detection of cmDNA allows earlier diagnosis of IWM in severely ill burn patients and earlier initiation of treatment. Further studies are needed to confirm the impact of earlier treatment initiation on patient outcome. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Conformational plasticity of RepB, the replication initiator protein of promiscuous streptococcal plasmid pMV158

PubMed Central

Boer, D. Roeland; Ruiz-Masó, José Angel; Rueda, Manuel; Petoukhov, Maxim V.; Machón, Cristina; Svergun, Dmitri I.; Orozco, Modesto; del Solar, Gloria; Coll, Miquel

2016-01-01

DNA replication initiation is a vital and tightly regulated step in all replicons and requires an initiator factor that specifically recognizes the DNA replication origin and starts replication. RepB from the promiscuous streptococcal plasmid pMV158 is a hexameric ring protein evolutionary related to viral initiators. Here we explore the conformational plasticity of the RepB hexamer by i) SAXS, ii) sedimentation experiments, iii) molecular simulations and iv) X-ray crystallography. Combining these techniques, we derive an estimate of the conformational ensemble in solution showing that the C-terminal oligomerisation domains of the protein form a rigid cylindrical scaffold to which the N-terminal DNA-binding/catalytic domains are attached as highly flexible appendages, featuring multiple orientations. In addition, we show that the hinge region connecting both domains plays a pivotal role in the observed plasticity. Sequence comparisons and a literature survey show that this hinge region could exists in other initiators, suggesting that it is a common, crucial structural element for DNA binding and manipulation. PMID:26875695

The potential role of ribosomal protein S5 on cell cycle arrest and initiation of murine erythroleukemia cell differentiation.

PubMed

Matragkou, Christina N; Papachristou, Eleni T; Tezias, Sotirios S; Tsiftsoglou, Asterios S; Choli-Papadopoulou, Theodora; Vizirianakis, Ioannis S

2008-07-01

Evidence now exists to indicate that some ribosomal proteins besides being structural components of the ribosomal subunits are involved in the regulation of cell differentiation and apoptosis. As we have shown earlier, initiation of erythroid differentiation of murine erythroleukemia (MEL) cells is associated with transcriptional inactivation of genes encoding ribosomal RNAs and ribosomal proteins S5 (RPS5) and L35a. In this study, we extended these observations and investigated whether transfection of MEL cells with RPS5 cDNA affects the onset of initiation of erythroid maturation and their entrance in cell cycle arrest. Stably transfected MEL cloned cells (MEL-C14 and MEL-C56) were established and assessed for their capacity to produce RPS5 RNA transcript and its translated product. The impact of RPS5 cDNA transfection on the RPS5 gene expression patterns and the accumulation of RPS5 protein in inducible transfected MEL cells were correlated with their ability to: (a) initiate differentiation, (b) enter cell cycle arrest at G(1)/G(0) phase, and (c) modulate the level of cyclin-dependent kinases CDK2, CDK4, and CDK6. The data presented indicate that deregulation of RPS5 gene expression (constitutive expression) affects RPS5 protein level and delays both the onset of initiation of erythroid maturation and entrance in cell cycle arrest in inducer-treated MEL cells. 2008 Wiley-Liss, Inc.

Distinct Mechanisms of Transcription Initiation by RNA Polymerases I and II.

PubMed

Engel, Christoph; Neyer, Simon; Cramer, Patrick

2018-05-20

RNA polymerases I and II (Pol I and Pol II) are the eukaryotic enzymes that catalyze DNA-dependent synthesis of ribosomal RNA and messenger RNA, respectively. Recent work shows that the transcribing forms of both enzymes are similar and the fundamental mechanisms of RNA chain elongation are conserved. However, the mechanisms of transcription initiation and its regulation differ between Pol I and Pol II. Recent structural studies of Pol I complexes with transcription initiation factors provided insights into how the polymerase recognizes its specific promoter DNA, how it may open DNA, and how initiation may be regulated. Comparison with the well-studied Pol II initiation system reveals a distinct architecture of the initiation complex and visualizes promoter- and gene-class-specific aspects of transcription initiation. On the basis of new structural studies, we derive a model of the Pol I transcription cycle and provide a molecular movie of Pol I transcription that can be used for teaching.

Suppression of the Escherichia coli dnaA46 mutation by changes in the activities of the pyruvate-acetate node links DNA replication regulation to central carbon metabolism.

PubMed

Tymecka-Mulik, Joanna; Boss, Lidia; Maciąg-Dorszyńska, Monika; Matias Rodrigues, João F; Gaffke, Lidia; Wosinski, Anna; Cech, Grzegorz M; Szalewska-Pałasz, Agnieszka; Węgrzyn, Grzegorz; Glinkowska, Monika

2017-01-01

To ensure faithful transmission of genetic material to progeny cells, DNA replication is tightly regulated, mainly at the initiation step. Escherichia coli cells regulate the frequency of initiation according to growth conditions. Results of the classical, as well as the latest studies, suggest that the DNA replication in E. coli starts at a predefined, constant cell volume per chromosome but the mechanisms coordinating DNA replication with cell growth are still not fully understood. Results of recent investigations have revealed a role of metabolic pathway proteins in the control of cell division and a direct link between metabolism and DNA replication has also been suggested both in Bacillus subtilis and E. coli cells. In this work we show that defects in the acetate overflow pathway suppress the temperature-sensitivity of a defective replication initiator-DnaA under acetogenic growth conditions. Transcriptomic and metabolic analyses imply that this suppression is correlated with pyruvate accumulation, resulting from alterations in the pyruvate dehydrogenase (PDH) activity. Consequently, deletion of genes encoding the pyruvate dehydrogenase subunits likewise resulted in suppression of the thermal-sensitive growth of the dnaA46 strain. We propose that the suppressor effect may be directly related to the PDH complex activity, providing a link between an enzyme of the central carbon metabolism and DNA replication.

Prevalence and Persistence of Cervical Human Papillomavirus Infection In HIV-Positive Women Initiating Highly-Active Antiretroviral Therapy

PubMed Central

Fife, Kenneth H.; Wu, Julia W.; Squires, Kathleen E.; Watts, D. Heather; Andersen, Janet W.; Brown, Darron R.

2009-01-01

Objective To determine the prevalence of HPV DNA in cervical specimens from treatment-naïve women initiating highly active antiretroviral therapy (HAART) and explore the longitudinal association of HPV DNA with CD4 count and HIV viral load (VL). Methods Women enrolled prior to HAART were evaluated at baseline, weeks 24, 48, and 96 with CD4 count, VL, and cervical swab for HPV DNA. Results The 146 subjects had a median CD4 count of 238 cells/μL and VL of 13,894 copies/mL. Ninety-seven (66%) subjects had HPV DNA detected in the baseline specimen including 90 subjects (62%) positive for one or more high risk HPV types. HPV DNA detection declined to 49% at week 96, and that of a high risk HPV type to 39%. The duration of follow-up was associated with decreased detection of HPV DNA of any type (p=0.045) and of high risk HPV types (p=0.003). There was at most a marginal association between HAART response and loss of detection of cervical HPV DNA. Conclusions Women initiating HAART had a high prevalence of cervical HPV DNA that declined over 96 weeks of HAART. The relationship of CD4 count and VL response to the decline of cervical HPV DNA was not strong. PMID:19387354

A novel chaotic image encryption scheme using DNA sequence operations

NASA Astrophysics Data System (ADS)

Wang, Xing-Yuan; Zhang, Ying-Qian; Bao, Xue-Mei

2015-10-01

In this paper, we propose a novel image encryption scheme based on DNA (Deoxyribonucleic acid) sequence operations and chaotic system. Firstly, we perform bitwise exclusive OR operation on the pixels of the plain image using the pseudorandom sequences produced by the spatiotemporal chaos system, i.e., CML (coupled map lattice). Secondly, a DNA matrix is obtained by encoding the confused image using a kind of DNA encoding rule. Then we generate the new initial conditions of the CML according to this DNA matrix and the previous initial conditions, which can make the encryption result closely depend on every pixel of the plain image. Thirdly, the rows and columns of the DNA matrix are permuted. Then, the permuted DNA matrix is confused once again. At last, after decoding the confused DNA matrix using a kind of DNA decoding rule, we obtain the ciphered image. Experimental results and theoretical analysis show that the scheme is able to resist various attacks, so it has extraordinarily high security.

Allostery through protein-induced DNA bubbles

DOE PAGES

Traverso, Joseph J.; Manoranjan, Valipuram S.; Bishop, A. R.; ...

2015-03-12

Allostery through DNA is increasingly recognized as an important modulator of DNA functions. Here, we show that the coalescence of protein-induced DNA bubbles can mediate allosteric interactions that drive protein aggregation. We propose that such allostery may regulate DNA's flexibility and the assembly of the transcription machinery. Mitochondrial transcription factor A (TFAM), a dual-function protein involved in mitochondrial DNA (mtDNA) packaging and transcription initiation, is an ideal candidate to test such a hypothesis owing to its ability to locally unwind the double helix. Numerical simulations demonstrate that the coalescence of TFAM-induced bubbles can explain experimentally observed TFAM oligomerization. The resultingmore » melted DNA segment, approximately 10 base pairs long, around the joints of the oligomers act as flexible hinges, which explains the efficiency of TFAM in compacting DNA. Since mitochondrial polymerase (mitoRNAP) is involved in melting the transcription bubble, TFAM may use the same allosteric interaction to both recruit mitoRNAP and initiate transcription.« less

Microsatellite DNA markers for delineating population structure and kinship among the endangered Kirtland's warbler (Dendroica kirtlandii)

USGS Publications Warehouse

King, T.L.; Eackles, M.S.; Henderson, A.P.; Bocetti, Carol I.; Currie, D.; Wunderle, J.M.

2005-01-01

We document the isolation and characterization of 23 microsatellite DNA markers for the endangered Kirtland's warbler (Dendroica kirtlandii), a Nearctic/Neotropical migrant passerine. This suite of markers revealed moderate to high levels of allelic diversity (averaging 7.7 alleles per locus) and heterozygosity (averaging 72%). Genotypic frequencies at 22 of 23 (95%) markers conformed to Hardy-Weinberg equilibrium expectations, and no linkage disequilibrium was observed in blood samples taken from 14 warblers found on the wintering grounds in the Bahamas archipelago. Multilocus genotypes resulting from this suite of markers should reduce the amount of resources required for initiating new genetic studies assessing breeding structure, parentage, demographics, and individual-level ecological interactions for D. kirtlandii. ?? 2005 Blackwell Publishing Ltd.

Mitochondrial DNA Depletion in Respiratory Chain-Deficient Parkinson Disease Neurons.

PubMed

Grünewald, Anne; Rygiel, Karolina A; Hepplewhite, Philippa D; Morris, Christopher M; Picard, Martin; Turnbull, Doug M

2016-03-01

To determine the extent of respiratory chain abnormalities and investigate the contribution of mtDNA to the loss of respiratory chain complexes (CI-IV) in the substantia nigra (SN) of idiopathic Parkinson disease (IPD) patients at the single-neuron level. Multiple-label immunofluorescence was applied to postmortem sections of 10 IPD patients and 10 controls to quantify the abundance of CI-IV subunits (NDUFB8 or NDUFA13, SDHA, UQCRC2, and COXI) and mitochondrial transcription factors (TFAM and TFB2M) relative to mitochondrial mass (porin and GRP75) in dopaminergic neurons. To assess the involvement of mtDNA in respiratory chain deficiency in IPD, SN neurons, isolated with laser-capture microdissection, were assayed for mtDNA deletions, copy number, and presence of transcription/replication-associated 7S DNA employing a triplex real-time polymerase chain reaction (PCR) assay. Whereas mitochondrial mass was unchanged in single SN neurons from IPD patients, we observed a significant reduction in the abundances of CI and II subunits. At the single-cell level, CI and II deficiencies were correlated in patients. The CI deficiency concomitantly occurred with low abundances of the mtDNA transcription factors TFAM and TFB2M, which also initiate transcription-primed mtDNA replication. Consistent with this, real-time PCR analysis revealed fewer transcription/replication-associated mtDNA molecules and an overall reduction in mtDNA copy number in patients. This effect was more pronounced in single IPD neurons with severe CI deficiency. Respiratory chain dysfunction in IPD neurons not only involves CI, but also extends to CII. These deficiencies are possibly a consequence of the interplay between nDNA and mtDNA-encoded factors mechanistically connected via TFAM. © 2016 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.

DNA condensation and size effects of DNA condensation agent

NASA Astrophysics Data System (ADS)

Liu, Yan-Hui; Jiang, Chong-Ming; Guo, Xin-Miao; Tang, Yan-Lin; Hu, Lin

2013-08-01

Based on the model of the strong correlation of counterions condensed on DNA molecule, by tailoring interaction potential, interduplex spacing and correlation spacing between condensed counterions on DNA molecule and interduplex spacing fluctuation strength, toroidal configuration, rod-like configuration and two-hole configurations are possible. The size effects of counterion structure on the toroidal structure can be detected by this model. The autocorrelation function of the tangent vectors is found as an effective way to detect the structure of toroidal conformations and the generic pathway of the process of DNA condensation. The generic pathway of all of the configurations involves an initial nucleation loop, and the next part of the DNA chain is folded on the top of the initial nucleation loop with different manners, in agreement with the recent experimental results.

DNA EMP AWARENESS COURSE NOTES. THIRD EDITION

DTIC Science & Technology

1977-10-01

education period for the many cases, this can be don durit,g che training and education of personnel. Past initial program planning phases. If this...8217 * e "White Paper " and assessment reports.protection level minimum nuclear maximum nuclear Identifying the nuclear weapon *threat levels (source region...threats to the system in context of the threat levels mission of the system is often called a "White Paper ". Delineation of these threats deter

Wheat DNA Primase (RNA Primer Synthesis in Vitro, Structural Studies by Photochemical Cross-Linking, and Modulation of Primase Activity by DNA Polymerases).

PubMed Central

Laquel, P.; Litvak, S.; Castroviejo, M.

1994-01-01

DNA primase synthesizes short RNA primers used by DNA polymerases to initiate DNA synthesis. Two proteins of approximately 60 and 50 kD were recognized by specific antibodies raised against yeast primase subunits, suggesting a high degree of analogy between wheat and yeast primase subunits. Gel-filtration chromatography of wheat primase showed two active forms of 60 and 110 to 120 kD. Ultraviolet-induced cross-linking with radioactive oligothymidilate revealed a highly labeled protein of 60 kD. After limited trypsin digestion of wheat (Triticum aestivum L.) primase, a major band of 48 kD and two minor bands of 38 and 17 kD were observed. In the absence of DNA polymerases, the purified primase synthesizes long RNA products. The size of the RNA product synthesized by wheat primase is considerably reduced by the presence of DNA polymerases, suggesting a modulatory effect of the association between these two enzymes. Lowering the primase concentration in the assay also favored short RNA primer synthesis. Several properties of the wheat DNA primase using oligoadenylate [oligo(rA)]-primed or unprimed polythymidilate templates were studied. The ability of wheat primase, without DNA polymerases, to elongate an oligo(rA) primer to long RNA products depends on the primer size, temperature, and the divalent cation concentration. Thus, Mn2+ ions led to long RNA products in a very wide range of concentrations, whereas with Mg2+ long products were observed around 15 mM. We studied the ability of purified wheat DNA polymerases to initiate DNA synthesis from an RNA primer: wheat DNA polymerase A showed the highest activity, followed by DNA polymerases B and CII, whereas DNA polymerase CI was unable to initiate DNA synthesis from an RNA primer. Results are discussed in terms of understanding the role of these polymerases in DNA replication in plants. PMID:12232187

The DnaA N-terminal domain interacts with Hda to facilitate replicase clamp-mediated inactivation of DnaA.

PubMed

Su'etsugu, Masayuki; Harada, Yuji; Keyamura, Kenji; Matsunaga, Chika; Kasho, Kazutoshi; Abe, Yoshito; Ueda, Tadashi; Katayama, Tsutomu

2013-12-01

DnaA activity for replication initiation of the Escherichia coli chromosome is negatively regulated by feedback from the DNA-loaded form of the replicase clamp. In this process, called RIDA (regulatory inactivation of DnaA), ATP-bound DnaA transiently assembles into a complex consisting of Hda and the DNA-clamp, which promotes inter-AAA+ domain association between Hda and DnaA and stimulates hydrolysis of DnaA-bound ATP, producing inactive ADP-DnaA. Using a truncated DnaA mutant, we previously demonstrated that the DnaA N-terminal domain is involved in RIDA. However, the precise role of the N-terminal domain in RIDA has remained largely unclear. Here, we used an in vitro reconstituted system to demonstrate that the Asn-44 residue in the N-terminal domain of DnaA is crucial for RIDA but not for replication initiation. Moreover, an assay termed PDAX (pull-down after cross-linking) revealed an unstable interaction between a DnaA-N44A mutant and Hda. In vivo, this mutant exhibited an increase in the cellular level of ATP-bound DnaA. These results establish a model in which interaction between DnaA Asn-44 and Hda stabilizes the association between the AAA+ domains of DnaA and Hda to facilitate DnaA-ATP hydrolysis during RIDA. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Acceptance of Domestic Cat Mitochondrial DNA in a Criminal Proceeding

PubMed Central

Lyons, Leslie A.; Grahn, Robert A.; Kun, Teri J.; Netzel, Linda R.; Wictum, Elizabeth E.; Halverson, Joy L.

2014-01-01

Shed hair from domestic animals readily adheres to clothing and other contact items, providing a source of transfer evidence for criminal investigations. Mitochondrial DNA is often the only option for DNA analysis of shed hair. Human mitochondrial DNA analysis has been accepted in the US court system since 1996. The murder trial of the State of Missouri versus Henry L. Polk, Jr. represents the first legal proceeding where cat mitochondrial DNA analysis was introduced into evidence. The mitochondrial DNA evidence was initially considered inadmissible due to concerns about the cat dataset and the scientific acceptance of the marker. Those concerns were subsequently addressed, and the evidence was deemed admissible. This report reviews the case in regards to the cat biological evidence and its ultimate admission as generally accepted and reliable. Expansion and saturation analysis of the cat mitochondrial DNA control region dataset supported the initial interpretation of the evidence. PMID:25086413

Strong minor groove base conservation in sequence logos implies DNA distortion or base flipping during replication and transcription initiation.

PubMed

Schneider, T D

2001-12-01

The sequence logo for DNA binding sites of the bacteriophage P1 replication protein RepA shows unusually high sequence conservation ( approximately 2 bits) at a minor groove that faces RepA. However, B-form DNA can support only 1 bit of sequence conservation via contacts into the minor groove. The high conservation in RepA sites therefore implies a distorted DNA helix with direct or indirect contacts to the protein. Here I show that a high minor groove conservation signature also appears in sequence logos of sites for other replication origin binding proteins (Rts1, DnaA, P4 alpha, EBNA1, ORC) and promoter binding proteins (sigma(70), sigma(D) factors). This finding implies that DNA binding proteins generally use non-B-form DNA distortion such as base flipping to initiate replication and transcription.

BINDING OF CARCINOGENS TO DNA AND COVALENT ADDUCTS DNA DAMAGE - PAH, AROMATIC AMINES, NITRO-AROMATIC COMPOUNDS, AND HALOGENATED COMPOUNDS

EPA Science Inventory

DNA adducts are the covalent addition products resulting from binding of reactive chemical species to DNA bases. The cancer initiating role of DNA adducts is well-established, and is clearly reflected in the high cancer incidence observed in individuals with deficiencies in any o...

Alteration in levels of unsaturated fatty acids in mutants of Escherichia coli defective in DNA replication.

PubMed

Suzuki, E; Kondo, T; Makise, M; Mima, S; Sakamoto, K; Tsuchiya, T; Mizushima, T

1998-07-01

We previously reported that mutations in the dnaA gene which encodes the initiator of chromosomal DNA replication in Escherichia coli caused an alteration in the levels of unsaturated fatty acids of phospholipids in membranes. In this study, we examined fatty acid compositions in other mutants which are defective in DNA replication. As in the case of temperature-sensitive dnaA mutants, temperature-sensitive dnaC and dnaE mutants, which have defects in initiation and elongation, respectively, of DNA replication showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) compared with the wild-type strain, especially at high temperatures. On the other hand, temperature-sensitive mutants defective in cellular processes other than DNA replication, such as RNA synthesis and cell division, did not show a lower level of unsaturation of fatty acids compared with the wild-type strain. These results suggest that the inhibition of DNA replication causes a lower level of unsaturation of fatty acids in Escherichia coli cells.

Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli.

PubMed

Suzuki, E; Kondo, T; Makise, M; Mima, S; Sakamoto, K; Tsuchiya, T; Mizushima, T

1998-04-01

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperature-sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42 degrees C (non-permissive temperature) and at 37 degrees C (semi-permissive temperature), but not at 28 degrees C (permissive temperature), compared with the wild-type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature-sensitive dnaA mutants showed similar results. On the other hand, a cold-sensitive dnaAcos mutant, in which over-initiation of DNA replication occurs at low temperature (28 degrees C), showed a higher level of unsaturation of fatty acids at 28 degrees C. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.

RNF168 forms a functional complex with RAD6 during the DNA damage response

PubMed Central

Liu, Chao; Wang, Degui; Wu, Jiaxue; Keller, Jennifer; Ma, Teng; Yu, Xiaochun

2013-01-01

Summary Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, E2 ubiquitin conjugating enzymes are crucial for catalyzing substrate ubiquitination that recruits downstream DNA repair factors to DNA lesions. To identify novel E2 conjugating enzymes important for initiating the DNA-damage-induced ubiquitination cascade, we screened most of the known E2 enzymes and found that RAD6A and RAD6B function together with RNF168 in the ionizing radiation (IR)-induced DNA damage response. Similarly to RNF168-deficient cells, RAD6A- or RAD6B-deficient cells exhibit a reduction in DNA-damage-induced protein ubiquitination. Correspondingly, DNA-damage-induced foci formation of DNA damage repair proteins, such as BRCA1 and 53BP1, is impaired in the absence of RAD6A or RAD6B. Moreover, the RNF168–RAD6 complex targeted histone H1.2 for ubiquitination in vitro and regulated DNA-damage-induced histone H1.2 ubiquitination in vivo. Collectively, these data demonstrate that RNF168, in complex with RAD6A or RAD6B, is activated in the DNA-damage-induced protein ubiquitination cascade. PMID:23525009

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression

PubMed Central

Löb, D.; Lengert, N.; Chagin, V. O.; Reinhart, M.; Casas-Delucchi, C. S.; Cardoso, M. C.; Drossel, B.

2016-01-01

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase. PMID:27052359

Mechanism of Origin DNA Recognition and Assembly of an Initiator-Helicase Complex by SV40 Large Tumor Antigen

PubMed Central

Chang, Y. Paul; Xu, Meng; Machado, Ana Carolina Dantas; Yu, Xian Jessica; Rohs, Remo; Chen, Xiaojiang S.

2013-01-01

SUMMARY The DNA tumor virus Simian virus 40 (SV40) is a model system for studying eukaryotic replication. SV40 large tumor antigen (LTag) is the initiator/helicase that is essential for genome replication. LTag recognizes and assembles at the viral replication origin. We determined the structure of two multidomain LTag subunits bound to origin DNA. The structure reveals that the origin binding domains (OBDs) and Zn and AAA+ domains are involved in origin recognition and assembly. Notably, the OBDs recognize the origin in an unexpected manner. The histidine residues of the AAA+ domains insert into a narrow minor groove region with enhanced negative electrostatic potential. Computational analysis indicates that this region is intrinsically narrow, demonstrating the role of DNA shape readout in origin recognition. Our results provide important insights into the assembly of the LTag initiator/ helicase at the replication origin and suggest that histidine contacts with the minor groove serve as a mechanism of DNA shape readout. PMID:23545501

Intragenic DNA methylation prevents spurious transcription initiation.

PubMed

Neri, Francesco; Rapelli, Stefania; Krepelova, Anna; Incarnato, Danny; Parlato, Caterina; Basile, Giulia; Maldotti, Mara; Anselmi, Francesca; Oliviero, Salvatore

2017-03-02

In mammals, DNA methylation occurs mainly at CpG dinucleotides. Methylation of the promoter suppresses gene expression, but the functional role of gene-body DNA methylation in highly expressed genes has yet to be clarified. Here we show that, in mouse embryonic stem cells, Dnmt3b-dependent intragenic DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation. Using different genome-wide approaches, we demonstrate that this Dnmt3b function is dependent on its enzymatic activity and recruitment to the gene body by H3K36me3. Furthermore, the spurious transcripts can either be degraded by the RNA exosome complex or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins. Elongating RNA polymerase II therefore triggers an epigenetic crosstalk mechanism that involves SetD2, H3K36me3, Dnmt3b and DNA methylation to ensure the fidelity of gene transcription initiation, with implications for intragenic hypomethylation in cancer.

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

NASA Technical Reports Server (NTRS)

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

1994-01-01

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

An isolated Hda-clamp complex is functional in the regulatory inactivation of DnaA and DNA replication.

PubMed

Kawakami, Hironori; Su'etsugu, Masayuki; Katayama, Tsutomu

2006-10-01

In Escherichia coli, a complex consisting of Hda and the DNA-loaded clamp-subunit of the DNA polymerase III holoenzyme promotes hydrolysis of DnaA-ATP. The resultant ADP-DnaA is inactive for initiation of chromosomal DNA replication, thereby repressing excessive initiations. As the cellular content of the clamp is 10-100 times higher than that of Hda, most Hda molecules might be complexed with the clamp in vivo. Although Hda predominantly forms irregular aggregates when overexpressed, in the present study we found that co-overexpression of the clamp with Hda enhances Hda solubility dramatically and we efficiently isolated the Hda-clamp complex. A single molecule of the complex appears to consist of two Hda molecules and a single clamp. The complex is competent in DnaA-ATP hydrolysis and DNA replication in the presence of DNA and the clamp deficient subassembly of the DNA polymerase III holoenzyme (pol III*). These findings indicate that the clamp contained in the complex is loaded onto DNA through an interaction with the pol III* and that the Hda activity is preserved in these processes. The complex consisting of Hda and the DNA-unloaded clamp may play a specific role in a process proceeding to the DnaA-ATP hydrolysis in vivo.

How MCM loading and spreading specify eukaryotic DNA replication initiation sites

PubMed Central

Hyrien, Olivier

2016-01-01

DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes. PMID:27635237

Molecular determinants of origin discrimination by Orc1 initiators in archaea.

PubMed

Dueber, Erin C; Costa, Alessandro; Corn, Jacob E; Bell, Stephen D; Berger, James M

2011-05-01

Unlike bacteria, many eukaryotes initiate DNA replication from genomic sites that lack apparent sequence conservation. These loci are identified and bound by the origin recognition complex (ORC), and subsequently activated by a cascade of events that includes recruitment of an additional factor, Cdc6. Archaeal organisms generally possess one or more Orc1/Cdc6 homologs, belonging to the Initiator clade of ATPases associated with various cellular activities (AAA(+)) superfamily; however, these proteins recognize specific sequences within replication origins. Atomic resolution studies have shown that archaeal Orc1 proteins contact double-stranded DNA through an N-terminal AAA(+) domain and a C-terminal winged-helix domain (WHD), but use remarkably few base-specific contacts. To investigate the biochemical effects of these associations, we mutated the DNA-interacting elements of the Orc1-1 and Orc1-3 paralogs from the archaeon Sulfolobus solfataricus, and tested their effect on origin binding and deformation. We find that the AAA(+) domain has an unpredicted role in controlling the sequence selectivity of DNA binding, despite an absence of base-specific contacts to this region. Our results show that both the WHD and ATPase region influence origin recognition by Orc1/Cdc6, and suggest that not only DNA sequence, but also local DNA structure help define archaeal initiator binding sites. © The Author(s) 2011. Published by Oxford University Press.

Free-energy landscape and characteristic forces for the initiation of DNA unzipping.

PubMed

Mentes, Ahmet; Florescu, Ana Maria; Brunk, Elizabeth; Wereszczynski, Jeff; Joyeux, Marc; Andricioaei, Ioan

2015-04-07

DNA unzipping, the separation of its double helix into single strands, is crucial in modulating a host of genetic processes. Although the large-scale separation of double-stranded DNA has been studied with a variety of theoretical and experimental techniques, the minute details of the very first steps of unzipping are still unclear. Here, we use atomistic molecular-dynamics simulations, coarse-grained simulations, and a statistical-mechanical model to study the initiation of DNA unzipping by an external force. Calculation of the potential of mean force profiles for the initial separation of the first few terminal basepairs in a DNA oligomer revealed that forces ranging between 130 and 230 pN are needed to disrupt the first basepair, and these values are an order of magnitude larger than those needed to disrupt basepairs in partially unzipped DNA. The force peak has an echo of ∼50 pN at the distance that unzips the second basepair. We show that the high peak needed to initiate unzipping derives from a free-energy basin that is distinct from the basins of subsequent basepairs because of entropic contributions, and we highlight the microscopic origin of the peak. To our knowledge, our results suggest a new window of exploration for single-molecule experiments. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans.

PubMed

Wyatt, Lauren H; Luz, Anthony L; Cao, Xiou; Maurer, Laura L; Blawas, Ashley M; Aballay, Alejandro; Pan, William K Y; Meyer, Joel N

2017-04-01

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Reported impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl 2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H 2 O 2 ), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl 2 , low-level DNA damage (∼0.25 lesions/10kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H 2 O 2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H 2 O 2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H 2 O 2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl 2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans

PubMed Central

Wyatt, Lauren H.; Luz, Anthony L.; Cao, Xiou; Maurer, Laura L.; Blawas, Ashley M.; Aballay, Alejandro; Pan, William K.; Meyer, Joel N.

2017-01-01

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H2O2), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl2, low-level DNA damage (~0.25 lesions/10 kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H2O2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H2O2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H2O2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion. PMID:28242054

Excision Repair-Initiated Enzyme-Assisted Bicyclic Cascade Signal Amplification for Ultrasensitive Detection of Uracil-DNA Glycosylase.

PubMed

Wang, Li-Juan; Ren, Ming; Zhang, Qianyi; Tang, Bo; Zhang, Chun-Yang

2017-04-18

Uracil-DNA glycosylase (UDG) is an important base excision repair (BER) enzyme responsible for the repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using excision repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-excision repair, (2) excision repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate isothermal exponential amplification, producing a large number of triggers. The resultant trigger may selectively hybridize with the signal probe which is modified with FAM and BHQ1, forming a RNA-DNA heterogeneous duplex. The subsequent hydrolysis of RNA-DNA duplex by RNase H leads to the generation of fluorescence signal. This assay exhibits ultrahigh sensitivity with a detection limit of 0.0001 U/mL, and it can even measure UDG activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of inhibitors, thereby providing a powerful tool for DNA repair enzyme-related biomedical research and clinical diagnosis.

Initiation of lambda DNA replication. The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein.

PubMed

Osipiuk, J; Georgopoulos, C; Zylicz, M

1993-03-05

It is known that the initiation of bacteriophage lambda replication requires the orderly assembly of the lambda O.lambda P.DnaB helicase protein preprimosomal complex at the ori lambda DNA site. The DnaK, DnaJ, and GrpE heat shock proteins act together to destabilize the lambda P.DnaB complex, thus freeing DnaB and allowing it to unwind lambda DNA near the ori lambda site. The first step of this disassembly reaction is the binding of DnaK to the lambda P protein. In this report, we examined the influence of the DnaJ and GrpE proteins on the stability of the lambda P.DnaK complex. We present evidence for the existence of the following protein-protein complexes: lambda P.DnaK, lambda P.DnaJ, DnaJ.DnaK, DnaK.GrpE, and lambda P.DnaK.GrpE. Our results suggest that the presence of GrpE alone destabilizes the lambda P.DnaK complex, whereas the presence of DnaJ alone stabilizes the lambda P.DnaK complex. Using immunoprecipitation, we show that in the presence of GrpE, DnaK exhibits a higher affinity for the lambda P.DnaJ complex than it does alone. Using cross-linking with glutaraldehyde, we show that oligomeric forms of DnaK exhibit a higher affinity for lambda P than monomeric DnaK. However, in the presence of GrpE, monomeric DnaK can efficiently bind lambda P protein. These findings help explain our previous results, namely that in the GrpE-dependent lambda DNA replication system, the DnaK protein requirement can be reduced up to 10-fold.

DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase*

PubMed Central

Bharti, Sanjay Kumar; Sommers, Joshua A.; Zhou, Jun; Kaplan, Daniel L.; Spelbrink, Johannes N.; Mergny, Jean-Louis; Brosh, Robert M.

2014-01-01

Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the “Pattern Finder” G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase. PMID:25193669

Chemical form of selenium differentially influences DNA repair pathways following exposure to lead nitrate.

PubMed

McKelvey, Shauna M; Horgan, Karina A; Murphy, Richard A

2015-01-01

Lead, an environmental toxin is known to induce a broad range of physiological and biochemical dysfunctions in humans through a number of mechanisms including the deactivation of antioxidants thus leading to generation of reactive oxygen species (ROS) and subsequent DNA damage. Selenium on the other hand has been proven to play an important role in the protection of cells from free radical damage and oxidative stress, though its effects are thought to be form and dose dependent. As the liver is the primary organ required for metabolite detoxification, HepG2 cells were chosen to assess the protective effects of various selenium compounds following exposure to the genotoxic agent lead nitrate. Initially DNA damage was quantified using a comet assay, gene expression patterns associated with DNA damage and signalling were also examined using PCR arrays and the biological pathways which were most significantly affected by selenium were identified. Interestingly, the organic type selenium compounds (selenium yeast and selenomethionine) conferred protection against lead induced DNA damage in HepG2 cells; this is evident by reduction in the quantity of DNA present in the comet tail of cells cultured in their presence with lead. This trend also followed through the gene expression changes noted in DNA damage pathways analysed. These results were in contrast with those of inorganic sodium selenite which promoted lead induced DNA damage evident in both the comet assay results and the gene expression analysis. Over all this study provided valuable insights into the effects which various selenium compounds had on the DNA damage and signalling pathway indicating the potential for using organic forms of selenium such as selenium enriched yeast to protect against DNA damaging agents. Copyright © 2014 Elsevier GmbH. All rights reserved.

Mitochondrial DNA Depletion in Respiratory Chain–Deficient Parkinson Disease Neurons

PubMed Central

Rygiel, Karolina A.; Hepplewhite, Philippa D.; Morris, Christopher M.; Picard, Martin; Turnbull, Doug M.

2016-01-01

Objective To determine the extent of respiratory chain abnormalities and investigate the contribution of mtDNA to the loss of respiratory chain complexes (CI–IV) in the substantia nigra (SN) of idiopathic Parkinson disease (IPD) patients at the single‐neuron level. Methods Multiple‐label immunofluorescence was applied to postmortem sections of 10 IPD patients and 10 controls to quantify the abundance of CI–IV subunits (NDUFB8 or NDUFA13, SDHA, UQCRC2, and COXI) and mitochondrial transcription factors (TFAM and TFB2M) relative to mitochondrial mass (porin and GRP75) in dopaminergic neurons. To assess the involvement of mtDNA in respiratory chain deficiency in IPD, SN neurons, isolated with laser‐capture microdissection, were assayed for mtDNA deletions, copy number, and presence of transcription/replication‐associated 7S DNA employing a triplex real‐time polymerase chain reaction (PCR) assay. Results Whereas mitochondrial mass was unchanged in single SN neurons from IPD patients, we observed a significant reduction in the abundances of CI and II subunits. At the single‐cell level, CI and II deficiencies were correlated in patients. The CI deficiency concomitantly occurred with low abundances of the mtDNA transcription factors TFAM and TFB2M, which also initiate transcription‐primed mtDNA replication. Consistent with this, real‐time PCR analysis revealed fewer transcription/replication‐associated mtDNA molecules and an overall reduction in mtDNA copy number in patients. This effect was more pronounced in single IPD neurons with severe CI deficiency. Interpretation Respiratory chain dysfunction in IPD neurons not only involves CI, but also extends to CII. These deficiencies are possibly a consequence of the interplay between nDNA and mtDNA‐encoded factors mechanistically connected via TFAM. ANN NEUROL 2016;79:366–378 PMID:26605748

Genetic variations in the DNA replication origins of human papillomavirus family correlate with their oncogenic potential.

PubMed

Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

2018-04-01

Human papillomaviruses (HPVs) encompass a large family of viruses that range from benign to highly carcinogenic. The crucial differences between benign and carcinogenic types of HPV remain unknown, except that the two HPV types differ in the frequency of DNA replication. We have systematically analyzed the mechanism of HPV DNA replication initiation in low-risk and high-risk HPVs. Our results demonstrate that HPV-encoded E2 initiator protein and its four binding sites in the replication origin play pivotal roles in determining the destiny of the HPV-infected cell. We have identified strain-specific single nucleotide variations in E2 binding sites found only in the high-risk HPVs. We have demonstrated that these variations result in attenuated formation of the E2-DNA complex. E2 binding to these sites is linked to the activation of the DNA replication origin as well as initiation of DNA replication. Both electrophoretic mobility shift assay and atomic force microscopy studies demonstrated that binding of E2 from either low- or high-risk HPVs with variant binding sequences lacked multimeric E2-DNA complex formation in vitro. These results provided a molecular basis of differential DNA replication in the two types of HPVs and pointed to a correlation with the development of cancer. Copyright © 2017. Published by Elsevier B.V.

A new structural framework for integrating replication protein A into DNA processing machinery

PubMed Central

Brosey, Chris A.; Yan, Chunli; Tsutakawa, Susan E.; Heller, William T.; Rambo, Robert P.; Tainer, John A.; Ivanov, Ivaylo; Chazin, Walter J.

2013-01-01

By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA’s DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA’s DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways. PMID:23303776

Replication intermediates of the linear mitochondrial DNA of Candida parapsilosis suggest a common recombination based mechanism for yeast mitochondria.

PubMed

Gerhold, Joachim M; Sedman, Tiina; Visacka, Katarina; Slezakova, Judita; Tomaska, Lubomir; Nosek, Jozef; Sedman, Juhan

2014-08-15

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria*

PubMed Central

Gerhold, Joachim M.; Sedman, Tiina; Visacka, Katarina; Slezakova, Judita; Tomaska, Lubomir; Nosek, Jozef; Sedman, Juhan

2014-01-01

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations. PMID:24951592

A new structural framework for integrating replication protein A into DNA processing machinery

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

Brosey, Chris; Yan, Chunli; Tsutakawa, Susan

2013-01-17

By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA's DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA's DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamicmore » on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.« less

Mitochondrial DNA and Cancer Epidemiology Workshop

Cancer.gov

A workshop to review the state-of-the science in the mitochondrial DNA field and its use in cancer epidemiology, and to develop a concept for a research initiative on mitochondrial DNA and cancer epidemiology.

WE-H-BRA-08: A Monte Carlo Cell Nucleus Model for Assessing Cell Survival Probability Based On Particle Track Structure Analysis

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

Lee, B; Georgia Institute of Technology, Atlanta, GA; Wang, C

Purpose: To correlate the damage produced by particles of different types and qualities to cell survival on the basis of nanodosimetric analysis and advanced DNA structures in the cell nucleus. Methods: A Monte Carlo code was developed to simulate subnuclear DNA chromatin fibers (CFs) of 30nm utilizing a mean-free-path approach common to radiation transport. The cell nucleus was modeled as a spherical region containing 6000 chromatin-dense domains (CDs) of 400nm diameter, with additional CFs modeled in a sparser interchromatin region. The Geant4-DNA code was utilized to produce a particle track database representing various particles at different energies and dose quantities.more » These tracks were used to stochastically position the DNA structures based on their mean free path to interaction with CFs. Excitation and ionization events intersecting CFs were analyzed using the DBSCAN clustering algorithm for assessment of the likelihood of producing DSBs. Simulated DSBs were then assessed based on their proximity to one another for a probability of inducing cell death. Results: Variations in energy deposition to chromatin fibers match expectations based on differences in particle track structure. The quality of damage to CFs based on different particle types indicate more severe damage by high-LET radiation than low-LET radiation of identical particles. In addition, the model indicates more severe damage by protons than of alpha particles of same LET, which is consistent with differences in their track structure. Cell survival curves have been produced showing the L-Q behavior of sparsely ionizing radiation. Conclusion: Initial results indicate the feasibility of producing cell survival curves based on the Monte Carlo cell nucleus method. Accurate correlation between simulated DNA damage to cell survival on the basis of nanodosimetric analysis can provide insight into the biological responses to various radiation types. Current efforts are directed at producing cell survival curves for high-LET radiation.« less

Ancestor of land plants acquired the DNA-3-methyladenine glycosylase (MAG) gene from bacteria through horizontal gene transfer.

PubMed

Fang, Huimin; Huangfu, Liexiang; Chen, Rujia; Li, Pengcheng; Xu, Shuhui; Zhang, Enying; Cao, Wei; Liu, Li; Yao, Youli; Liang, Guohua; Xu, Chenwu; Zhou, Yong; Yang, Zefeng

2017-08-24

The origin and evolution of land plants was an important event in the history of life and initiated the establishment of modern terrestrial ecosystems. From water to terrestrial environments, plants needed to overcome the enhanced ultraviolet (UV) radiation and many other DNA-damaging agents. Evolving new genes with the function of DNA repair is critical for the origin and radiation of land plants. In bacteria, the DNA-3-methyladenine glycosylase (MAG) recognizes of a variety of base lesions and initiates the process of the base excision repair for damaged DNA. The homologs of MAG gene are present in all major lineages of streptophytes, and both the phylogenic and sequence similarity analyses revealed that green plant MAG gene originated through an ancient horizontal gene transfer (HGT) event from bacteria. Experimental evidence demonstrated that the expression of the maize ZmMAG gene was induced by UV and zeocin, both of which are known as DNA-damaging agents. Further investigation revealed that Streptophyta MAG genes had undergone positive selection during the initial evolutionary period in the ancestor of land plants. Our findings demonstrated that the ancient HGT of MAG to the ancestor of land plants probably played an important role in preadaptation to DNA-damaging agents in terrestrial environments.

Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

PubMed Central

Erliandri, Indri; Fu, Haiqing; Nakano, Megumi; Kim, Jung-Hyun; Miga, Karen H.; Liskovykh, Mikhail; Earnshaw, William C.; Masumoto, Hiroshi; Kouprina, Natalay; Aladjem, Mirit I.; Larionov, Vladimir

2014-01-01

In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed Human Artificial Chromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, suggesting that CENP-B may have a role in regulating the replication of centromeric regions. Mapping of replication initiation sites in the HAC revealed that replication preferentially initiated in transcriptionally active regions. PMID:25228468

Sewage sludge does not induce genotoxicity and carcinogenesis.

PubMed

Silva, Paula Regina Pereira; Barbisan, Luis Fernando; Dagli, Maria Lúcia Zaidan; Saldiva, Paulo Hilário Nascimento

2012-07-01

Through a series of experiments, the genotoxic/mutagenic and carcinogenic potential of sewage sludge was assessed. Male Wistar rats were randomly assigned to four groups: Group 1 - negative control; Group 2 - liver carcinogenesis initiated by diethylnitrosamine (DEN; 200 mg/kg i.p.); Group 3 and G4-liver carcinogenesis initiated by DEN and fed 10,000 ppm or 50,000 ppm of sewage sludge. The animals were submitted to a 70% partial hepatectomy at the 3(rd) week. Livers were processed for routine histological analysis and immunohistochemistry, in order to detect glutathione S-transferase positive altered hepatocyte foci (GST-P(+) AHF). Peripheral blood samples for the comet assay were obtained from the periorbital plexus immediately prior to sacrificing. Polychromatic erythrocytes (PCEs) were analyzed in femoral bone-marrow smears, and the frequencies of those micronucleated (MNPCEs) registered. There was no sewage-sludge-induced increase in frequency of either DNA damage in peripheral blood leucocytes, or MNPCEs in the femoral bone marrow. Also, there was no increase in the levels of DNA damage, in the frequency of MNPCEs, and in the development of GST-P AHF when compared with the respective control group.

Induced DNA demethylation by targeting Ten-Eleven Translocation 2 to the human ICAM-1 promoter

PubMed Central

Chen, Hui; Kazemier, Hinke G; de Groote, Marloes L.; Ruiters, Marcel H. J.; Xu, Guo-Liang; Rots, Marianne G.

2014-01-01

Increasing evidence indicates that active DNA demethylation is involved in several processes in mammals, resulting in developmental stage-specificity and cell lineage-specificity. The recently discovered Ten-Eleven Translocation (TET) dioxygenases are accepted to be involved in DNA demethylation by initiating 5-mC oxidation. Aberrant DNA methylation profiles are associated with many diseases. For example in cancer, hypermethylation results in silencing of tumor suppressor genes. Such silenced genes can be re-expressed by epigenetic drugs, but this approach has genome-wide effects. In this study, fusions of designer DNA binding domains to TET dioxygenase family members (TET1, -2 or -3) were engineered to target epigenetically silenced genes (ICAM-1, EpCAM). The effects on targeted CpGs’ methylation and on expression levels of the target genes were assessed. The results indicated demethylation of targeted CpG sites in both promoters for targeted TET2 and to a lesser extent for TET1, but not for TET3. Interestingly, we observed re-activation of transcription of ICAM-1. Thus, our work suggests that we provided a mechanism to induce targeted DNA demethylation, which facilitates re-activation of expression of the target genes. Furthermore, this Epigenetic Editing approach is a powerful tool to investigate functions of epigenetic writers and erasers and to elucidate consequences of epigenetic marks. PMID:24194590

Evaluation of various glyphosate concentrations on DNA damage in human Raji cells and its impact on cytotoxicity.

PubMed

Townsend, Michelle; Peck, Connor; Meng, Wei; Heaton, Matthew; Robison, Richard; O'Neill, Kim

2017-04-01

Glyphosate is a highly used active compound in agriculturally based pesticides. The literature regarding the toxicity of glyphosate to human cells has been highly inconsistent. We studied the resulting DNA damage and cytotoxicity of various glyphosate concentrations on human cells to evaluate DNA damaging potential. Utilizing human Raji cells, DNA damage was quantified using the comet assay, while cytotoxicity was further analyzed using MTT viability assays. Several glyphosate concentrations were assessed, ranging from 15 mM to 0.1 μM. We found that glyphosate treatment is lethal to Raji cells at concentrations above 10 mM, yet has no cytotoxic effects at concentrations at or below 100 μM. Treatment concentrations of 1 mM and 5 mM induce statistically significant DNA damage to Raji cells following 30-60 min of treatment, however, cells show a slow recovery from initial damage and cell viability is unaffected after 2 h. At these same concentrations, cells treated with additional compound did not recover and maintained high levels of DNA damage. While the cytotoxicity of glyphosate appears to be minimal for physiologically relevant concentrations, the compound has a definitive cytotoxic nature in human cells at high concentrations. Our data also suggests a mammalian metabolic pathway for the degradation of glyphosate may be present. Copyright © 2017 Elsevier Inc. All rights reserved.

A ranking index for quality assessment of forensic DNA profiles forensic DNA profiles

PubMed Central

2010-01-01

Background Assessment of DNA profile quality is vital in forensic DNA analysis, both in order to determine the evidentiary value of DNA results and to compare the performance of different DNA analysis protocols. Generally the quality assessment is performed through manual examination of the DNA profiles based on empirical knowledge, or by comparing the intensities (allelic peak heights) of the capillary electrophoresis electropherograms. Results We recently developed a ranking index for unbiased and quantitative quality assessment of forensic DNA profiles, the forensic DNA profile index (FI) (Hedman et al. Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles, Biotechniques 47 (2009) 951-958). FI uses electropherogram data to combine the intensities of the allelic peaks with the balances within and between loci, using Principal Components Analysis. Here we present the construction of FI. We explain the mathematical and statistical methodologies used and present details about the applied data reduction method. Thereby we show how to adapt the ranking index for any Short Tandem Repeat-based forensic DNA typing system through validation against a manual grading scale and calibration against a specific set of DNA profiles. Conclusions The developed tool provides unbiased quality assessment of forensic DNA profiles. It can be applied for any DNA profiling system based on Short Tandem Repeat markers. Apart from crime related DNA analysis, FI can therefore be used as a quality tool in paternal or familial testing as well as in disaster victim identification. PMID:21062433

Water versus DNA: New insights into proton track-structure modeling in radiobiology and radiotherapy

DOE PAGES

Champion, Christophe; Quinto, Michele A.; Monti, Juan M.; ...

2015-09-25

Water is a common surrogate of DNA for modelling the charged particle-induced ionizing processes in living tissue exposed to radiations. The present study aims at scrutinizing the validity of this approximation and then revealing new insights into proton-induced energy transfers by a comparative analysis between water and realistic biological medium. In this context, a self-consistent quantum mechanical modelling of the ionization and electron capture processes is reported within the continuum distorted wave-eikonal initial state framework for both isolated water molecules and DNA components impacted by proton beams. Their respective probability of occurrence-expressed in terms of total cross sections-as well asmore » their energetic signature (potential and kinetic) are assessed in order to clearly emphasize the differences existing between realistic building blocks of living matter and the controverted water-medium surrogate. Thus the consequences in radiobiology and radiotherapy will be discussed in particular in view of treatment planning refinement aiming at better radiotherapy strategies.« less

Assembling programmable FRET-based photonic networks using designer DNA scaffolds

PubMed Central

Buckhout-White, Susan; Spillmann, Christopher M; Algar, W. Russ; Khachatrian, Ani; Melinger, Joseph S.; Goldman, Ellen R.; Ancona, Mario G.; Medintz, Igor L.

2014-01-01

DNA demonstrates a remarkable capacity for creating designer nanostructures and devices. A growing number of these structures utilize Förster resonance energy transfer (FRET) as part of the device's functionality, readout or characterization, and, as device sophistication increases so do the concomitant FRET requirements. Here we create multi-dye FRET cascades and assess how well DNA can marshal organic dyes into nanoantennae that focus excitonic energy. We evaluate 36 increasingly complex designs including linear, bifurcated, Holliday junction, 8-arm star and dendrimers involving up to five different dyes engaging in four-consecutive FRET steps, while systematically varying fluorophore spacing by Förster distance (R0). Decreasing R0 while augmenting cross-sectional collection area with multiple donors significantly increases terminal exciton delivery efficiency within dendrimers compared with the first linear constructs. Förster modelling confirms that best results are obtained when there are multiple interacting FRET pathways rather than independent channels by which excitons travel from initial donor(s) to final acceptor. PMID:25504073

Water versus DNA: new insights into proton track-structure modelling in radiobiology and radiotherapy.

PubMed

Champion, C; Quinto, M A; Monti, J M; Galassi, M E; Weck, P F; Fojón, O A; Hanssen, J; Rivarola, R D

2015-10-21

Water is a common surrogate of DNA for modelling the charged particle-induced ionizing processes in living tissue exposed to radiations. The present study aims at scrutinizing the validity of this approximation and then revealing new insights into proton-induced energy transfers by a comparative analysis between water and realistic biological medium. In this context, a self-consistent quantum mechanical modelling of the ionization and electron capture processes is reported within the continuum distorted wave-eikonal initial state framework for both isolated water molecules and DNA components impacted by proton beams. Their respective probability of occurrence-expressed in terms of total cross sections-as well as their energetic signature (potential and kinetic) are assessed in order to clearly emphasize the differences existing between realistic building blocks of living matter and the controverted water-medium surrogate. Consequences in radiobiology and radiotherapy will be discussed in particular in view of treatment planning refinement aiming at better radiotherapy strategies.

RAD51 interconnects between DNA replication, DNA repair and immunity.

PubMed

Bhattacharya, Souparno; Srinivasan, Kalayarasan; Abdisalaam, Salim; Su, Fengtao; Raj, Prithvi; Dozmorov, Igor; Mishra, Ritu; Wakeland, Edward K; Ghose, Subroto; Mukherjee, Shibani; Asaithamby, Aroumougame

2017-05-05

RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System

PubMed Central

Cooper, Lauren A.; Stringer, Anne M.

2018-01-01

ABSTRACT In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5′ end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing. PMID:29666291

Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein

PubMed Central

Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

2004-01-01

In Escherichia coli, interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and β proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with β in vitro. A new β-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified β-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind β. A 10-amino-acid peptide containing the E. coli Hda β-binding motif was shown to compete with Hda for binding to β in an Hda-β interaction assay. These results establish that the interaction of Hda with β is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication. PMID:15150238

Interaction of the sliding clamp beta-subunit and Hda, a DnaA-related protein.

PubMed

Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

2004-06-01

In Escherichia coli, interactions between the replication initiation protein DnaA, the beta subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro. A new beta-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified beta-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind beta. A 10-amino-acid peptide containing the E. coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay. These results establish that the interaction of Hda with beta is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

Packaging of Dinoroseobacter shibae DNA into Gene Transfer Agent Particles Is Not Random.

PubMed

Tomasch, Jürgen; Wang, Hui; Hall, April T K; Patzelt, Diana; Preusse, Matthias; Petersen, Jörn; Brinkmann, Henner; Bunk, Boyke; Bhuju, Sabin; Jarek, Michael; Geffers, Robert; Lang, Andrew S; Wagner-Döbler, Irene

2018-01-01

Gene transfer agents (GTAs) are phage-like particles which contain a fragment of genomic DNA of the bacterial or archaeal producer and deliver this to a recipient cell. GTA gene clusters are present in the genomes of almost all marine Rhodobacteraceae (Roseobacters) and might be important contributors to horizontal gene transfer in the world's oceans. For all organisms studied so far, no obvious evidence of sequence specificity or other nonrandom process responsible for packaging genomic DNA into GTAs has been found. Here, we show that knock-out of an autoinducer synthase gene of Dinoroseobacter shibae resulted in overproduction and release of functional GTA particles (DsGTA). Next-generation sequencing of the 4.2-kb DNA fragments isolated from DsGTAs revealed that packaging was not random. DNA from low-GC conjugative plasmids but not from high-GC chromids was excluded from packaging. Seven chromosomal regions were strongly overrepresented in DNA isolated from DsGTA. These packaging peaks lacked identifiable conserved sequence motifs that might represent recognition sites for the GTA terminase complex. Low-GC regions of the chromosome, including the origin and terminus of replication, were underrepresented in DNA isolated from DsGTAs. DNA methylation reduced packaging frequency while the level of gene expression had no influence. Chromosomal regions found to be over- and underrepresented in DsGTA-DNA were regularly spaced. We propose that a "headful" type of packaging is initiated at the sites of coverage peaks and, after linearization of the chromosomal DNA, proceeds in both directions from the initiation site. GC-content, DNA-modifications, and chromatin structure might influence at which sides GTA packaging can be initiated. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Packaging of Dinoroseobacter shibae DNA into Gene Transfer Agent Particles Is Not Random

PubMed Central

Wang, Hui; Hall, April T K; Patzelt, Diana; Preusse, Matthias; Petersen, Jörn; Brinkmann, Henner; Bunk, Boyke; Bhuju, Sabin; Jarek, Michael; Geffers, Robert; Lang, Andrew S; Wagner-Döbler, Irene

2018-01-01

Abstract Gene transfer agents (GTAs) are phage-like particles which contain a fragment of genomic DNA of the bacterial or archaeal producer and deliver this to a recipient cell. GTA gene clusters are present in the genomes of almost all marine Rhodobacteraceae (Roseobacters) and might be important contributors to horizontal gene transfer in the world’s oceans. For all organisms studied so far, no obvious evidence of sequence specificity or other nonrandom process responsible for packaging genomic DNA into GTAs has been found. Here, we show that knock-out of an autoinducer synthase gene of Dinoroseobacter shibae resulted in overproduction and release of functional GTA particles (DsGTA). Next-generation sequencing of the 4.2-kb DNA fragments isolated from DsGTAs revealed that packaging was not random. DNA from low-GC conjugative plasmids but not from high-GC chromids was excluded from packaging. Seven chromosomal regions were strongly overrepresented in DNA isolated from DsGTA. These packaging peaks lacked identifiable conserved sequence motifs that might represent recognition sites for the GTA terminase complex. Low-GC regions of the chromosome, including the origin and terminus of replication, were underrepresented in DNA isolated from DsGTAs. DNA methylation reduced packaging frequency while the level of gene expression had no influence. Chromosomal regions found to be over- and underrepresented in DsGTA-DNA were regularly spaced. We propose that a “headful” type of packaging is initiated at the sites of coverage peaks and, after linearization of the chromosomal DNA, proceeds in both directions from the initiation site. GC-content, DNA-modifications, and chromatin structure might influence at which sides GTA packaging can be initiated. PMID:29325123

Hepatitis B virus (HBV)-specific short hairpin RNA is capable of reducing the formation of HBV covalently closed circular (CCC) DNA but has no effect on established CCC DNA in vitro.

PubMed

Starkey, Jason L; Chiari, Estelle F; Isom, Harriet C

2009-01-01

Hepatitis B virus (HBV) covalently closed circular (CCC) DNA is the source of HBV transcripts and persistence in chronically infected patients. The novel aspect of this study was to determine the effect of RNA interference (RNAi) on HBV CCC DNA when administered prior to establishment of HBV replication or during chronic HBV infection. HBV replication was initiated in HepG2 cells by transduction with HBV baculovirus. Subculture of HBV-expressing HepG2 cells at 10 days post-transduction generates a system in which HBV replication is ongoing and HBV is expressed largely from CCC DNA, thus simulating chronic HBV infection. HepG2 cells were transduced with short hairpin RNA (shRNA)-expressing baculovirus prior to initiation of HBV replication or during chronic HBV replication, and the levels of HBV RNA, HBV surface antigens (HBsAg) and replicative intermediates (RI), extracellular (EC) and CCC DNA species were measured. HBsAg, HBV RNA and DNA levels were markedly reduced until day 8 whether cells were transduced with shRNA prior to or during a chronic infection; however, the CCC DNA species were only affected when shRNA was administered prior to initiation of infection. We conclude that RNAi may have a therapeutic value for controlling HBV replication at the level of RI and EC DNA and for reducing establishment of CCC DNA during HBV infection. Our data support previous findings demonstrating the stability of HBV CCC DNA following antiviral therapy. This study also reports the development of a novel HBV baculovirus subculture system that can be used to evaluate antiviral effects on chronic HBV replication.

IgA response and protection following nasal vaccination of chickens with Newcastle disease virus DNA vaccine nanoencapsulated with Ag@SiO2 hollow nanoparticles

PubMed Central

Zhao, Kai; Rong, Guangyu; Hao, Yan; Yu, Lu; Kang, Hong; Wang, Xin; Wang, Xiaohua; Jin, Zheng; Ren, Zhiyu; Li, Zejun

2016-01-01

Newcastle disease caused by ND virus (NDV) is a highly contagious disease of birds. Vaccine for effective protection of poultry animals from NDV infection is urgently needed. Mucosal immunity plays a very important role in the antiviral immune response. In this study, a NDV F gene-containing DNA vaccine encapsulated in Ag@SiO2 hollow nanoparticles (pFDNA-Ag@SiO2-NPs) with an average diameter of 500 nm were prepared to assess the mucosal immune response. These nanoparticles exhibited low cytotoxicity and did not destroy the bioactivity of plasmid DNA, which could be expressed in vitro. The plasmid DNA was sustainably released after an initial burst release. In vivo immunization showed that the intranasal immunization of chickens with pFDNA-Ag@SiO2-NPs induced high titers of serum antibody, significantly promoted lymphocyte proliferation and induced higher expression levels of IL-2 and IFN-γ in a dose-dependent manner. These results indicated that the Ag@SiO2 hollow nanoparticles could serve as an efficient and safe delivery carrier for NDV DNA vaccine to induce mucosal immunity. This study has provided promising results for the further development of mucosal vaccines encapsulated in inorganic nanoparticles. PMID:27170532

IgA response and protection following nasal vaccination of chickens with Newcastle disease virus DNA vaccine nanoencapsulated with Ag@SiO2 hollow nanoparticles

NASA Astrophysics Data System (ADS)

Zhao, Kai; Rong, Guangyu; Hao, Yan; Yu, Lu; Kang, Hong; Wang, Xin; Wang, Xiaohua; Jin, Zheng; Ren, Zhiyu; Li, Zejun

2016-05-01

Newcastle disease caused by ND virus (NDV) is a highly contagious disease of birds. Vaccine for effective protection of poultry animals from NDV infection is urgently needed. Mucosal immunity plays a very important role in the antiviral immune response. In this study, a NDV F gene-containing DNA vaccine encapsulated in Ag@SiO2 hollow nanoparticles (pFDNA-Ag@SiO2-NPs) with an average diameter of 500 nm were prepared to assess the mucosal immune response. These nanoparticles exhibited low cytotoxicity and did not destroy the bioactivity of plasmid DNA, which could be expressed in vitro. The plasmid DNA was sustainably released after an initial burst release. In vivo immunization showed that the intranasal immunization of chickens with pFDNA-Ag@SiO2-NPs induced high titers of serum antibody, significantly promoted lymphocyte proliferation and induced higher expression levels of IL-2 and IFN-γ in a dose-dependent manner. These results indicated that the Ag@SiO2 hollow nanoparticles could serve as an efficient and safe delivery carrier for NDV DNA vaccine to induce mucosal immunity. This study has provided promising results for the further development of mucosal vaccines encapsulated in inorganic nanoparticles.

Reverse transcription of phage RNA and its fragment directed by synthetic heteropolymeric primers

PubMed Central

Frolova, L. Yu.; Metelyev, V. G.; Ratmanova, K. I.; Smirnov, V. D.; Shabarova, Z. A.; Prokofyev, M. A.; Berzin, V. M.; Jansone, I. V.; Gren, E. J.; Kisselev, L. L.

1977-01-01

DNA synthesis catalysed by RNA-directed DNA-polymerase (reverse transcriptase) was found to proceed on the RNA template of an MS2 phage in the presence of heteropolymeric synthetic octa- and nonadeoxyribonucleotide primers complementary to the intercistronic region (coat protein binding site) and the region of the coat protein cistron, respectively. The product of synthesis consists of discrete DNA fractions of different length, including transcripts longer than 1,000 nucleotides. The coat protein inhibits DNA synthesis if it is initiated at its binding site, but has no effect on DNA synthesis initiated at the coat protein cistron. It has been suggested that, in this system, the initiation of DNA synthesis by synthetic primers is topographically specific. The MS2 coat protein binding site (an RNA fragment of 59 nucleotides) serves as a template for polydeoxyribonucleotide synthesis in the presence of octanucleotide primer and reverse transcriptase. The product of synthesis is homogenous and its length corresponds to the length of the template. The effective and complete copying of the fragment having a distinct secondary structure proves that the secondary structure does not interfere, in principle, with RNA being a template in the system of reverse transcription. PMID:71713

FTS is responsible for radiation-induced nuclear phosphorylation of EGFR and repair of DNA damage in cervical cancer cells.

PubMed

Muthusami, Sridhar; Prabakaran, D S; Yu, Jae-Ran; Park, Woo-Yoon

2015-02-01

Radiation-induced nuclear stabilization and phosphorylation of epidermal growth factor receptor (EGFR) confers radioresistance. Understanding of the factor(s) regulating the nuclear stabilization and phosphorylation of EGFR is important for the modulation of radioresistance. Present study was designed to delineate the regulation of EGFR nuclear stabilization and phosphorylation by fused toes homolog (FTS), an oncoprotein, which is responsible for the radioresistance in cervical cancer cells. A cervical cancer cell line, ME180 was used. Radiation-induced change in the levels of EGFR, p-EGFR and FTS were evaluated in the cytoplasm and nucleus using Western blot analyses. FTS was silenced using siRNA-based approach. Interaction between EGFR and FTS was assessed using immunofluorescence and immunoprecipitation analyses. Double-strand breaks (DSB) of DNA were assessed using γ H2AX. Radiation increased the levels of EGFR and FTS in the cytoplasm and nucleus. EGFR and FTS are in physical association with each other and are co-localized in the cells. FTS silencing largely reduced the nuclear stabilization and phosphorylation of EGFR and DNA-protein kinase along with increased initial and residual DSBs. EGFR and FTS physically associate with each other and FTS silencing radiosensitizes ME180 cells through impaired nuclear EGFR signaling.

Mechanism of Promoter Melting by the Xeroderma Pigmentosum Complementation Group B Helicase of Transcription Factor IIH Revealed by Protein-DNA Photo-Cross-Linking

PubMed Central

Douziech, Maxime; Coin, Frédéric; Chipoulet, Jean-Marc; Arai, Yoko; Ohkuma, Yoshiaki; Egly, Jean-Marc; Coulombe, Benoit

2000-01-01

The p89/xeroderma pigmentosum complementation group B (XPB) ATPase-helicase of transcription factor IIH (TFIIH) is essential for promoter melting prior to transcription initiation by RNA polymerase II (RNAPII). By studying the topological organization of the initiation complex using site-specific protein-DNA photo-cross-linking, we have shown that p89/XPB makes promoter contacts both upstream and downstream of the initiation site. The upstream contact, which is in the region where promoter melting occurs (positions −9 to +2), requires tight DNA wrapping around RNAPII. The addition of hydrolyzable ATP tethers the template strand at positions −5 and +1 to RNAPII subunits. A mutation in p89/XPB found in a xeroderma pigmentosum patient impairs the ability of TFIIH to associate correctly with the complex and thereby melt promoter DNA. A model for open complex formation is proposed. PMID:11027286

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

PubMed

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

2016-01-02

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

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

PubMed Central

Rothrock, Michael J.; Hiett, Kelli L.; Gamble, John; Caudill, Andrew C.; Cicconi-Hogan, Kellie M.; Caporaso, J. Gregory

2014-01-01

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the “gold standard” enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples. PMID:25548939

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

PubMed Central

Zhao, Wei; Jardine, Paul J.

2015-01-01

ABSTRACT During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. IMPORTANCE During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs. PMID:26423956

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor.

PubMed

Zhao, Wei; Jardine, Paul J; Grimes, Shelley

2015-12-01

During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Escherichia coli DnaA forms helical structures along the longitudinal cell axis distinct from MreB filaments.

PubMed

Boeneman, Kelly; Fossum, Solveig; Yang, Yanhua; Fingland, Nicholas; Skarstad, Kirsten; Crooke, Elliott

2009-05-01

DnaA initiates chromosomal replication in Escherichia coli at a well-regulated time in the cell cycle. To determine how the spatial distribution of DnaA is related to the location of chromosomal replication and other cell cycle events, the localization of DnaA in living cells was visualized by confocal fluorescence microscopy. The gfp gene was randomly inserted into a dnaA-bearing plasmid via in vitro transposition to create a library that included internally GFP-tagged DnaA proteins. The library was screened for the ability to rescue dnaA(ts) mutants, and a candidate gfp-dnaA was used to replace the dnaA gene of wild-type cells. The resulting cells produce close to physiological levels of GFP-DnaA from the endogenous promoter as their only source of DnaA and somewhat under-initiate replication with moderate asynchrony. Visualization of GFP-tagged DnaA in living cells revealed that DnaA adopts a helical pattern that spirals along the long axis of the cell, a pattern also seen in wild-type cells by immunofluorescence with affinity purified anti-DnaA antibody. Although the DnaA helices closely resemble the helices of the actin analogue MreB, co-visualization of GFP-tagged DnaA and RFP-tagged MreB demonstrates that DnaA and MreB adopt discrete helical structures along the length of the longitudinal cell axis.

Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate

NASA Technical Reports Server (NTRS)

Winters, T. A.; Russell, P. S.; Kohli, M.; Dar, M. E.; Neumann, R. D.; Jorgensen, T. J.

1999-01-01

Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.

Replication protein A: directing traffic at the intersection of replication and repair.

PubMed

Oakley, Greg G; Patrick, Steve M

2010-06-01

Since the initial discovery of replication protein A (RPA) as a DNA replication factor, much progress has been made on elucidating critical roles for RPA in other DNA metabolic pathways. RPA has been shown to be required for DNA replication, DNA repair, DNA recombination, and the DNA damage response pathway with roles in checkpoint activation. This review summarizes the current understanding of RPA structure, phosphorylation and protein-protein interactions in mediating these DNA metabolic processes.

DNA Replication Arrest and DNA Damage Responses Induced by Alkylating Minor Groove Binders

DTIC Science & Technology

2003-05-01

Alkylating minor groove DNA binder adozelesin is capable of inhibiting DNA replication in treated cells through a trans-acting mechanism. The trans... replication in vitro. Using purified proteins in DNA replication initiation assays, we found that RPA purified from cells treated with adozelesin in not...adozelesin has the same single-stranded DNA binding activity and support nucleotide excision repair as normal RPA, but is not able to support SV40 DNA

Multiple Regulatory Systems Coordinate DNA Replication with Cell Growth in Bacillus subtilis

PubMed Central

Murray, Heath; Koh, Alan

2014-01-01

In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes. PMID:25340815

Multiple regulatory systems coordinate DNA replication with cell growth in Bacillus subtilis.

PubMed

Murray, Heath; Koh, Alan

2014-10-01

In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.

Electrostatic interactions during acidic phospholipid reactivation of DnaA protein, the Escherichia coli initiator of chromosomal replication.

PubMed

Kitchen, J L; Li, Z; Crooke, E

1999-05-11

The initiation of Escherichia coli chromosomal replication by DnaA protein is strongly influenced by the tight binding of the nucleotides ATP and ADP. Anionic phospholipids in a fluid bilayer promote the conversion of inactive ADP-DnaA protein to replicatively active ATP-DnaA protein in vitro, and thus likely play a key role in regulating DnaA activity. Previous studies have revealed that, during this reactivation, a specific region of DnaA protein inserts into the hydrophobic portion of the lipid bilayer in an acidic phospholipid-dependent manner. To elucidate the requirement for acidic phospholipids in the reactivation process, the contribution of electrostatic forces in the interaction of DnaA and lipid was examined. DnaA-lipid binding required anionic phospholipids, and DnaA-lipid binding as well as lipid-mediated release of DnaA-bound nucleotide were inhibited by increased ionic strength, suggesting the involvement of electrostatic interactions in these processes. As the vesicular content of acidic phospholipids was increased, both nucleotide release and DnaA-lipid binding increased in a linear, parallel manner. Given that DnaA-membrane binding, the insertion of DnaA into the membrane, and the consequent nucleotide release all require anionic phospholipids, the acidic headgroup may be necessary to recruit DnaA protein to the membrane for insertion and subsequent reactivation for replication.

Specific functions of the Rep and Rep' proteins of porcine circovirus during copy-release and rolling-circle DNA replication

USDA-ARS?s Scientific Manuscript database

The roles of two porcine circovirus replication initiator proteins, Rep and Rep', in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replica...

Starting a DNA barcode reference library for shallow water polychaetes from the southern European Atlantic coast.

PubMed

Lobo, Jorge; Teixeira, Marcos A L; Borges, Luisa M S; Ferreira, Maria S G; Hollatz, Claudia; Gomes, Pedro T; Sousa, Ronaldo; Ravara, Ascensão; Costa, Maria H; Costa, Filipe O

2016-01-01

Annelid polychaetes have been seldom the focus of dedicated DNA barcoding studies, despite their ecological relevance and often dominance, particularly in soft-bottom estuarine and coastal marine ecosystems. Here, we report the first assessment of the performance of DNA barcodes in the discrimination of shallow water polychaete species from the southern European Atlantic coast, focusing on specimens collected in estuaries and coastal ecosystems of Portugal. We analysed cytochrome oxidase I DNA barcodes (COI-5P) from 164 specimens, which were assigned to 51 morphospecies. To our data set from Portugal, we added available published sequences selected from the same species, genus or family, to inspect for taxonomic congruence among studies and collection location. The final data set comprised 290 specimens and 79 morphospecies, which generated 99 Barcode Index Numbers (BINs) within Barcode of Life Data Systems (BOLD). Among these, 22 BINs were singletons, 47 other BINs were concordant, confirming the initial identification based on morphological characters, and 30 were discordant, most of which consisted on multiple BINs found for the same morphospecies. Some of the most prominent cases in the latter category include Hediste diversicolor (O.F. Müller, 1776) (7), Eulalia viridis (Linnaeus, 1767) (2) and Owenia fusiformis (delle Chiaje, 1844) (5), all of them reported from Portugal and frequently used in ecological studies as environmental quality indicators. Our results for these species showed discordance between molecular lineages and morphospecies, or added additional relatively divergent lineages. The potential inaccuracies in environmental assessments, where underpinning polychaete species diversity is poorly resolved or clarified, demand additional and extensive investigation of the DNA barcode diversity in this group, in parallel with alpha taxonomy efforts. © 2015 John Wiley & Sons Ltd.

RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells

PubMed Central

Im, Jun-Sub; Park, Soon-Young; Cho, Won-Ho; Bae, Sung-Ho; Hurwitz, Jerard; Lee, Joon-Kyu

2015-01-01

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells. PMID:25602958

Mesenchymal stem cells cancel azoxymethane-induced tumor initiation.

PubMed

Nasuno, Masanao; Arimura, Yoshiaki; Nagaishi, Kanna; Isshiki, Hiroyuki; Onodera, Kei; Nakagaki, Suguru; Watanabe, Shuhei; Idogawa, Masashi; Yamashita, Kentaro; Naishiro, Yasuyoshi; Adachi, Yasushi; Suzuki, Hiromu; Fujimiya, Mineko; Imai, Kohzoh; Shinomura, Yasuhisa

2014-04-01

The role of mesenchymal stem cells (MSCs) in tumorigenesis remains controversial. Therefore, our goal was to determine whether exogenous MSCs possess intrinsic antineoplastic or proneoplastic properties in azoxymethane (AOM)-induced carcinogenesis. Three in vivo models were studied: an AOM/dextran sulfate sodium colitis-associated carcinoma model, an aberrant crypt foci model, and a model to assess the acute apoptotic response of a genotoxic carcinogen (AARGC). We also performed in vitro coculture experiments. As a result, we found that MSCs partially canceled AOM-induced tumor initiation but not tumor promotion. Moreover, MSCs inhibited the AARGC in colonic epithelial cells because of the removal of O(6)-methylguanine (O(6) MeG) adducts through O(6) MeG-DNA methyltransferase activation. Furthermore, MSCs broadly affected the cell-cycle machinery, potentially leading to G1 arrest in vivo. Coculture of IEC-6 rat intestinal cells with MSCs not only arrested the cell cycle at the G1 phase, but also induced apoptosis. The anti-carcinogenetic properties of MSCs in vitro required transforming growth factor (TGF)-β signaling because such properties were completely abrogated by absorption of TGF-β under indirect coculture conditions. MSCs inhibited AOM-induced tumor initiation by preventing the initiating cells from sustaining DNA insults and subsequently inducing G1 arrest in the initiated cells that escaped from the AARGC. Furthermore, tumor initiation perturbed by MSCs might potentially dysregulate WNT and TGF-β-Smad signaling pathways in subsequent tumorigenesis. Obtaining a better understanding of MSC functions in colon carcinogenesis is essential before commencing the broader clinical application of promising MSC-based therapies for cancer-prone patients with inflammatory bowel disease. © AlphaMed Press.

"Bad Luck Mutations": DNA Mutations Are not the Whole Answer to Understanding Cancer Risk.

PubMed

Trosko, James E; Carruba, Giuseppe

2017-01-01

It has been proposed that many human cancers are generated by intrinsic mechanisms that produce "Bad Luck" mutations by the proliferation of organ-specific adult stem cells. There have been serious challenges to this interpretation, including multiple extrinsic factors thought to be correlated with mutations found in cancers associated with these exposures. While support for both interpretations provides some validity, both interpretations ignore several concepts of the multistage, multimechanism process of carcinogenesis, namely, (1) mutations can be generated by both "errors of DNA repair" and "errors of DNA replication," during the "initiation" process of carcinogenesis; (2) "initiated" stem cells must be clonally amplified by nonmutagenic, intrinsic or extrinsic epigenetic mechanisms; (3) organ-specific stem cell numbers can be modified during in utero development, thereby altering the risk to cancer later in life; and (4) epigenetic tumor promoters are characterized by species, individual genetic-, gender-, developmental state-specificities, and threshold levels to be active; sustained and long-term exposures; and exposures in the absence of antioxidant "antipromoters." Because of the inevitability of some of the stem cells generating "initiating" mutations by either "errors of DNA repair" or "errors of DNA replication," a tumor is formed depending on the promotion phase of carcinogenesis. While it is possible to reduce our frequencies of mutagenic "initiated" cells, one can never reduce it to zero. Because of the extended period of the promotion phase of carcinogenesis, strategies to reduce the appearance of cancers must involve the interruption of the promotion of these initiated cells.

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

Damian, Luminita, E-mail: luminitadamian@microcal.eu.com; Universite de Toulouse, UPS, IPBS, F-31077 Toulouse; IUB, School of Engineering and Science, D-28727 Bremen

Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of interaction between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the binding was not affected by the nature of the template tested and the dissociation constants were in the same range when ssDNA (K{sub d}more » = 3.62 {+-} 2.1 x 10{sup -8} M) or the RNA corresponding sequence (K{sub d} = 2.7 {+-} 0.82 x 10{sup -8} M) bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferences which block the initiation complex formation. These results suggest that the limiting step in translation of ssDNA is the elongation process.« less

Synthesis of surface-anchored DNA-polymer bioconjugates using reversible addition-fragmentation chain transfer polymerization.

PubMed

He, Peng; He, Lin

2009-07-13

We report here an approach to grafting DNA-polymer bioconjugates on a planar solid support using reversible addition-fragmentation chain transfer (RAFT) polymerization. In particular, a trithiocarbonate compound as the RAFT chain transfer agent (CTA) is attached to the distal point of a surface-immobilized oligonucleotide. Initiation of RAFT polymerization leads to controlled growth of polymers atop DNA molecules on the surface. Growth kinetics of poly(monomethoxy-capped oligo(ethylene glycol) methacrylate) atop DNA molecules is investigated by monitoring the change of polymer film thickness as a function of reaction time. The reaction conditions, including the polymerization temperature, the initiator concentration, the CTA surface density, and the selection of monomers, are varied to examine their impacts on the grafting efficiency of DNA-polymer conjugates. Comparing to polymer growth atop small molecules, the experimental results suggest that DNA molecules significantly accelerate polymer growth, which is speculated as a result of the presence of highly charged DNA backbones and purine/pyrimidine moieties surrounding the reaction sites.

Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes.

PubMed

Klymus, Katy E; Marshall, Nathaniel T; Stepien, Carol A

2017-01-01

Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity.

Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes

PubMed Central

Klymus, Katy E.; Marshall, Nathaniel T.

2017-01-01

Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity. PMID:28542313

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity

PubMed Central

Pokrzywinski, Kaytee L.; Biel, Thomas G.; Kryndushkin, Dmitry; Rao, V. Ashutosh

2016-01-01

Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis. PMID:28030582

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity.

PubMed

Pokrzywinski, Kaytee L; Biel, Thomas G; Kryndushkin, Dmitry; Rao, V Ashutosh

2016-01-01

Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis.

Novel essential residues of Hda for interaction with DnaA in the regulatory inactivation of DnaA: unique roles for Hda AAA Box VI and VII motifs.

PubMed

Nakamura, Kenta; Katayama, Tsutomu

2010-04-01

Escherichia coli ATP-DnaA initiates chromosomal replication. For preventing extra-initiations, a complex of ADP-Hda and the DNA-loaded replicase clamp promotes DnaA-ATP hydrolysis, yielding inactive ADP-DnaA. However, the Hda-DnaA interaction mode remains unclear except that the Hda Box VII Arg finger (Arg-153) and DnaA sensor II Arg-334 within each AAA(+) domain are crucial for the DnaA-ATP hydrolysis. Here, we demonstrate that direct and functional interaction of ADP-Hda with DnaA requires the Hda residues Ser-152, Phe-118 and Asn-122 as well as Hda Arg-153 and DnaA Arg-334. Structural analyses suggest intermolecular interactions between Hda Ser-152 and DnaA Arg-334 and between Hda Phe-118 and the DnaA Walker B motif region, in addition to an intramolecular interaction between Hda Asn-122 and Arg-153. These interactions likely sustain a specific association of ADP-Hda and DnaA, promoting DnaA-ATP hydrolysis. Consistently, ATP-DnaA and ADP-DnaA interact with the ADP-Hda-DNA-clamp complex with similar affinities. Hda Phe-118 and Asn-122 are contained in the Box VI region, and their hydrophobic and electrostatic features are basically conserved in the corresponding residues of other AAA(+) proteins, suggesting a conserved role for Box VI. These findings indicate novel interaction mechanisms for Hda-DnaA as well as a potentially fundamental mechanism in AAA(+) protein interactions.

New insights into replication origin characteristics in metazoans

PubMed Central

Puy, Aurore; Rialle, Stéphanie; Kaplan, Noam; Segal, Eran

2012-01-01

We recently reported the identification and characterization of DNA replication origins (Oris) in metazoan cell lines. Here, we describe additional bioinformatic analyses showing that the previously identified GC-rich sequence elements form origin G-rich repeated elements (OGREs) that are present in 67% to 90% of the DNA replication origins from Drosophila to human cells, respectively. Our analyses also show that initiation of DNA synthesis takes place precisely at 160 bp (Drosophila) and 280 bp (mouse) from the OGRE. We also found that in most CpG islands, an OGRE is positioned in opposite orientation on each of the two DNA strands and detected two sites of initiation of DNA synthesis upstream or downstream of each OGRE. Conversely, Oris not associated with CpG islands have a single initiation site. OGRE density along chromosomes correlated with previously published replication timing data. Ori sequences centered on the OGRE are also predicted to have high intrinsic nucleosome occupancy. Finally, OGREs predict G-quadruplex structures at Oris that might be structural elements controlling the choice or activation of replication origins. PMID:22373526

The FA pathway counteracts oxidative stress through selective protection of antioxidant defense gene promoters.

PubMed

Du, Wei; Rani, Reena; Sipple, Jared; Schick, Jonathan; Myers, Kasiani C; Mehta, Parinda; Andreassen, Paul R; Davies, Stella M; Pang, Qishen

2012-05-03

Oxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with BM failure and cancer. Here we show that major antioxidant defense genes are down-regulated in FA patients, and that gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of the antioxidant defense genes. Assessment of promoter activity and DNA damage repair kinetics shows that increased initial damage, rather than a reduced repair rate, contributes to the augmented oxidative DNA damage. Mechanistically, FA proteins act in concert with the chromatin-remodeling factor BRG1 to protect the promoters of antioxidant defense genes from oxidative damage. Specifically, BRG1 binds to the promoters of the antioxidant defense genes at steady state. On challenge with oxidative stress, FA proteins are recruited to promoter DNA, which correlates with significant increase in the binding of BRG1 within promoter regions. In addition, oxidative stress-induced FANCD2 ubiquitination is required for the formation of a FA-BRG1-promoter complex. Taken together, these data identify a role for the FA pathway in cellular antioxidant defense.

Assembling and auditing a comprehensive DNA barcode reference library for European marine fishes.

PubMed

Oliveira, L M; Knebelsberger, T; Landi, M; Soares, P; Raupach, M J; Costa, F O

2016-12-01

A large-scale comprehensive reference library of DNA barcodes for European marine fishes was assembled, allowing the evaluation of taxonomic uncertainties and species genetic diversity that were otherwise hidden in geographically restricted studies. A total of 4118 DNA barcodes were assigned to 358 species generating 366 Barcode Index Numbers (BIN). Initial examination revealed as much as 141 BIN discordances (more than one species in each BIN). After implementing an auditing and five-grade (A-E) annotation protocol, the number of discordant species BINs was reduced to 44 (13% grade E), while concordant species BINs amounted to 271 (78% grades A and B) and 14 other had insufficient data (grade D). Fifteen species displayed comparatively high intraspecific divergences ranging from 2·6 to 18·5% (grade C), which is biologically paramount information to be considered in fish species monitoring and stock assessment. On balance, this compilation contributed to the detection of 59 European fish species probably in need of taxonomic clarification or re-evaluation. The generalized implementation of an auditing and annotation protocol for reference libraries of DNA barcodes is recommended. © 2016 The Fisheries Society of the British Isles.

5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley

PubMed Central

Solís, María-Teresa; El-Tantawy, Ahmed-Abdalla; Cano, Vanesa; Risueño, María C.; Testillano, Pilar S.

2015-01-01

Microspores are reprogrammed by stress in vitro toward embryogenesis. This process is an important tool in breeding to obtain double-haploid plants. DNA methylation is a major epigenetic modification that changes in differentiation and proliferation. We have shown changes in global DNA methylation during microspore reprogramming. 5-Azacytidine (AzaC) cannot be methylated and leads to DNA hypomethylation. AzaC is a useful demethylating agent to study DNA dynamics, with a potential application in microspore embryogenesis. This work analyzes the effects of short and long AzaC treatments on microspore embryogenesis initiation and progression in two species, the dicot Brassica napus and the monocot Hordeum vulgare. This involved the quantitative analyses of proembryo and embryo production, the quantification of DNA methylation, 5-methyl-deoxy-cytidine (5mdC) immunofluorescence and confocal microscopy, and the analysis of chromatin organization (condensation/decondensation) by light and electron microscopy. Four days of AzaC treatments (2.5 μM) increased embryo induction, response associated with a decrease of DNA methylation, modified 5mdC, and heterochromatin patterns compared to untreated embryos. By contrast, longer AzaC treatments diminished embryo production. Similar effects were found in both species, indicating that DNA demethylation promotes microspore reprogramming, totipotency acquisition, and embryogenesis initiation, while embryo differentiation requires de novo DNA methylation and is prevented by AzaC. This suggests a role for DNA methylation in the repression of microspore reprogramming and possibly totipotency acquisition. Results provide new insights into the role of epigenetic modifications in microspore embryogenesis and suggest a potential benefit of inhibitors, such as AzaC, to improve the process efficiency in biotechnology and breeding programs. PMID:26161085

Replication of the Escherichia coli chromosome in RNase HI-deficient cells: multiple initiation regions and fork dynamics.

PubMed

Maduike, Nkabuije Z; Tehranchi, Ashley K; Wang, Jue D; Kreuzer, Kenneth N

2014-01-01

DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on RNA-DNA hybrids (R-loops), which are normally removed by enzymes such as RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The DNA copy number profiles of various RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for genome stability, offering a new understanding of how RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons. © 2013 John Wiley & Sons Ltd.

Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

PubMed Central

George, Tresa; Wen, Qin; Griffiths, Richard; Ganesh, Anil; Meuth, Mark; Sanders, Cyril M.

2009-01-01

Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity. PMID:19700773

The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA

PubMed Central

Gai, Dahai; Wang, Damian; Li, Shu-Xing; Chen, Xiaojiang S

2016-01-01

DNA replication is a fundamental biological process. The initial step in eukaryotic DNA replication is the assembly of the pre-initiation complex, including the formation of two head-to-head hexameric helicases around the replication origin. How these hexameric helicases interact with their origin dsDNA remains unknown. Here, we report the co-crystal structure of the SV40 Large-T Antigen (LT) hexameric helicase bound to its origin dsDNA. The structure shows that the six subunits form a near-planar ring that interacts with the origin, so that each subunit makes unique contacts with the DNA. The origin dsDNA inside the narrower AAA+ domain channel shows partial melting due to the compression of the two phosphate backbones, forcing Watson-Crick base-pairs within the duplex to flip outward. This structure provides the first snapshot of a hexameric helicase binding to origin dsDNA, and suggests a possible mechanism of origin melting by LT during SV40 replication in eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.18129.001 PMID:27921994

Lesion search and recognition by thymine DNA glycosylase revealed by single molecule imaging

PubMed Central

Buechner, Claudia N.; Maiti, Atanu; Drohat, Alexander C.; Tessmer, Ingrid

2015-01-01

The ability of DNA glycosylases to rapidly and efficiently detect lesions among a vast excess of nondamaged DNA bases is vitally important in base excision repair (BER). Here, we use single molecule imaging by atomic force microscopy (AFM) supported by a 2-aminopurine fluorescence base flipping assay to study damage search by human thymine DNA glycosylase (hTDG), which initiates BER of mutagenic and cytotoxic G:T and G:U mispairs in DNA. Our data reveal an equilibrium between two conformational states of hTDG–DNA complexes, assigned as search complex (SC) and interrogation complex (IC), both at target lesions and undamaged DNA sites. Notably, for both hTDG and a second glycosylase, hOGG1, which recognizes structurally different 8-oxoguanine lesions, the conformation of the DNA in the SC mirrors innate structural properties of their respective target sites. In the IC, the DNA is sharply bent, as seen in crystal structures of hTDG lesion recognition complexes, which likely supports the base flipping required for lesion identification. Our results support a potentially general concept of sculpting of glycosylases to their targets, allowing them to exploit the energetic cost of DNA bending for initial lesion sensing, coupled with continuous (extrahelical) base interrogation during lesion search by DNA glycosylases. PMID:25712093

Photoinduced Oxidative DNA Damage Revealed by an Agarose Gel Nicking Assay: A Biophysical Chemistry Laboratory Experiment

NASA Astrophysics Data System (ADS)

Shafirovich, Vladimir; Singh, Carolyn; Geacintov, Nicholas E.

2003-11-01

Oxidative damage of DNA molecules associated with electron-transfer reactions is an important phenomenon in living cells, which can lead to mutations and contribute to carcinogenesis and the aging processes. This article describes the design of several simple experiments to explore DNA damage initiated by photoinduced electron-transfer reactions sensitized by the acridine derivative, proflavine (PF). A supercoiled DNA agarose gel nicking assay is employed as a sensitive probe of DNA strand cleavage. A low-cost experimental and computer-interfaced imaging apparatus is described allowing for the digital recording and analysis of the gel electrophoresis results. The first experiment describes the formation of direct strand breaks in double-stranded DNA induced by photoexcitation of the intercalated PF molecules. The second experiment demonstrates that the addition of the well-known electron acceptor, methylviologen, gives rise to a significant enhancement of the photochemical DNA strand cleavage effect. This occurs by an electron transfer step to methylviologen that renders the inital photoinduced charge separation between photoexcited PF and DNA irreversible. The third experiment demonstrates that the action spectrum of the DNA photocleavage matches the absorption spectrum of DNA-bound, intercalated PF molecules, which differs from that of free PF molecules. This result demonstrates that the photoinduced DNA strand cleavage is initiated by intercalated rather than free PF molecules.

Indicators of replicative damage in equine tendon fibroblast monolayers

PubMed Central

2013-01-01

Background Superficial digital flexor tendon (SDFT) injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. Relevant in vitro studies of this process require fibroblasts not already responding to stresses caused by the cell culture protocols. We investigated indicators of replicative damage in SDFT fibroblast monolayers, effects of this on their reparative ability, and measures that can be taken to reduce it. Results We found significant evidence of replicative stress, initially observing consistently large numbers of binucleate (BN) cells. A more variable but prominent feature was the presence of numerous gammaH2AX (γH2AX) puncta in nuclei, this being a histone protein that is phosphorylated in response to DNA double-stranded breaks (DSBs). Enrichment for injury detection and cell cycle arrest factors (p53 (ser15) and p21) occurred most frequently in BN cells; however, their numbers did not correlate with DNA damage levels and it is likely that the two processes have different causative mechanisms. Such remarkable levels of injury and binucleation are usually associated with irradiation, or treatment with cytoskeletal-disrupting agents. Both DSBs and BN cells were greatest in subconfluent (replicating) monolayers. The DNA-damaged cells co-expressed the replication markers TPX2/repp86 and centromere protein F. Once damaged in the early stages of culture establishment, fibroblasts continued to express DNA breaks with each replicative cycle. However, significant levels of cell death were not measured, suggesting that DNA repair was occurring. Comet assays showed that DNA repair was delayed in proportion to levels of genotoxic stress. Conclusions Researchers using tendon fibroblast monolayers should assess their “health” using γH2AX labelling. Continued use of early passage cultures expressing initially high levels of γH2AX puncta should be avoided for mechanistic studies and ex-vivo therapeutic applications, as this will not be resolved with further replicative cycling. Low density cell culture should be avoided as it enriches for both DNA damage and mitotic defects (polyploidy). As monolayers differing only slightly in baseline DNA damage levels showed markedly variable responses to a further injury, studies of effects of various stressors on tendon cells must be very carefully controlled. PMID:24025445

Radiation-induced genomic instability

NASA Technical Reports Server (NTRS)

Kronenberg, A.

1994-01-01

Quantitative assessment of the heritable somatic effects of ionizing radiation exposures has relied upon the assumption that radiation-induced lesions were 'fixed' in the DNA prior to the first postirradiation mitosis. Lesion conversion was thought to occur during the initial round of DNA replication or as a consequence of error-prone enzymatic processing of lesions. The standard experimental protocols for the assessment of a variety of radiation-induced endpoints (cell death, specific locus mutations, neoplastic transformation and chromosome aberrations) evaluate these various endpoints at a single snapshot in time. In contrast with the aforementioned approaches, some studies have specifically assessed radiation effects as a function of time following exposure. Evidence has accumulated in support of the hypothesis that radiation exposure induces a persistent destabilization of the genome. This instability has been observed as a delayed expression of lethal mutations, as an enhanced rate of accumulation of non-lethal heritable alterations, and as a progressive intraclonal chromosomal heterogeneity. The genetic controls and biochemical mechanisms underlying radiation-induced genomic instability have not yet been delineated. The aim is to integrate the accumulated evidence that suggests that radiation exposure has a persistent effect on the stability of the mammalian genome.

Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

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

Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan

Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase activemore » site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.« less

Evidence of animal mtDNA recombination between divergent populations of the potato cyst nematode Globodera pallida.

PubMed

Hoolahan, Angelique H; Blok, Vivian C; Gibson, Tracey; Dowton, Mark

2012-03-01

Recombination is typically assumed to be absent in animal mitochondrial genomes (mtDNA). However, the maternal mode of inheritance means that recombinant products are indistinguishable from their progenitor molecules. The majority of studies of mtDNA recombination assess past recombination events, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. Here we use the potato cyst nematode, Globodera pallida, to investigate past and contemporary recombination. Past recombination was assessed within and between populations of G. pallida, and contemporary recombination was assessed in the progeny of experimental crosses of these populations. Breeding of genetically divergent organisms may cause paternal mtDNA leakage, resulting in heteroplasmy and facilitating the detection of recombination. To assess contemporary recombination we looked for evidence of recombination between the mtDNA of the parental populations within the mtDNA of progeny. Past recombination was detected between a South American population and several UK populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal mtDNA leakage occurred, and the mtDNA of these populations subsequently recombined. This evidence challenges two dogmas of animal mtDNA evolution; no recombination and maternal inheritance. No contemporary recombination between the parental populations was detected in the progeny of the experimental crosses. This supports current arguments that mtDNA recombination events are rare. More sensitive detection methods may be required to adequately assess contemporary mtDNA recombination in animals.

Toward Applications for DNA Nanotechnology-More Bricks To Build With.

PubMed

Dietz, Hendrik

2016-06-16

Another brick in the wall: DNA nanotechnology has come a long way since its initial beginnings. This would not be possible without the continued development of methods for DNA assembly and new uses for DNA as a material. This Special Issue highlights some of the newest building blocks for nanodevices based on DNA. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular Weight of Deoxyribonucleic Acid Synthesized During Initiation of Chromosome Replication in Escherichia coli

PubMed Central

Kuempel, Peter L.

1972-01-01

Alkaline sucrose gradients were used to study the molecular weight of deoxyribonucleic acid (DNA) synthesized during the initiation of chromosome replication in Escherichia coli 15 TAU-bar. The experiments were conducted to determine whether newly synthesized, replication origin DNA is attached to higher-molecular-weight parental DNA. Little of the DNA synthesized after readdition of required amino acids to cells previously deprived of the amino acids was present in DNA with a molecular weight comparable to that of the parental DNA. The newly synthesized, low-molecular-weight DNA rapidly appeared in higher-molecular-weight material, but there was an upper limit to the size of this intermediate-molecular-weight DNA. This limit was not observed when exponentially growing cells converted newly synthesized DNA to higher-molecular-weight material. The size of the intermediate-molecular-weight DNA was related to the age of the replication forks, and the size increased as the replication forks moved further from the replication origin. The results indicate that the newly synthesized replication origin DNA is not attached to parental DNA, but it is rapidly attached to the growing strands that extend from the replication fork to the replication origin, or to the other replication fork if replication is bidirectional. Experiments are reported which demonstrate that the DNA investigated was from the vicinity of the replication origin and was not plasmid DNA or DNA from random positions on the chromosome. PMID:4562387

Genome-wide uniformity of human ‘open’ pre-initiation complexes

PubMed Central

Lai, William K.M.; Pugh, B. Franklin

2017-01-01

Transcription of protein-coding and noncoding DNA occurs pervasively throughout the mammalian genome. Their sites of initiation are generally inferred from transcript 5′ ends and are thought to be either locally dispersed or focused. How these two modes of initiation relate is unclear. Here, we apply permanganate treatment and chromatin immunoprecipitation (PIP-seq) of initiation factors to identify the precise location of melted DNA separately associated with the preinitiation complex (PIC) and the adjacent paused complex (PC). This approach revealed the two known modes of transcription initiation. However, in contrast to prevailing views, they co-occurred within the same promoter region: initiation originating from a focused PIC, and broad nucleosome-linked initiation. PIP-seq allowed transcriptional orientation of Pol II to be determined, which may be useful near promoters where sufficient sense/anti-sense transcript mapping information is lacking. PIP-seq detected divergently oriented Pol II at both coding and noncoding promoters, as well as at enhancers. Their occupancy levels were not necessarily coupled in the two orientations. DNA sequence and shape analysis of initiation complex sites suggest that both sequence and shape contribute to specificity, but in a context-restricted manner. That is, initiation sites have the locally “best” initiator (INR) sequence and/or shape. These findings reveal a common core to pervasive Pol II initiation throughout the human genome. PMID:27927716

Diverged composition and regulation of the Trypanosoma brucei origin recognition complex that mediates DNA replication initiation

PubMed Central

Marques, Catarina A.; Tiengwe, Calvin; Lemgruber, Leandro; Damasceno, Jeziel D.; Scott, Alan; Paape, Daniel; Marcello, Lucio; McCulloch, Richard

2016-01-01

Abstract Initiation of DNA replication depends upon recognition of genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played by a six-protein origin recognition complex (ORC). Why eukaryotes evolved a multisubunit initiator, and the roles of each component, remains unclear. In Trypanosoma brucei, an ancient unicellular eukaryote, only one ORC-related initiator, TbORC1/CDC6, has been identified by sequence homology. Here we show that three TbORC1/CDC6-interacting factors also act in T. brucei nuclear DNA replication and demonstrate that TbORC1/CDC6 interacts in a high molecular complex in which a diverged Orc4 homologue and one replicative helicase subunit can also be found. Analysing the subcellular localization of four TbORC1/CDC6-interacting factors during the cell cycle reveals that one factor, TbORC1B, is not a static constituent of ORC but displays S-phase restricted nuclear localization and expression, suggesting it positively regulates replication. This work shows that ORC architecture and regulation are diverged features of DNA replication initiation in T. brucei, providing new insight into this key stage of eukaryotic genome copying. PMID:26951375

Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes

PubMed Central

Duchi, Diego; Gryte, Kristofer; Robb, Nicole C; Morichaud, Zakia; Sheppard, Carol; Wigneshweraraj, Sivaramesh

2018-01-01

Abstract Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP–DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation. PMID:29177430

Data Release: DNA barcodes of plant species collected for the Global Genome Initiative for Gardens Program, National Museum of Natural History, Smithsonian Institution

PubMed Central

Zúñiga, Jose D.; Gostel, Morgan R.; Mulcahy, Daniel G.; Barker, Katharine; Asia Hill; Sedaghatpour, Maryam; Vo, Samantha Q.; Funk, Vicki A.; Coddington, Jonathan A.

2017-01-01

Abstract The Global Genome Initiative has sequenced and released 1961 DNA barcodes for genetic samples obtained as part of the Global Genome Initiative for Gardens Program. The dataset includes barcodes for 29 plant families and 309 genera that did not have sequences flagged as barcodes in GenBank and sequences from officially recognized barcoding genetic markers meet the data standard of the Consortium for the Barcode of Life. The genetic samples were deposited in the Smithsonian Institution’s National Museum of Natural History Biorepository and their records were made public through the Global Genome Biodiversity Network’s portal. The DNA barcodes are now available on GenBank. PMID:29118648

Conditions Determining Initiation of DNA Synthesis in 3T3 Cells*

PubMed Central

Dulbecco, R.; Stoker, M. G. P.

1970-01-01

Experiments were designed to discriminate between inhibition of growth due to contacts or exhaustion of serum factors. The cell layer was wounded and the migrating cells were followed by time-lapse cinematography; DNA synthesis in the same cells was recognized by means of 3H-thymidine labeling and radioautography. In this way, the complete history of individual cells migrating to the wound could be described. The results show that topographical relationships between cells play an important role in controlling initiation of DNA synthesis. It is still unclear whether initiation is promoted by release from contacts or by the increased ability of the cells to utilize serum factors because of their changes in shapes and activities. PMID:5273897

Crystallization and preliminary crystallographic characterization of the origin-binding domain of the bacteriophage λ O replication initiator

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

Struble, E. B., E-mail: evi.struble@nist.gov; Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205; Center for Advanced Research in Biotechnology/NIST, 9600 Gudelsky Drive, Rockville, MD 20850

2007-06-01

Crystallization and preliminary diffraction data of the N-terminal 19–139 fragment of the origin-binding domain of bacteriophage λ O replication initiator are reported. The bacteriophage λ O protein binds to the λ replication origin (oriλ) and serves as the primary replication initiator for the viral genome. The binding energy derived from the binding of O to oriλ is thought to help drive DNA opening to facilitate initiation of DNA replication. Detailed understanding of this process is severely limited by the lack of high-resolution structures of O protein or of any lambdoid phage-encoded paralogs either with or without DNA. The production ofmore » crystals of the origin-binding domain of λ O that diffract to 2.5 Å is reported. Anomalous dispersion methods will be used to solve this structure.« less

Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP-ribosyl)ation

PubMed Central

Hodgson, Andrea; Wier, Eric M.; Wen, Matthew G.; Kamenyeva, Olena; Xia, Xue; Koo, Lily Y.

2016-01-01

The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production. PMID:27635653

Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes

PubMed Central

Tóth, Júlia; Bollins, Jack; Szczelkun, Mark D.

2015-01-01

DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This ‘DNA sliding’ is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding. PMID:26538601

DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA.

PubMed

Keyamura, Kenji; Katayama, Tsutomu

2011-08-19

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA*

PubMed Central

Keyamura, Kenji; Katayama, Tsutomu

2011-01-01

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis. PMID:21708944

Tissue-specific profile of DNA replication in the swimming larvae of Ciona intestinalis.

PubMed

Nakayama, Akie; Satoh, Nori; Sasakura, Yasunori

2005-03-01

The cell cycle is strictly regulated during development and its regulation is essential for organ formation and developmental timing. Here we observed the pattern of DNA replication in swimming larvae of an ascidian, Ciona intestinalis. Usually, Ciona swimming larvae obtain competence for metamorphosis at about 4-5 h after hatching, and these competent larvae initiate metamorphosis soon after they adhere to substrate with their papillae. In these larvae, three major tissues (epidermis, endoderm and mesenchyme) showed extensive DNA replication with distinct pattern and timing, suggesting tissue-specific cell cycle regulation. However, DNA replication did not continue in aged larvae which kept swimming for several days, suggesting that the cell cycle is arrested in these larvae at a certain time to prevent further growth of adult organ rudiments until the initiation of metamorphosis. Inhibition of the cell cycle by aphidicolin during the larval stage affects only the speed of metamorphosis, and not the formation of adult organ rudiments or the timing of the initiation of metamorphosis. However, after the completion of tail resorption, DNA replication is necessary for further metamorphic events. Our data showed that DNA synthesis in the larval trunk is not directly associated with the organization of adult organs, but it contributes to the speed of metamorphosis after settlement.

Characterization of 5' end of human thromboxane receptor gene. Organizational analysis and mapping of protein kinase C--responsive elements regulating expression in platelets.

PubMed

D'Angelo, D D; Davis, M G; Houser, W A; Eubank, J J; Ritchie, M E; Dorn, G W

1995-09-01

Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5' flanking region of the thromboxane receptor gene. The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximately 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into platelet-like K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximately 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly suggest that the mechanism for previously described upregulation of platelet thromboxane receptors after acute myocardial infarction is increased thromboxane receptor gene transcription in platelet-progenitor cells.

Use of swabs for sampling epithelial cells for molecular genetics analyses in Enteroctopus

USGS Publications Warehouse

Hollenback, Nathan; Scheel, David; Gravley, Megan C.; Sage, George K.; Toussaint, Rebecca K.; Talbot, Sandra L.

2017-01-01

We evaluated the efficacy of using swabs to collect cells from the epidermis of octopus as a non-invasive DNA source for classical genetic studies, and demonstrated value of the technique by incorporating it into an effort to determine, within a day, the lineage of captured, live Enteroctopus (E. dofleini or a cryptic lineage). The cryptic lineage was targeted for captive behavioral and morphological studies, while once genetically identified, the non-target lineage could be more rapidly released back to the wild. We used commercially available sterile foamtipped swabs and a high-salt preservation buffer to collect and store paired swab and muscle (arm tip) tissue sampled from live Enteroctopus collected from Prince William Sound, Alaska. We performed a one-day extraction of DNA from epithelial swab samples and amplification of two diagnostic microsatellite loci to determine the lineage of each of the 21 individuals. Following this rapid lineage assessment, which allowed us to release non-target individuals within a day of laboratory work, we compared paired swab and muscle tissue samples from each individual to assess quantity of DNA yields and consistency of genotyping results, followed by assessment of locus-by-locus reliability of DNA extracts from swabs. Epithelial swabs yielded, on average, lower quantities of DNA (170.32 ± 74.72 (SD) ng/μL) relative to DNA obtained from tissues collected using invasive or destructive techniques (310.95 ± 147.37 (SD) ng/μL. We observed some decrease in yields of DNA from extractions of swab samples conducted 19 and 31 months after initial extractions when samples were stored at room temperature in lysis buffer. All extractions yielded quantities of DNA sufficient to amplify and score all loci, which included fragment data from 10 microsatellite loci (nine polymorphic loci and monomorphic locus EdoμA106), and nucleotide sequence data from a 528 base pair portion of the nuclear octopine dehydrogenase gene. All results from genotyping and sequencing using paired swab and muscle tissue extracts were concordant, and experimental reliability levels for multilocus genotypes generated from swab samples exceeded 97%. This technique is useful for studies in which invasive sampling is not optimal, and in remote field situations since samples can be stored at ambient temperatures for at least 31 months. The use of epithelial swabs is thus a noninvasive technique appropriate for sampling genetic material from live octopuses for use in classical genetic studies as well as supporting experimental and behavioral studies.

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

PubMed Central

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

2017-01-01

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

Investigating the epigenetic effects of a prototype smoke-derived carcinogen in human cells.

PubMed

Tommasi, Stella; Kim, Sang-in; Zhong, Xueyan; Wu, Xiwei; Pfeifer, Gerd P; Besaratinia, Ahmad

2010-05-12

Global loss of DNA methylation and locus/gene-specific gain of DNA methylation are two distinct hallmarks of carcinogenesis. Aberrant DNA methylation is implicated in smoking-related lung cancer. In this study, we have comprehensively investigated the modulation of DNA methylation consequent to chronic exposure to a prototype smoke-derived carcinogen, benzo[a]pyrene diol epoxide (B[a]PDE), in genomic regions of significance in lung cancer, in normal human cells. We have used a pulldown assay for enrichment of the CpG methylated fraction of cellular DNA combined with microarray platforms, followed by extensive validation through conventional bisulfite-based analysis. Here, we demonstrate strikingly similar patterns of DNA methylation in non-transformed B[a]PDE-treated cells vs control using high-throughput microarray-based DNA methylation profiling confirmed by conventional bisulfite-based DNA methylation analysis. The absence of aberrant DNA methylation in our model system within a timeframe that precedes cellular transformation suggests that following carcinogen exposure, other as yet unknown factors (secondary to carcinogen treatment) may help initiate global loss of DNA methylation and region-specific gain of DNA methylation, which can, in turn, contribute to lung cancer development. Unveiling the initiating events that cause aberrant DNA methylation in lung cancer has tremendous public health relevance, as it can help define future strategies for early detection and prevention of this highly lethal disease.

Investigating the Epigenetic Effects of a Prototype Smoke-Derived Carcinogen in Human Cells

PubMed Central

Tommasi, Stella; Kim, Sang-in; Zhong, Xueyan; Wu, Xiwei; Pfeifer, Gerd P.; Besaratinia, Ahmad

2010-01-01

Global loss of DNA methylation and locus/gene-specific gain of DNA methylation are two distinct hallmarks of carcinogenesis. Aberrant DNA methylation is implicated in smoking-related lung cancer. In this study, we have comprehensively investigated the modulation of DNA methylation consequent to chronic exposure to a prototype smoke-derived carcinogen, benzo[a]pyrene diol epoxide (B[a]PDE), in genomic regions of significance in lung cancer, in normal human cells. We have used a pulldown assay for enrichment of the CpG methylated fraction of cellular DNA combined with microarray platforms, followed by extensive validation through conventional bisulfite-based analysis. Here, we demonstrate strikingly similar patterns of DNA methylation in non-transformed B[a]PDE-treated cells vs control using high-throughput microarray-based DNA methylation profiling confirmed by conventional bisulfite-based DNA methylation analysis. The absence of aberrant DNA methylation in our model system within a timeframe that precedes cellular transformation suggests that following carcinogen exposure, other as yet unknown factors (secondary to carcinogen treatment) may help initiate global loss of DNA methylation and region-specific gain of DNA methylation, which can, in turn, contribute to lung cancer development. Unveiling the initiating events that cause aberrant DNA methylation in lung cancer has tremendous public health relevance, as it can help define future strategies for early detection and prevention of this highly lethal disease. PMID:20485678

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

PubMed

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

2009-02-01

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

Strategy for Sensitive and Specific Detection of Yersinia pestis in Skeletons of the Black Death Pandemic

PubMed Central

Seifert, Lisa; Harbeck, Michaela; Thomas, Astrid; Hoke, Nadja; Zöller, Lothar; Wiechmann, Ingrid; Grupe, Gisela; Scholz, Holger C.; Riehm, Julia M.

2013-01-01

Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14th century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study. PMID:24069445

EGFR T790M mutation testing of non-small cell lung cancer tissue and blood samples artificially spiked with circulating cell-free tumor DNA: results of a round robin trial.

PubMed

Fassunke, Jana; Ihle, Michaela Angelika; Lenze, Dido; Lehmann, Annika; Hummel, Michael; Vollbrecht, Claudia; Penzel, Roland; Volckmar, Anna-Lena; Stenzinger, Albrecht; Endris, Volker; Jung, Andreas; Lehmann, Ulrich; Zeugner, Silke; Baretton, Gustavo; Kreipe, Hans; Schirmacher, Peter; Kirchner, Thomas; Dietel, Manfred; Büttner, Reinhard; Merkelbach-Bruse, Sabine

2017-10-01

The European Commision (EC) recently approved osimertinib for the treatment of adult patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) harboring EGFR T790M mutations. Besides tissue-based testing, blood samples containing cell-free circulating tumor DNA (ctDNA) can be used to interrogate T790M status. Herein, we describe the conditions and results of a round robin trial (RRT) for T790M mutation testing in NSCLC tissue specimens and peripheral blood samples spiked with cell line DNA mimicking tumor-derived ctDNA. The underlying objectives of this two-staged external quality assessment (EQA) approach were (a) to evaluate the accuracy of T790M mutations testing across multiple centers and (b) to investigate if a liquid biopsy-based testing for T790M mutations in spiked blood samples is feasible in routine diagnostic. Based on a successfully completed internal phase I RRT, an open RRT for EGFR T790M mutation testing in tumor tissue and blood samples was initiated. In total, 48 pathology centers participated in the EQA. Of these, 47 (97.9%) centers submitted their analyses within the pre-defined time frame and 44 (tissue), respectively, 40 (plasma) successfully passed the test. The overall success rates in the RRT phase II were 91.7% (tissue) and 83.3% (blood), respectively. Thirty-eight out of 48 participants (79.2%) successfully passed both parts of the RRT. The RRT for blood-based EGFR testing initiated in Germany is, to the best of our knowledge, the first of his kind in Europe. In summary, our results demonstrate that blood-based genotyping for EGFR resistance mutations can be successfully integrated in routine molecular diagnostics complementing the array of molecular methods already available at pathology centers in Germany.

Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.

PubMed

Seifert, Lisa; Harbeck, Michaela; Thomas, Astrid; Hoke, Nadja; Zöller, Lothar; Wiechmann, Ingrid; Grupe, Gisela; Scholz, Holger C; Riehm, Julia M

2013-01-01

Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14(th) century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study.

Histone Modification Associated with Initiation of DNA Replication | Center for Cancer Research

Cancer.gov

Before cells are able to divide, they must first duplicate their chromosomes accurately. DNA replication and packaging of DNA into chromosomes by histone proteins need to be coordinated by the cell to ensure proper transmission of genetic and epigenetic information to the next generation. Mammalian DNA replication begins at specific chromosomal sites, called replication

Novel synthetic (S,S) and (R,R)-secoisolariciresinol diglucosides (SDGs) protect naked plasmid and genomic DNA From gamma radiation damage.

PubMed

Mishra, Om P; Pietrofesa, Ralph; Christofidou-Solomidou, Melpo

2014-07-01

Secoisolariciresinol diglucoside (SDG) is the major lignan in wholegrain flaxseed. However, extraction methods are complex and are associated with low yield and high costs. Using a novel synthetic pathway, our group succeeded in chemically synthesizing SDG (S,S and R,R enantiomers), which faithfully recapitulates the properties of their natural counterparts, possessing strong antioxidant and free radical scavenging properties. This study further extends initial findings by now investigating the DNA-radioprotective properties of the synthetic SDG enantiomers compared to the commercial SDG. DNA radioprotection was assessed by cell-free systems such as: (a) plasmid relaxation assay to determine the extent of the supercoiled (SC) converted to open-circular (OC) plasmid DNA (pBR322) after exposure of the plasmid to gamma radiation; and (b) determining the extent of genomic DNA fragmentation. Exposure of plasmid DNA to 25 Gy of γ radiation resulted in decreased supercoiled form and increased open-circular form, indicating radiation-induced DNA damage. Synthetic SDG (S,S) and SDG (R,R), and commercial SDG at concentrations of 25-250 μM significantly and equipotently reduced the radiation-induced supercoiled to open-circular plasmid DNA in a dose-dependent conversion. In addition, exposure of calf thymus DNA to 50 Gy of gamma radiation resulted in DNA fragments of low-molecular weight (<6,000 bps), which was prevented in a dose-dependence manner by all synthetic and natural SDG enantomers, at concentrations as low as 0.5 μM. These novel results demonstrated that synthetic SDG (S,S) and SDG (R,R) isomers and commercial SDG possess DNA-radioprotective properties. Such properties along with their antioxidant and free radical scavenging activity, reported earlier, suggest that SDGs are promising candidates for radioprotection for normal tissue damage as a result of accidental exposure during radiation therapy for cancer treatment.

Novel Synthetic (S,S) and (R,R)-Secoisolariciresinol Diglucosides (SDGs) Protect Naked Plasmid and Genomic DNA From Gamma Radiation Damage

PubMed Central

Mishra, Om P.; Pietrofesa, Ralph; Christofidou-Solomidou, Melpo

2014-01-01

Secoisolariciresinol diglucoside (SDG) is the major lignan in wholegrain flaxseed. However, extraction methods are complex and are associated with low yield and high costs. Using a novel synthetic pathway, our group succeeded in chemically synthesizing SDG (S,S and R,R enantiomers), which faithfully recapitulates the properties of their natural counterparts, possessing strong antioxidant and free radical scavenging properties. This study further extends initial findings by now investigating the DNA-radioprotective properties of the synthetic SDG enantiomers compared to the commercial SDG. DNA radioprotection was assessed by cell-free systems such as: (a) plasmid relaxation assay to determine the extent of the supercoiled (SC) converted to open-circular (OC) plasmid DNA (pBR322) after exposure of the plasmid to gamma radiation; and (b) determining the extent of genomic DNA fragmentation. Exposure of plasmid DNA to 25 Gy of γ radiation resulted in decreased supercoiled form and increased open-circular form, indicating radiation-induced DNA damage. Synthetic SDG (S,S) and SDG (R,R), and commercial SDG at concentrations of 25–250 μM significantly and equipotently reduced the radiation-induced supercoiled to open-circular plasmid DNA in a dose-dependent conversion. In addition, exposure of calf thymus DNA to 50 Gy of gamma radiation resulted in DNA fragments of low-molecular weight (<6,000 bps), which was prevented in a dose-dependence manner by all synthetic and natural SDG enantomers, at concentrations as low as 0.5 μM. These novel results demonstrated that synthetic SDG (S,S) and SDG (R,R) isomers and commercial SDG possess DNA-radioprotective properties. Such properties along with their antioxidant and free radical scavenging activity, reported earlier, suggest that SDGs are promising candidates for radioprotection for normal tissue damage as a result of accidental exposure during radiation therapy for cancer treatment. PMID:24945894

Functional Architecture of T7 RNA Polymerase Transcription Complexes

PubMed Central

Nayak, Dhananjaya; Guo, Qing; Sousa, Rui

2007-01-01

Summary T7 RNA polymerase is the best-characterized member of a widespread family of single-subunit RNA polymerases. Crystal structures of T7 RNA polymerase initiation and elongation complexes have provided a wealth of detailed information on RNA polymerase interactions with the promoter and transcription bubble, but the absence of DNA downstream of the melted region of the template in the initiation complex structure, and the absence of DNA upstream of the transcription bubble in the elongation complex structure means that our picture of the functional architecture of T7 RNA polymerase transcription complexes remains incomplete. Here we use the site-specifically tethered chemical nucleases and functional characterization of directed T7 RNAP mutants to both reveal the architecture of the duplex DNA that flanks the transcription bubble in the T7 RNAP initiation and elongation complexes, and to define the function of the interactions made by these duplex elements. We find that downstream duplex interactions made with a cluster of lysines (K711/K713/K714) are present during both elongation and initiation where they contribute to stabilizing a bend in the downstream DNA that is important for promoter opening. The upstream DNA in the elongation complex is also found to be sharply bent at the upstream edge of the transcription bubble, thereby allowing formation of upstream duplex:polymerase interactions that contribute to elongation complex stability. PMID:17580086

Spectroscopic study of a DNA brush synthesized in situ by surface initiated enzymatic polymerization.

PubMed

Khan, M Nuruzzaman; Tjong, Vinalia; Chilkoti, Ashutosh; Zharnikov, Michael

2013-08-29

We used a combination of synchrotron-based X-ray photoelectron spectroscopy (XPS) and angle-resolved near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to study the chemical integrity, purity, and possible internal alignment of single-strand (ss) adenine deoxynucleotide (poly(A)) DNA brushes. The brushes were synthesized by surface-initiated enzymatic polymerization (SIEP) on a 25-mer of adenine self-assembled monolayer (SAM) on gold (A25-SH), wherein the terminal 3'-OH of the A25-SH serve as the initiation sites for SIEP of poly(A). XPS and NEXAFS spectra of poly(A) brushes were found to be almost identical to those of A25-SH initiator, with no unambiguous traces of contamination. Apart from the well-defined chemical integrity and contamination-free character, the brushes were found to have a high degree of orientational order, with an upright orientation of individual strands, despite their large thickness up to ~55 nm, that corresponds to a chain length of at least several hundred nucleotides for individual ssDNA molecules. The orientational order exhibited by these poly(A) DNA brushes, mediated presumably by base stacking, was found to be independent of the brush thickness as long as the packing density was high enough. The well-defined character and orientational ordering of the ssDNA brushes make them a potentially promising system for different applications.

Interactions of the SAP Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Carra, Claudio; Huff, Janice; Pluth, Janice M.; Cucinotta, Francis A.

2007-01-01

NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku70/80 complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The SAP domain of Ku70 (residues 556-609), contains an a helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the SAP/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the SAP domain of Ku70 and a 10 base pairs DNA duplex. Large-scale conformational changes were observed and some putative binding modes were suggested based on energetic analysis. These modes are consistent with previous experimental investigations. In addition, the results indicate that cooperation of SAP with other domains of Ku70/80 is necessary to explain the high affinity of binding as observed in experiments.

The Checkpoint Kinase 1 Inhibitor Prexasertib Induces Regression of Preclinical Models of Human Neuroblastoma.

PubMed

Lowery, Caitlin D; VanWye, Alle B; Dowless, Michele; Blosser, Wayne; Falcon, Beverly L; Stewart, Julie; Stephens, Jennifer; Beckmann, Richard P; Bence Lin, Aimee; Stancato, Louis F

2017-08-01

Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G 2 -M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma. Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line-derived xenograft mouse models of neuroblastoma. Results: Within 24 hours, single-agent prexasertib promoted γH2AX-positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA-PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature. Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354-63. ©2017 AACR . ©2017 American Association for Cancer Research.

A Prospective Blinded Evaluation of Urine-DNA Testing for Detection of Urothelial Bladder Carcinoma in Patients with Gross Hematuria.

PubMed

Dahmcke, Christina M; Steven, Kenneth E; Larsen, Louise K; Poulsen, Asger L; Abdul-Al, Ahmad; Dahl, Christina; Guldberg, Per

2016-12-01

Retrospective studies have provided proof of principle that bladder cancer can be detected by testing for the presence of tumor DNA in urine. We have conducted a prospective blinded study to determine whether a urine-based DNA test can replace flexible cystoscopy in the initial assessment of gross hematuria. A total of 475 consecutive patients underwent standard urological examination including flexible cystoscopy and computed tomography urography, and provided urine samples immediately before (n=461) and after (n=444) cystoscopy. Urine cells were collected using a filtration device and tested for eight DNA mutation and methylation biomarkers. Clinical evaluation identified 99 (20.8%) patients with urothelial bladder tumors. With this result as a reference and based on the analysis of all urine samples, the DNA test had a sensitivity of 97.0%, a specificity of 76.9%, a positive predictive value of 52.5%, and a negative predictive value of 99.0%. In three patients with a positive urine-DNA test without clinical evidence of cancer, a tumor was detected at repeat cystoscopy within 16 mo. Our results suggest that urine-DNA testing can be used to identify a large subgroup of patients with gross hematuria in whom cystoscopy is not required. We tested the possibility of using a urine-based DNA test to check for bladder cancer in patients with visible blood in the urine. Our results show that the test efficiently detects bladder cancer and therefore may be used to greatly reduce the number of patients who would need to undergo cystoscopy. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

A comparison of pedigree- and DNA-based measures for identifying inbreeding depression in the critically endangered Attwater's Prairie-chicken.

PubMed

Hammerly, Susan C; Morrow, Michael E; Johnson, Jeff A

2013-11-01

The primary goal of captive breeding programmes for endangered species is to prevent extinction, a component of which includes the preservation of genetic diversity and avoidance of inbreeding. This is typically accomplished by minimizing mean kinship in the population, thereby maintaining equal representation of the genetic founders used to initiate the captive population. If errors in the pedigree do exist, such an approach becomes less effective for minimizing inbreeding depression. In this study, both pedigree- and DNA-based methods were used to assess whether inbreeding depression existed in the captive population of the critically endangered Attwater's Prairie-chicken (Tympanuchus cupido attwateri), a subspecies of prairie grouse that has experienced a significant decline in abundance and concurrent reduction in neutral genetic diversity. When examining the captive population for signs of inbreeding, variation in pedigree-based inbreeding coefficients (f(pedigree)) was less than that obtained from DNA-based methods (f(DNA)). Mortality of chicks and adults in captivity were also positively correlated with parental relatedness (r(DNA)) and f(DNA), respectively, while no correlation was observed with pedigree-based measures when controlling for additional variables such as age, breeding facility, gender and captive/release status. Further, individual homozygosity by loci (HL) and parental rDNA values were positively correlated with adult mortality in captivity and the occurrence of a lethal congenital defect in chicks, respectively, suggesting that inbreeding may be a contributing factor increasing the frequency of this condition among Attwater's Prairie-chickens. This study highlights the importance of using DNA-based methods to better inform management decisions when pedigrees are incomplete or errors may exist due to uncertainty in pairings. © 2013 John Wiley & Sons Ltd.

Wolbachia and DNA barcoding insects: patterns, potential, and problems.

PubMed

Smith, M Alex; Bertrand, Claudia; Crosby, Kate; Eveleigh, Eldon S; Fernandez-Triana, Jose; Fisher, Brian L; Gibbs, Jason; Hajibabaei, Mehrdad; Hallwachs, Winnie; Hind, Katharine; Hrcek, Jan; Huang, Da-Wei; Janda, Milan; Janzen, Daniel H; Li, Yanwei; Miller, Scott E; Packer, Laurence; Quicke, Donald; Ratnasingham, Sujeevan; Rodriguez, Josephine; Rougerie, Rodolphe; Shaw, Mark R; Sheffield, Cory; Stahlhut, Julie K; Steinke, Dirk; Whitfield, James; Wood, Monty; Zhou, Xin

2012-01-01

Wolbachia is a genus of bacterial endosymbionts that impacts the breeding systems of their hosts. Wolbachia can confuse the patterns of mitochondrial variation, including DNA barcodes, because it influences the pathways through which mitochondria are inherited. We examined the extent to which these endosymbionts are detected in routine DNA barcoding, assessed their impact upon the insect sequence divergence and identification accuracy, and considered the variation present in Wolbachia COI. Using both standard PCR assays (Wolbachia surface coding protein--wsp), and bacterial COI fragments we found evidence of Wolbachia in insect total genomic extracts created for DNA barcoding library construction. When >2 million insect COI trace files were examined on the Barcode of Life Datasystem (BOLD) Wolbachia COI was present in 0.16% of the cases. It is possible to generate Wolbachia COI using standard insect primers; however, that amplicon was never confused with the COI of the host. Wolbachia alleles recovered were predominantly Supergroup A and were broadly distributed geographically and phylogenetically. We conclude that the presence of the Wolbachia DNA in total genomic extracts made from insects is unlikely to compromise the accuracy of the DNA barcode library; in fact, the ability to query this DNA library (the database and the extracts) for endosymbionts is one of the ancillary benefits of such a large scale endeavor--which we provide several examples. It is our conclusion that regular assays for Wolbachia presence and type can, and should, be adopted by large scale insect barcoding initiatives. While COI is one of the five multi-locus sequence typing (MLST) genes used for categorizing Wolbachia, there is limited overlap with the eukaryotic DNA barcode region.

Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells.

PubMed

Yan, Z; DeGregori, J; Shohet, R; Leone, G; Stillman, B; Nevins, J R; Williams, R S

1998-03-31

Cdc6 has a critical regulatory role in the initiation of DNA replication in yeasts, but its function in mammalian cells has not been characterized. We show here that Cdc6 is expressed selectively in proliferating but not quiescent mammalian cells, both in culture and within tissues of intact animals. During the transition from a growth-arrested to a proliferative state, transcription of mammalian Cdc6 is regulated by E2F proteins, as revealed by a functional analysis of the human Cdc6 promoter and by the ability of exogenously expressed E2F proteins to stimulate the endogenous Cdc6 gene. Immunodepletion of Cdc6 by microinjection of anti-Cdc6 antibody blocks initiation of DNA replication in a human tumor cell line. We conclude that expression of human Cdc6 is regulated in response to mitogenic signals though transcriptional control mechanisms involving E2F proteins, and that Cdc6 is required for initiation of DNA replication in mammalian cells.

Formation and processing of DNA damage substrates for the hNEIL enzymes.

PubMed

Fleming, Aaron M; Burrows, Cynthia J

2017-06-01

Reactive oxygen species (ROS) are harnessed by the cell for signaling at the same time as being detrimental to cellular components such as DNA. The genome and transcriptome contain instructions that can alter cellular processes when oxidized. The guanine (G) heterocycle in the nucleotide pool, DNA, or RNA is the base most prone to oxidation. The oxidatively-derived products of G consistently observed in high yields from hydroxyl radical, carbonate radical, or singlet oxygen oxidations under conditions modeling the cellular reducing environment are discussed. The major G base oxidation products are 8-oxo-7,8-dihydroguanine (OG), 5-carboxamido-5-formamido-2-iminohydantoin (2Ih), spiroiminodihydantoin (Sp), and 5-guanidinohydantoin (Gh). The yields of these products show dependency on the oxidant and the reaction context that includes nucleoside, single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and G-quadruplex DNA (G4-DNA) structures. Upon formation of these products in cells, they are recognized by the DNA glycosylases in the base excision repair (BER) pathway. This review focuses on initiation of BER by the mammalian Nei-like1-3 (NEIL1-3) glycosylases for removal of 2Ih, Sp, and Gh. The unique ability of the human NEILs to initiate removal of the hydantoins in ssDNA, bulge-DNA, bubble-DNA, dsDNA, and G4-DNA is outlined. Additionally, when Gh exists in a G4 DNA found in a gene promoter, NEIL-mediated repair is modulated by the plasticity of the G4-DNA structure provided by additional G-runs flanking the sequence. On the basis of these observations and cellular studies from the literature, the interplay between DNA oxidation and BER to alter gene expression is discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

eDNA and specific primers for early detection of invasive species--A case study on the bivalve Rangia cuneata, currently spreading in Europe.

PubMed

Ardura, Alba; Zaiko, Anastasija; Martinez, Jose L; Samulioviene, Aurelija; Semenova, Anna; Garcia-Vazquez, Eva

2015-12-01

Intense human activities facilitate the successful spread and establishment of non-indigenous aquatic organisms in marine and freshwater ecosystems. In some cases such intrusions result in noticeable and adverse changes in the recipient environments. In the Baltic Sea, the discovery and rapid initial spread of the North American wedge clam Rangia cuneata represents a new wave of invasion which may trigger unpredictable changes of the local benthic communities. In this study we present a species-specific DNA-based marker developed in silico and experimentally tested on environmental samples. Marker specificity and sensitivity were assessed in vitro from water samples containing different mixtures of the target species and other five bivalves currently present in the region: the native Cerastoderma glaucum, Macoma balthica and Mytilus trossulus, the invasive Dreissena polymorpha and the cryptogenic Mya arenaria. Cross-species amplification was not found in any case. The method allows to detecting at least 0.4 ng of R. cuneata DNA per μl, and 0.1 g of tissue per liter of water. Finally, the marker performance was assessed in water samples from the Baltic Sea and Vistula Lagoon. The coincidence between independent visual observations of R. cuneata and positive PCR amplification of the marker from the water samples confirmed the efficiency of this highly reproducible, fast, and technically easy method. R. cuneata traces can be detected from environmental DNA even when the population is sparse and small, enabling rapid management responses and allowing to track the invasion dynamics. Copyright © 2015 Elsevier Ltd. All rights reserved.

Direct role for the RNA polymerase domain of T7 primase in primer delivery

PubMed Central

Zhu, Bin; Lee, Seung-Joo; Richardson, Charles C.

2010-01-01

Gene 4 protein (gp4) encoded by bacteriophage T7 contains a C-terminal helicase and an N-terminal primase domain. After synthesis of tetraribonucleotides, gp4 must transfer them to the polymerase for use as primers to initiate DNA synthesis. In vivo gp4 exists in two molecular weight forms, a 56-kDa form and the full-length 63-kDa form. The 56-kDa gp4 lacks the N-terminal Cys4 zinc-binding motif important in the recognition of primase sites in DNA. The 56-kDa gp4 is defective in primer synthesis but delivers a wider range of primers to initiate DNA synthesis compared to the 63-kDa gp4. Suppressors exist that enable the 56-kDa gp4 to support the growth of T7 phage lacking gene 4 (T7Δ4). We have identified 56-kDa DNA primases defective in primer delivery by screening for their ability to support growth of T7Δ4 phage in the presence of this suppressor. Trp69 is critical for primer delivery. Replacement of Trp69 with lysine in either the 56- or 63-kDa gp4 results in defective primer delivery with other functions unaffected. DNA primase harboring lysine at position 69 fails to stabilize the primer on DNA. Thus, a primase subdomain not directly involved in primer synthesis is involved in primer delivery. The stabilization of the primer by DNA primase is necessary for DNA polymerase to initiate synthesis. PMID:20439755

Studies on sex-organ development. Changes in chromatin structure during spermatogenesis in maturing rooster testis as demonstrated by the initiation pattern of ribonucleic acid synthesis in vitro.

PubMed Central

Mezquita, C; Teng, C S

1978-01-01

To probe the structural change in the genome of the differentiating germ cell of the maturing rooster testis, the chromatin from nuclei at various stages of differentiation were transcribed with prokaryotic RNA polymerase from Escherichia coli or with eukaryotic RNA polymerase II from wheat germ. The transcription was performed under conditions of blockage of RNA chain reinitiation in vitro with rifampicin or rifampicin AF/013. With the E. coli enzyme, the changes in (1) the titration curve for the enzyme-chromatin interaction, (2) the number of initiation sites, (3) the rate of elongation of RNA chains, and (4) the kinetics of the formation of stable initiation complexes revealed the unmasking of DNA in elongated spermatids and the masking of DNA in spermatozoa. In both cases the stability of the DNA duplex in the initiation region for RNA synthesis greatly increased. In contrast with the E. coli enzyme, the wheat-germ RNA polymerase II was relatively inefficient at transcribing chromatin of elongated spermatids. Such behaviour can be predicted if unmasked double-stranded DNA is present in elongated spermatids. PMID:346018

Modeling Thermal Inactivation of Bacillus Spores

DTIC Science & Technology

2009-03-01

Ungers, G. “The Negative Control Mecha- nism for E . Coli DNA Replication,” Proceedings of the National Academy of Sci- ences of the USA, 63: 1410-1417...damage by: d [DNA] dt = − k1 [DNA]− k2 [H2O] [DNA] where [DNA] =information content of DNA k1 =rate coefficient associated with [DNA] breakdown during...ax im um a va ila bl e w at er p er u ni t v ol um e Core Cortex Absorbed water Bound water Total water Figure 3.2: Initial Distribution of

A complex of RAG-1 and RAG-2 proteins persists on DNA after single-strand cleavage at V(D)J recombination signal sequences.

PubMed Central

Grawunder, U; Lieber, M R

1997-01-01

The recombination activating gene (RAG) 1 and 2 proteins are required for initiation of V(D)J recombination in vivo and have been shown to be sufficient to introduce DNA double-strand breaks at recombination signal sequences (RSSs) in a cell-free assay in vitro. RSSs consist of a highly conserved palindromic heptamer that is separated from a slightly less conserved A/T-rich nonamer by either a 12 or 23 bp spacer of random sequence. Despite the high sequence specificity of RAG-mediated cleavage at RSSs, direct binding of the RAG proteins to these sequences has been difficult to demonstrate by standard methods. Even when this can be demonstrated, questions about the order of events for an individual RAG-RSS complex will require methods that monitor aspects of the complex during transitions from one step of the reaction to the next. Here we have used template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) in order to assess occupancy of the reaction intermediates by the RAG complex during the reaction. In addition, this approach allows analysis of the accessibility of end products of a RAG-catalyzed cleavage reaction for N nucleotide addition. The results indicate that RAG proteins form a long-lived complex with the RSS once the initial nick is generated, because the 3'-OH group at the nick remains obstructed for TdT-catalyzed N nucleotide addition. In contrast, the 3'-OH group generated at the signal end after completion of the cleavage reaction can be efficiently tailed by TdT, suggesting that the RAG proteins disassemble from the signal end after DNA double-strand cleavage has been completed. Therefore, a single RAG complex maintains occupancy from the first step (nick formation) to the second step (cleavage). In addition, the results suggest that N region diversity at V(D)J junctions within rearranged immunoglobulin and T cell receptor gene loci can only be introduced after the generation of RAG-catalyzed DNA double-strand breaks, i.e. during the DNA end joining phase of the V(D)J recombination reaction. PMID:9060432

Computational Micromodel for Epigenetic Mechanisms

PubMed Central

Raghavan, Karthika; Ruskin, Heather J.; Perrin, Dimitri; Goasmat, Francois; Burns, John

2010-01-01

Characterization of the epigenetic profile of humans since the initial breakthrough on the human genome project has strongly established the key role of histone modifications and DNA methylation. These dynamic elements interact to determine the normal level of expression or methylation status of the constituent genes in the genome. Recently, considerable evidence has been put forward to demonstrate that environmental stress implicitly alters epigenetic patterns causing imbalance that can lead to cancer initiation. This chain of consequences has motivated attempts to computationally model the influence of histone modification and DNA methylation in gene expression and investigate their intrinsic interdependency. In this paper, we explore the relation between DNA methylation and transcription and characterize in detail the histone modifications for specific DNA methylation levels using a stochastic approach. PMID:21152421

DNA recombination-initiation plays a role in the extremely biased inheritance of yeast [rho-] mitochondrial DNA that contains the replication origin ori5.

PubMed

Ling, Feng; Hori, Akiko; Shibata, Takehiko

2007-02-01

Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho(-)] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho(-)] mtDNA harboring ori5, one of the replication origins of normal ([rho(+)]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho(-)] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho(-)] mtDNA are simultaneously suppressed by the ntg1 null mutation. These results support a model in which, like homologous recombination, rolling-circle HS [ori5] [rho(-)] mtDNA replication is initiated by double-stranded breakage in ori5, followed by Mhr1-mediated homologous pairing of the processed nascent DNA ends with circular mtDNA. The hypersuppressiveness of HS [ori5] [rho(-)] mtDNA depends on a replication advantage furnished by the higher density of ori5 sequences and on a segregation advantage furnished by the higher genome copy number on transmitted concatemers.

Is DNA Alive? A Study of Conceptual Change Through Targeted Instruction

NASA Astrophysics Data System (ADS)

Witzig, Stephen B.; Freyermuth, Sharyn K.; Siegel, Marcelle A.; Izci, Kemal; Pires, J. Chris

2013-08-01

We are involved in a project to incorporate innovative assessments within a reform-based large-lecture biochemistry course for nonmajors. We not only assessed misconceptions but purposefully changed instruction throughout the semester to confront student ideas. Our research questions targeted student conceptions of deoxyribonucleic acid (DNA) along with understanding in what ways classroom discussions/activities influence student conceptions. Data sources included pre-/post-assessments, semi-structured interviews, and student work on exams/assessments. We found that students held misconceptions about the chemical nature of DNA, with 63 % of students claiming that DNA is alive prior to instruction. The chemical nature of DNA is an important fundamental concept in science fields. We confronted this misconception throughout the semester collecting data from several instructional interventions. Case studies of individual students revealed how various instructional strategies/assessments allowed students to construct and demonstrate the scientifically accepted understanding of the chemical nature of DNA. However, the post-assessment exposed that 40 % of students still held misconceptions about DNA, indicating the persistent nature of this misconception. Implications for teaching and learning are discussed.

Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages

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

Sun, Siyang; Gao, Song; Kondabagil, Kiran

2012-04-04

Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a 'small terminase' and a 'large terminase' component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the centralmore » domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation.« less

AFM Imaging of Hybridization Chain Reaction Mediated Signal Transmission between Two DNA Origami Structures.

PubMed

Helmig, Sarah; Gothelf, Kurt Vesterager

2017-10-23

Signal transfer is central to the controlled exchange of information in biology and advanced technologies. Therefore, the development of reliable, long-range signal transfer systems for artificial nanoscale assemblies is of great scientific interest. We have designed such a system for the signal transfer between two connected DNA nanostructures, using the hybridization chain reaction (HCR). Two sets of metastable DNA hairpins, one of which is immobilized at specific points along tracks on DNA origami structures, are polymerized to form a continuous DNA duplex, which is visible using atomic force microscopy (AFM). Upon addition of a designed initiator, the initiation signal is efficiently transferred more than 200 nm from a specific location on one origami structure to an end point on another origami structure. The system shows no significant loss of signal when crossing from one nanostructure to another and, therefore, has the potential to be applied to larger multi-component DNA assemblies. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Function and horizontal transfer of the small terminase subunit of the tailed bacteriophage Sf6 DNA packaging nanomotor

PubMed Central

Leavitt, Justin C.; Gilcrease, Eddie B.; Wilson, Kassandra; Casjens, Sherwood R.

2013-01-01

Bacteriophage Sf6 DNA packaging series initiate at many locations across a 2 kbp region. Our in vivo studies that show that Sf6 small terminase subunit (TerS) protein recognizes a specific packaging (pac) site near the center of this region, that this site lies within the portion of the Sf6 gene that encodes the DNA-binding domain of TerS protein, that this domain of the TerS protein is responsible for the imprecision in Sf6 packaging initiation, and that the DNA-binding domain of TerS must be covalently attached to the domain that interacts with the rest of the packaging motor. The TerS DNA-binding domain is self-contained in that it apparently does not interact closely with the rest of the motor and it binds to a recognition site that lies within the DNA that encodes the domain. This arrangement has allowed the horizontal exchange of terS genes among phages to be very successful. PMID:23562538

RPO41-independent maintenance of [rho-] mitochondrial DNA in Saccharomyces cerevisiae.

PubMed

Fangman, W L; Henly, J W; Brewer, B J

1990-01-01

A subset of promoters in the mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae has been proposed to participate in replication initiation, giving rise to a primer through site-specific cleavage of an RNA transcript. To test whether transcription is essential for mtDNA maintenance, we examined two simple mtDNA deletion ([rho-]) genomes in yeast cells. One genome (HS3324) contains a consensus promoter (ATATAAGTA) for the mitochondrial RNA polymerase encoded by the nuclear gene RPO41, and the other genome (4a) does not. As anticipated, in RPO41 cells transcripts from the HS3324 genome were more abundant than were transcripts from the 4a genome. When the RPO41 gene was disrupted, both [rho-] genomes were efficiently maintained. The level of transcripts from HS3324 mtDNA was decreased greater than 400-fold in cells carrying the RPO41 disrupted gene; however, the low-level transcripts from 4a mtDNA were undiminished. These results indicate that replication of [rho-] genomes can be initiated in the absence of wild-type levels of the RPO41-encoded RNA polymerase.

Inhibitory effect(s) of polymeric black tea polyphenols on the formation of B(a)P-derived DNA adducts in mouse skin.

PubMed

Krishnan, Rajesh; Maru, Girish B

2005-01-01

The biological activities and chemopreventive properties of green tea polyphenols have been demonstrated, while similar information regarding newly formed major polymeric polyphenols in black tea are not available. Cancer chemoprevention may be achieved by the inhibition of any stage of carcinogenesis. In the present study, we investigated the anti-initiating effects of five polymeric black tea polyphenol (PBP) fractions, by determining their effects on the formation of [3H]-B(a)P-derived DNA adducts as well as the activity of cytochrome P-450 isozymes CYP 1A1 and 1A2 in vitro employing rat liver microsomes. PBP 1-3 inhibited both the microsome catalyzed [3H]-B(a)P-derived DNA adduct formation as well as the activity of CYP 1A1 and 1A2 as assessed by the decreased formation of resorufin from the respective substrates. Further investigation revealed that topical pretreatment(s) of mice with PBP 1-5 (200 mug/day x 4) resulted in a significant decrease in the levels of single topical B(a)P (1 mg/mouse) - induced DNA adducts in epidermal DNA determined by employing 32P-post labeling analysis. Overall, our results suggest that black tea-derived PBPs have one of the chemopreventive properties shown by monomeric green tea polyphenols.

AQUATIC PLANT SPECIATION AFFECTED BY DIVERSIFYING SELECTION OF ORGANELLE DNA REGIONS(1).

PubMed

Kato, Syou; Misawa, Kazuharu; Takahashi, Fumio; Sakayama, Hidetoshi; Sano, Satomi; Kosuge, Keiko; Kasai, Fumie; Watanabe, Makoto M; Tanaka, Jiro; Nozaki, Hisayoshi

2011-10-01

Many of the genes that control photosynthesis are carried in the chloroplast. These genes differ among species. However, evidence has yet to be reported revealing the involvement of organelle genes in the initial stages of plant speciation. To elucidate the molecular basis of aquatic plant speciation, we focused on the unique plant species Chara braunii C. C. Gmel. that inhabits both shallow and deep freshwater habitats and exhibits habitat-based dimorphism of chloroplast DNA (cpDNA). Here, we examined the "shallow" and "deep" subpopulations of C. braunii using two nuclear DNA (nDNA) markers and cpDNA. Genetic differentiation between the two subpopulations was measured in both nDNA and cpDNA regions, although phylogenetic analyses suggested nuclear gene flow between subpopulations. Neutrality tests based on Tajima's D demonstrated diversifying selection acting on organelle DNA regions. Furthermore, both "shallow" and "deep" haplotypes of cpDNA detected in cultures originating from bottom soils of three deep environments suggested that migration of oospores (dormant zygotes) between the two habitats occurs irrespective of the complete habitat-based dimorphism of cpDNA from field-collected vegetative thalli. Therefore, the two subpopulations are highly selected by their different aquatic habitats and show prezygotic isolation, which represents an initial process of speciation affected by ecologically based divergent selection of organelle genes. © 2011 Phycological Society of America.

Chemical and Biological Sensing Using Hybridization Chain Reaction.

PubMed

Augspurger, Erik E; Rana, Muhit; Yigit, Mehmet V

2018-05-25

Since the advent of its theoretical discovery more than 30 years ago, DNA nanotechnology has been used in a plethora of diverse applications in both the fundamental and applied sciences. The recent prominence of DNA-based technologies in the scientific community is largely due to the programmable features stored in its nucleobase composition and sequence, which allow it to assemble into highly advanced structures. DNA nanoassemblies are also highly controllable due to the precision of natural and artificial base-pairing, which can be manipulated by pH, temperature, metal ions, and solvent types. This programmability and molecular-level control have allowed scientists to create and utilize DNA nanostructures in one, two, and three dimensions (1D, 2D, and 3D). Initially, these 2D and 3D DNA lattices and shapes attracted a broad scientific audience because they are fundamentally captivating and structurally elegant; however, transforming these conceptual architectural blueprints into functional materials is essential for further advancements in the DNA nanotechnology field. Herein, the chemical and biological sensing applications of a 1D DNA self-assembly process known as hybridization chain reaction (HCR) are reviewed. HCR is a one-dimensional (1D) double stranded (ds) DNA assembly process initiated only in the presence of a specific short ssDNA (initiator) and two kinetically trapped DNA hairpin structures. HCR is considered an enzyme-free isothermal amplification process, which shows substantial promise and offers a wide range of applications for in situ chemical and biological sensing. Due to its modular nature, HCR can be programmed to activate only in the presence of highly specific biological and/or chemical stimuli. HCR can also be combined with different types of molecular reporters and detection approaches for various analytical readouts. While the long dsDNA HCR product may not be as structurally attractive as the 2D and 3D DNA networks, HCR is highly instrumental for applied biological, chemical, and environmental sciences, and has therefore been studied to foster a variety of objectives. In this review, we have focused on nucleic acid, protein, metabolite, and heavy metal ion detection using this 1D DNA nanotechnology via fluorescence, electrochemical, and nanoparticle-based methodologies.

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication

PubMed Central

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-01

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. PMID:27679476

Cdc6-Induced Conformational Changes in ORC Bound to Origin DNA Revealed by Cryo-Electron Microscopy

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

Sun J.; Li H.; Kawakami, H.

2012-03-07

The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here, we report a single-particle cryo-EM-derived structure of the supramolecular assembly comprising Saccharomyces cerevisiae ORC, the replication initiation factor Cdc6, and double-stranded ARS1 origin DNA in the presence of ATP{gamma}S. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, in particular reorienting the Orc1 N-terminal BAH domain. Segmentation of the 3D map of ORC-Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 proteinmore » suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus, ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends.« less

Development of a nanostructured DNA delivery scaffold via electrospinning of PLGA and PLA-PEG block copolymers

NASA Technical Reports Server (NTRS)

Luu, Y. K.; Kim, K.; Hsiao, B. S.; Chu, B.; Hadjiargyrou, M.; Hadjiargyou, M. (Principal Investigator)

2003-01-01

The present work utilizes electrospinning to fabricate synthetic polymer/DNA composite scaffolds for therapeutic application in gene delivery for tissue engineering. The scaffolds are non-woven, nano-fibered, membranous structures composed predominantly of poly(lactide-co-glycolide) (PLGA) random copolymer and a poly(D,L-lactide)-poly(ethylene glycol) (PLA-PEG) block copolymer. Release of plasmid DNA from the scaffolds was sustained over a 20-day study period, with maximum release occurring at approximately 2 h. Cumulative release profiles indicated amounts released were approximately 68-80% of the initially loaded DNA. Variations in the PLGA to PLA-PEG block copolymer ratio vastly affected the overall structural morphology, as well as both the rate and efficiency of DNA release. Results indicated that DNA released directly from these electrospun scaffolds was indeed intact, capable of cellular transfection, and successfully encoded the protein beta-galactosidase. When tested under tensile loads, the electrospun polymer/DNA composite scaffolds exhibited tensile moduli of approximately 35 MPa, with approximately 45% strain initially. These values approximate those of skin and cartilage. Taken together, this work represents the first successful demonstration of plasmid DNA incorporation into a polymer scaffold using electrospinning.

Fidelity of DNA Replication in Normal and Malignant Human Brest Cells.

DTIC Science & Technology

1995-08-31

cellular DNA replication machinery, we have initiated experiments that utilize a multiprotein DNA replication complex (MRC) isolated from breast cancer...gene in an in vitro DNA replication assay. By utilizing the target gene in a bacterial mutant selection assay we have begun to determine the...frequency with which mutational sequence errors occur as a result of the in vitro DNA replication mediated by the breast cancer cell MRC and the normal breast

Mechanism-based classification of PAH mixtures to predict carcinogenic potential

DOE PAGES

Tilton, Susan C.; Siddens, Lisbeth K.; Krueger, Sharon K.; ...

2015-04-22

We have previously shown that relative potency factors and DNA adduct measurements are inadequate for predicting carcinogenicity of certain polycyclic aromatic hydrocarbons (PAHs) and PAH mixtures, particularly those that function through alternate pathways or exhibit greater promotional activity compared to benzo[ a]pyrene (BaP). Therefore, we developed a pathway based approach for classification of tumor outcome after dermal exposure to PAH/mixtures. FVB/N mice were exposed to dibenzo[ def,p]chrysene (DBC), BaP or environmental PAH mixtures (Mix 1-3) following a two-stage initiation/promotion skin tumor protocol. Resulting tumor incidence could be categorized by carcinogenic potency as DBC>>BaP=Mix2=Mix3>Mix1=Control, based on statistical significance. Gene expression profilesmore » measured in skin of mice collected 12 h post-initiation were compared to tumor outcome for identification of short-term bioactivity profiles. A Bayesian integration model was utilized to identify biological pathways predictive of PAH carcinogenic potential during initiation. Integration of probability matrices from four enriched pathways (p<0.05) for DNA damage, apoptosis, response to chemical stimulus and interferon gamma signaling resulted in the highest classification accuracy with leave-one-out cross validation. This pathway-driven approach was successfully utilized to distinguish early regulatory events during initiation prognostic for tumor outcome and provides proof-of-concept for using short-term initiation studies to classify carcinogenic potential of environmental PAH mixtures. As a result, these data further provide a ‘source-to outcome’ model that could be used to predict PAH interactions during tumorigenesis and provide an example of how mode-of-action based risk assessment could be employed for environmental PAH mixtures.« less

Mechanism-Based Classification of PAH Mixtures to Predict Carcinogenic Potential.

PubMed

Tilton, Susan C; Siddens, Lisbeth K; Krueger, Sharon K; Larkin, Andrew J; Löhr, Christiane V; Williams, David E; Baird, William M; Waters, Katrina M

2015-07-01

We have previously shown that relative potency factors and DNA adduct measurements are inadequate for predicting carcinogenicity of certain polycyclic aromatic hydrocarbons (PAHs) and PAH mixtures, particularly those that function through alternate pathways or exhibit greater promotional activity compared to benzo[a]pyrene (BaP). Therefore, we developed a pathway-based approach for classification of tumor outcome after dermal exposure to PAH/mixtures. FVB/N mice were exposed to dibenzo[def,p]chrysene (DBC), BaP, or environmental PAH mixtures (Mix 1-3) following a 2-stage initiation/promotion skin tumor protocol. Resulting tumor incidence could be categorized by carcinogenic potency as DBC > BaP = Mix2 = Mix3 > Mix1 = Control, based on statistical significance. Gene expression profiles measured in skin of mice collected 12 h post-initiation were compared with tumor outcome for identification of short-term bioactivity profiles. A Bayesian integration model was utilized to identify biological pathways predictive of PAH carcinogenic potential during initiation. Integration of probability matrices from four enriched pathways (P < .05) for DNA damage, apoptosis, response to chemical stimulus, and interferon gamma signaling resulted in the highest classification accuracy with leave-one-out cross validation. This pathway-driven approach was successfully utilized to distinguish early regulatory events during initiation prognostic for tumor outcome and provides proof-of-concept for using short-term initiation studies to classify carcinogenic potential of environmental PAH mixtures. These data further provide a 'source-to-outcome' model that could be used to predict PAH interactions during tumorigenesis and provide an example of how mode-of-action-based risk assessment could be employed for environmental PAH mixtures. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Specific functions of the Rep and Rep׳ proteins of porcine circovirus during copy-release and rolling-circle DNA replication.

PubMed

Cheung, Andrew K

2015-07-01

The roles of two porcine circovirus replication initiator proteins, Rep and Rep׳, in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replication) and generates the single-stranded circular (ssc) genome from the displaced DNA strand. In the process, a minus-genome primer (MGP) necessary for complementary-strand synthesis, from ssc to ccc, is synthesized. Rep׳ cleaves the growing nascent-strand to regenerate the parent ccc molecule. In the process, a Rep׳-DNA hybrid containing the right palindromic sequence (at the origin of DNA replication) is generated. Analysis of the virus particle showed that it is composed of four components: ssc, MGP, capsid protein and a novel Rep-related protein (designated Protein-3). Copyright © 2015. Published by Elsevier Inc.

5',5'''-P1, P4 diadenosine tetraphosphate (Ap4A): a putative initiator of DNA replication.

PubMed

Baril, E F; Coughlin, S A; Zamecnik, P C

1985-01-01

The proposal that Ap4A acts as an inducer of DNA replication is based primarily on two pieces of evidence (7). The intracellular levels of Ap4A increase ten- to 1000-fold as cells progress into S phase and the introduction of Ap4A into nonproliferating cells stimulated DNA synthesis. There is also some additional suggestive evidence such as the binding of Ap4A to a protein that is associated with multiprotein forms of the replicative DNA polymerase alpha and the ability of this enzyme to use Ap4A as a primer for DNA synthesis in vitro with single-stranded DNA templates. These observations have stimulated interest in the cellular metabolism of Ap4A. This is well since there is a great need for additional experimentation in order to clearly establish Ap4A as an inducer of DNA replication. Microinjection experiments of Ap4A into quiescent cells are needed in order to ascertain if Ap4A will stimulate DNA replication and possibly cell division in intact cells. Studies of the effects of nonhydrolyzable analogs of Ap4A on DNA replication in intact quiescent cells could also prove valuable. Although Ap4A can function as a primer for in vitro DNA synthesis by DNA polymerase alpha this may not be relevant in regard to its in vivo role in DNA replication. Ap4A in vivo could interact with key protein(s) in DNA replication and in this way act as an effector molecule in the initiation of DNA replication. In this regard the interaction of Ap4A with a protein associated with a multiprotein form of DNA polymerase alpha isolated from S-phase cells is of interest. More experiments are required to determine if there is a specific target protein(s) for Ap4A in vivo and what its role in DNA replication is. The cofractionation of tryptophanyl-tRNA synthetase with the replicative DNA polymerase alpha from animal and plant cells is of interest. The DNA polymerase alpha from synchronized animal cells also interacted with Ap4A. Although the plant cell alpha-like DNA polymerase did not interact with Ap4A this DNA polymerase was not a multiprotein form of polymerase alpha and the synchrony of the wheat germ embryos was not known. A possible tie between protein-synthesizing systems and the regulation of proteins involved in DNA replication may exist. The requirement of protein synthesis for the initiation of DNA replication has long been known. Also, it is well established that many temperature-sensitive mutants for tRNA synthetases are also DNA-synthesizing mutants. More investigation in this area may be warranted.(ABSTRACT TRUNCATED AT 400 WORDS)

Sewage sludge does not induce genotoxicity and carcinogenesis

PubMed Central

Silva, Paula Regina Pereira; Barbisan, Luis Fernando; Dagli, Maria Lúcia Zaidan; Saldiva, Paulo Hilário Nascimento

2012-01-01

Through a series of experiments, the genotoxic/mutagenic and carcinogenic potential of sewage sludge was assessed. Male Wistar rats were randomly assigned to four groups: Group 1 - negative control; Group 2 - liver carcinogenesis initiated by diethylnitrosamine (DEN; 200 mg/kg i.p.); Group 3 and G4-liver carcinogenesis initiated by DEN and fed 10,000 ppm or 50,000 ppm of sewage sludge. The animals were submitted to a 70% partial hepatectomy at the 3rd week. Livers were processed for routine histological analysis and immunohistochemistry, in order to detect glutathione S-transferase positive altered hepatocyte foci (GST-P+ AHF). Peripheral blood samples for the comet assay were obtained from the periorbital plexus immediately prior to sacrificing. Polychromatic erythrocytes (PCEs) were analyzed in femoral bone-marrow smears, and the frequencies of those micronucleated (MNPCEs) registered. There was no sewage-sludge-induced increase in frequency of either DNA damage in peripheral blood leucocytes, or MNPCEs in the femoral bone marrow. Also, there was no increase in the levels of DNA damage, in the frequency of MNPCEs, and in the development of GST-P AHF when compared with the respective control group. PMID:23055806

Health investigations of depleted-uranium clean-up workers.

PubMed

Milacic, Snezana

2008-01-01

The soil contaminated by depleted uranium (238U) ammunition during the NATO bombing of Serbia and Montenegro was cleaned-up for four months in 2002. A team of 11 clean-up workers (expert members) were medically examined three times: before decontamination as a preliminary medical check-up, immediately after decontamination, and four years after cleaning up contaminated ground. This short report presents investigations and health risk assessments of clean-up workers in radioactive decontamination operations and an assessment of the environmental health perspectives for citizens living in surrounding areas. The method of initial health disorders was used, analyzing the most sensitive biological materials, such as blood cells or chromosome damage, DNA strand breaks, radio-toxicological examination of urine. The total number of blood cells did not change, but variations of the relative number (percentage) of cells in the leukocyte formula were observed. The total number of DNA alterations was higher immediately after decontamination than before decontamination. Four years after decontamination the number of DNA alterations had decreased. However, the number of damaged cells (lymphocytes containing chromosome lesions) was higher in both medical examinations, immediately after and four years after decontamination. Disease or tumours due to 238U did not develop in the group of depleted uranium clean-up workers during the investigation period of four years. Further monitoring of haematological and chromosomal effects and the health condition of workers is necessary.

Initial analysis of sperm DNA methylome in Holstein bulls

USDA-ARS?s Scientific Manuscript database

Aberrant DNA methylation patterns have been associated with abnormal semen parameters, idiopathic male infertility and early embryonic loss in mammals. Using Holstein bulls with high (Bull1) or low (Bull2) fertility rates, we created two representative sperm DNA methylomes at a single-base resolutio...

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

PubMed Central

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

2011-01-01

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 β-lyase activity; (b) monofunctional; and (c) bifunctional with an associated β,δ-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

Development of a calcium phosphate co-precipitate/poly(lactide-co-glycolide) DNA delivery system: release kinetics and cellular transfection studies.

PubMed

Kofron, Michelle D; Laurencin, Cato T

2004-06-01

One of the most common non-viral methods for the introduction of foreign deoxyribonucleic acid (DNA) into cultured cells is calcium phosphate co-precipitate transfection. This technique involves the encapsulation of DNA within a calcium phosphate co-precipitate, particulate addition to in vitro cell culture, endocytosis of the co-precipitate, and exogenous DNA expression by the transfected cell. In this study, we fabricated a novel non-viral gene transfer system by adsorbing DNA, encapsulated in calcium phosphate (DNA/Ca-P) co-precipitates, to biodegradable two- and three-dimensional poly(lactide-co-glycolide) matrices (2D-DNA/Ca-P/PLAGA, 3D-DNA/Ca-P/PLAGA). Co-precipitate release studies demonstrated an initial burst release over the first 48 h. By day 7, approximately 96% of the initially adsorbed DNA/Ca-P co-precipitate had been released. This was followed by low levels of co-precipitate release for 42 days. Polymerase chain reaction was used to demonstrate the ability of the released DNA containing co-precipitates to transfect SaOS-2 cells cultured in vitro on the 3D-DNA/Ca-P/PLAGA matrix and maintenance of the structural integrity of the exogenous DNA. In summary, a promising system for the incorporation and controlled delivery of exogenous genes encapsulated within a calcium phosphate co-precipitate from biodegradable polymeric matrices has been developed and may have applicability to the delivery of therapeutic genes and the transfection of other cell types.

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

PubMed

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

2017-07-27

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

High- to low-dose extrapolation: critical determinants involved in the dose response of carcinogenic substances.

PubMed

Swenberg, J A; Richardson, F C; Boucheron, J A; Deal, F H; Belinsky, S A; Charbonneau, M; Short, B G

1987-12-01

Recent investigations on mechanism of carcinogenesis have demonstrated important quantitative relationships between the induction of neoplasia, the molecular dose of promutagenic DNA adducts and their efficiency for causing base-pair mismatch, and the extent of cell proliferation in target organ. These factors are involved in the multistage process of carcinogenesis, including initiation, promotion, and progression. The molecular dose of DNA adducts can exhibit supralinear, linear, or sublinear relationships to external dose due to differences in absorption, biotransformation, and DNA repair at high versus low doses. In contrast, increased cell proliferation is a common phenomena that is associated with exposures to relatively high doses of toxic chemicals. As such, it enhances the carcinogenic response at high doses, but has little effect at low doses. Since data on cell proliferation can be obtained for any exposure scenario and molecular dosimetry studies are beginning to emerge on selected chemical carcinogens, methods are needed so that these critical factors can be utilized in extrapolation from high to low doses and across species. The use of such information may provide a scientific basis for quantitative risk assessment.

High- to low-dose extrapolation: critical determinants involved in the dose response of carcinogenic substances.

PubMed Central

Swenberg, J A; Richardson, F C; Boucheron, J A; Deal, F H; Belinsky, S A; Charbonneau, M; Short, B G

1987-01-01

Recent investigations on mechanism of carcinogenesis have demonstrated important quantitative relationships between the induction of neoplasia, the molecular dose of promutagenic DNA adducts and their efficiency for causing base-pair mismatch, and the extent of cell proliferation in target organ. These factors are involved in the multistage process of carcinogenesis, including initiation, promotion, and progression. The molecular dose of DNA adducts can exhibit supralinear, linear, or sublinear relationships to external dose due to differences in absorption, biotransformation, and DNA repair at high versus low doses. In contrast, increased cell proliferation is a common phenomena that is associated with exposures to relatively high doses of toxic chemicals. As such, it enhances the carcinogenic response at high doses, but has little effect at low doses. Since data on cell proliferation can be obtained for any exposure scenario and molecular dosimetry studies are beginning to emerge on selected chemical carcinogens, methods are needed so that these critical factors can be utilized in extrapolation from high to low doses and across species. The use of such information may provide a scientific basis for quantitative risk assessment. PMID:3447904

Buccal viral DNA as a trigger for brincidofovir therapy in the mousepox model of smallpox.

PubMed

Crump, Ryan; Korom, Maria; Buller, R Mark; Parker, Scott

2017-03-01

Orthopoxviruses continue to pose a significant threat to the population as potential agents of bioterrorism. An intentional release of natural or engineered variola virus (VARV) or monkeypox viruses would cause mortality and morbidity in the target population. To address this, antivirals have been developed and evaluated in animal models of smallpox and monkeypox. One such antiviral, brincidofovir (BCV, previously CMX001), has demonstrated high levels of efficacy against orthopoxviruses in animal models and is currently under clinical evaluation for prevention and treatment of diseases caused by cytomegaloviruses and adenoviruses. In this study we use the mousepox model of smallpox to evaluate the relationship between the magnitude of the infectious virus dose and an efficacious BCV therapy outcome when treatment is initiated concomitant with detection of ectromelia virus viral DNA (vDNA) in mouse buccal swabs. We found that vDNA could be detected in buccal swabs of some, but not all infected mice over a range of challenge doses by day 3 or 4 postexposure, when initiation of BCV treatment was efficacious, suggesting that detection of vDNA in buccal swabs could be used as a trigger to initiate BCV treatment of an entire potentially exposed population. However, buccal swabs of some mice did not become positive until 5 days postexposure, when initiation of BCV therapy failed to protect mice that received high doses of virus. And finally, the data suggest that the therapeutic window for efficacious BCV treatment decreases as the virus infectious dose increases. Extrapolating these findings to VARV, the data suggest that treatment should be initiated as soon as possible after exposure and not rely on a diagnostic tool such as the measurement of vDNA in buccal cavity swabs; however, consideration should be given to the fact that the behavior/disease-course of VARV in humans is different from that of ectromelia virus in the mouse. Copyright © 2016 Elsevier B.V. All rights reserved.

Sustained efficacy and immunogenicity of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine: analysis of a randomised placebo-controlled trial up to 6.4 years.

PubMed

Romanowski, B; de Borba, P Colares; Naud, P S; Roteli-Martins, C M; De Carvalho, N S; Teixeira, J C; Aoki, F; Ramjattan, B; Shier, R M; Somani, R; Barbier, S; Blatter, M M; Chambers, C; Ferris, D; Gall, S A; Guerra, F A; Harper, D M; Hedrick, J A; Henry, D C; Korn, A P; Kroll, R; Moscicki, A-B; Rosenfeld, W D; Sullivan, B J; Thoming, C S; Tyring, S K; Wheeler, C M; Dubin, G; Schuind, A; Zahaf, T; Greenacre, Mary; Sgriobhadair, An

2009-12-12

Prophylactic human papillomavirus (HPV) vaccines have to provide sustained protection. We assessed efficacy, immunogenicity, and safety of the HPV-16/18 AS04-adjuvanted vaccine up to 6.4 years. Women aged 15-25 years, with normal cervical cytology, who were HPV-16/18 seronegative and oncogenic HPV DNA-negative (14 types) at screening participated in a double-blind, randomised, placebo-controlled initial study (n=1113; 560 vaccine group vs 553 placebo group) and follow-up study (n=776; 393 vs 383). 27 sites in three countries participated in the follow-up study. Cervical samples were tested every 6 months for HPV DNA. Management of abnormal cytologies was prespecified, and HPV-16/18 antibody titres were assessed. The primary objective was to assess long-term vaccine efficacy in the prevention of incident cervical infection with HPV 16 or HPV 18, or both. We report the analyses up to 6.4 years of this follow-up study and combined with the initial study. For the primary endpoint, the efficacy analysis was done in the according-to-protocol (ATP) cohort; the analysis of cervical intraepithelial neoplasia grade 2 and above (CIN2+) was done in the total vaccinated cohort (TVC). The study is registered with ClinicalTrials.gov, number NCT00120848. For the combined analysis of the initial and follow-up studies, the ATP efficacy cohort included 465 women in the vaccine group and 454 in the placebo group; the TVC included 560 women in the vaccine group and 553 in the placebo group. Vaccine efficacy against incident infection with HPV 16/18 was 95.3% (95% CI 87.4-98.7) and against 12-month persistent infection was 100% (81.8-100). Vaccine efficacy against CIN2+ was 100% (51.3-100) for lesions associated with HPV-16/18 and 71.9% (20.6-91.9) for lesions independent of HPV DNA. Antibody concentrations by ELISA remained 12-fold or more higher than after natural infection (both antigens). Safety outcomes were similar between groups: during the follow-up study, 30 (8%) participants reported a serious adverse event in the vaccine group versus 37 (10%) in the placebo group. None was judged related or possibly related to vaccination, and no deaths occurred. Our findings show excellent long-term efficacy, high and sustained immunogenicity, and favourable safety of the HPV-16/18 AS04-adjuvanted vaccine up to 6.4 years. GlaxoSmithKline Biologicals (Belgium).

Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

PubMed

Pu, Xinzhu; Wang, Zemin; Klaunig, James E

2015-08-06

Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

A Novel RNA Polymerase I Transcription Initiation Factor, TIF-IE, Commits rRNA Genes by Interaction with TIF-IB, Not by DNA Binding

PubMed Central

Al-Khouri, Anna Maria; Paule, Marvin R.

2002-01-01

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE. PMID:11784852

A novel RNA polymerase I transcription initiation factor, TIF-IE, commits rRNA genes by interaction with TIF-IB, not by DNA binding.

PubMed

Al-Khouri, Anna Maria; Paule, Marvin R

2002-02-01

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.

Modes of Overinitiation, dnaA Gene Expression, and Inhibition of Cell Division in a Novel Cold-Sensitive hda Mutant of Escherichia coli▿

PubMed Central

Fujimitsu, Kazuyuki; Su'etsugu, Masayuki; Yamaguchi, Yoko; Mazda, Kensaku; Fu, Nisi; Kawakami, Hironori; Katayama, Tsutomu

2008-01-01

The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the β clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25°C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25°C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42°C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25°C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25°C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway. PMID:18502852

Modes of overinitiation, dnaA gene expression, and inhibition of cell division in a novel cold-sensitive hda mutant of Escherichia coli.

PubMed

Fujimitsu, Kazuyuki; Su'etsugu, Masayuki; Yamaguchi, Yoko; Mazda, Kensaku; Fu, Nisi; Kawakami, Hironori; Katayama, Tsutomu

2008-08-01

The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the beta clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25 degrees C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25 degrees C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42 degrees C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25 degrees C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25 degrees C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.

Threading dynamics of a polymer through parallel pores: Potential applications to DNA size separation

NASA Astrophysics Data System (ADS)

Åkerman, Björn

1997-04-01

DNA orientation measurements by linear dichroism (LD) spectroscopy and single molecule imaging by fluorescence microscopy are used to investigate the effect of DNA size (71-740 kilo base pairs) and field strength E (1-5.9 V/cm) on the conformation dynamics during the field-driven threading of DNA molecules through a set of parallel pores in agarose gels, with average pore radii between 380 Å and 1400 Å. Locally relaxed but globally oriented DNA molecules are subjected to a perpendicular field, and the observed LD time profile is compared with a recent theory for the threading [D. Long and J.-L. Viovy, Phys. Rev. E 53, 803 (1996)] which assumes the same initial state. As predicted the DNA is driven by the ends into a U-form, leading to an overshoot in the LD. The overshoot-time scales as E-(1.2-1.4) as predicted, but grows more slowly with DNA size than the predicted linear dependence. For long molecules loops form initially in the threading process but are finally consumed by the ends, and the process of transfer of DNA segments, from the loops to the arms of the U, leads to a shoulder in the LD as predicted. The critical size below which loops do not form (as indicated by the LD shoulder being absent) is between 71 and 105 kbp (0.5% agarose, 5.9 V/cm), and considerably larger than predicted because in the initial state the DNA molecules are housed in gel cavities with effective pore sizes about four times larger than the average pore size. From the data, the separation of DNA by exploiting the threading dynamics in pulsed fields [D. Long et al., CR Acad. Sci. Paris, Ser. IIb 321, 239 (1995)] is shown to be feasible in principle in an agarose-based system.

DNA (DEOXYRIBONUCLEIC ACID) SYNTHESIS FOLLOWING MICROINJECTION OF HETEROLOGOUS SPERM AND SOMATIC CELL NUCLEI INTO HAMSTER OOCYTES

EPA Science Inventory

The authors have investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in 3H-thymidine after being parthenogenetically activated by sha...

New DNA-binding radioprotectors

NASA Astrophysics Data System (ADS)

Martin, Roger

The normal tissue damage associated with cancer radiotherapy has motivated the development at Peter Mac of a new class of DNA-binding radioprotecting drugs that could be applied top-ically to normal tissues at risk. Methylproamine (MP), the lead compound, reduces radiation induced cell kill at low concentrations. For example, experiments comparing the clonogenic survival of transformed human keratinocytes treated with 30 micromolar MP before and dur-ing various doses of ionising radiation, with the radiation dose response for untreated cells, indicate a dose reduction factor (DRF) of 2. Similar survival curve experiments using various concentrations of MP, with parallel measurements of uptake of MP into cell nuclei, have en-abled the relationship between drug uptake and extent of radioprotection to be established. Radioprotection has also been demonstrated after systemic administration to mice, for three different endpoints, namely lung, jejunum and bone marrow (survival at 30 days post-TBI). The results of pulse radiolysis studies indicated that the drugs act by reduction of transient radiation-induced oxidative species on DNA. This hypothesis was substantiated by the results of experiments in which MP radioprotection of radiation-induced DNA double-strand breaks, assessed as -H2AX foci, in the human keratinocyte cell line. For both endpoints, the extent of radioprotection increased with MP concentration up to a maximal value. These results are consistent with the hypothesis that radioprotection by MP is mediated by attenuation of the extent of initial DNA damage. However, although MP is a potent radioprotector, it becomes cytotoxic at higher concentrations. This limitation has been addressed in an extensive program of lead optimisation and some promising analogues have emerged from which the next lead will be selected. Given the clinical potential of topical radioprotection, the new analogues are being assessed in terms of delivery to mouse oral mucosa. This is facilitated by the fact that, like the parent minor groove-binding drug Hoechst 33342, DNA-bound MP (and its analogues) are fluorescent, enabling quantitative assessment of delivery of topically applied drug to basal cells in the mouse oral mucosa. Comparison with the data from prior in vitro experiments described above, indicate that topical delivery is sufficient to confer radioprotection. Although the primary motivation for this project relates to Radiation Oncology, the new ra-dioprotectors obviously have more general potential.

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast

PubMed Central

Sanchez, Joseph C.; Kwan, Elizabeth X.; Raghuraman, M. K.; Brewer, Bonita J.

2017-01-01

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways—DNA replication and ribosome biogenesis. PMID:29036220

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

PubMed

Sanchez, Joseph C; Kwan, Elizabeth X; Pohl, Thomas J; Amemiya, Haley M; Raghuraman, M K; Brewer, Bonita J

2017-10-01

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.

The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.

PubMed

Monot, Clément; Kuciak, Monika; Viollet, Sébastien; Mir, Ashfaq Ali; Gabus, Caroline; Darlix, Jean-Luc; Cristofari, Gaël

2013-05-01

L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

HIV sequence diversity during the early phase of infection is associated with HIV DNA reductions during antiretroviral therapy.

PubMed

Wang, Nidan; Li, Yijia; Han, Yang; Xie, Jing; Li, Taisheng

2017-06-01

The association between baseline human immunodeficiency virus (HIV) sequence diversity and HIV DNA decay after the initiation of antiretroviral therapy (ART) remains uncharacterized during the early stages of HIV infection. Samples were obtained from a cohort of 17 patients with early HIV infection (<6 months after infection) who initiated ART, and the C2V5 region of the HIV-1 envelope (env) gene was amplified via single genome amplification (SGA) to determine the peripheral plasma HIV quasispecies. We categorized HIV quasispecies into two groups according to baseline viral sequence genetic distance, which was determined by the Poisson-Fitter tool. Total HIV DNA in peripheral blood mononuclear cells (PBMCs), viral load, and T cell subsets were measured prior to and after the initiation of ART. The median SGA sequence number was 17 (range 6-28). At baseline, we identified 7 patients with homogeneous viral populations (designated the Homogeneous group) and 10 patients with heterogeneous viral populations (designated the Heterogeneous group) based on SGA sequences. Both groups exhibited similar HIV DNA decay rates during the first 6 months of ART (P > 0.99), but the Homogenous group experienced more prominent decay than the Heterogeneous group after 6 months (P = 0.037). The Heterogeneous group had higher CD4 cell counts after ART initiation; however, both groups had comparable recovery in terms of CD4/CD8 ratios and CD8 T cell activation levels. Viral population homogeneity upon the initiation of ART is associated with a decrease in HIV DNA levels during ART. J. Med. Virol. 89:982-988, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Octyl Methoxycinnamate Modulates Gene Expression and Prevents Cyclobutane Pyrimidine Dimer Formation but not Oxidative DNA Damage in UV-Exposed Human Cell Lines

PubMed Central

Duale, Nur; Olsen, Ann-Karin; Christensen, Terje; Butt, Shamas T.; Brunborg, Gunnar

2010-01-01

Octyl methoxycinnamate (OMC) is one of the most widely used sunscreen ingredients. To analyze biological effects of OMC, an in vitro approach was used implying ultraviolet (UV) exposure of two human cell lines, a primary skin fibroblast (GM00498) and a breast cancer (MCF-7) cell lines. End points include cell viability assessment, assay of cyclobutane pyrimidine dimers (CPDs) and oxidated DNA lesions using alkaline elution and lesion-specific enzymes, and gene expression analysis of a panel of 17 DNA damage–responsive genes. We observed that OMC provided protection against CPDs, and the degree of protection correlated with the OMC-mediated reduction in UV dose. No such protection was found with respect to oxidative DNA lesions. Upon UV exposure in the presence of OMC, the gene expression studies showed significant differential changes in some of the genes studied and the expression of p53 protein was also changed. For some genes, the change in expression seemed to be delayed in time by OMC. The experimental approach applied in this study, using a panel of 17 genes in an in vitro cellular system together with genotoxicity assays, may be useful in the initial screening of active ingredients in sunscreens. PMID:20071424

From structure to mechanism—understanding initiation of DNA replication

PubMed Central

Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L. Maximilian; Schneider, Sarah; Speck, Christian

2017-01-01

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2–7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. PMID:28717046

Initiation of DNA replication: functional and evolutionary aspects

PubMed Central

Bryant, John A.; Aves, Stephen J.

2011-01-01

Background The initiation of DNA replication is a very important and highly regulated step in the cell division cycle. It is of interest to compare different groups of eukaryotic organisms (a) to identify the essential molecular events that occur in all eukaryotes, (b) to start to identify higher-level regulatory mechanisms that are specific to particular groups and (c) to gain insights into the evolution of initiation mechanisms. Scope This review features a wide-ranging literature survey covering replication origins, origin recognition and usage, modification of origin usage (especially in response to plant hormones), assembly of the pre-replication complex, loading of the replisome, genomics, and the likely origin of these mechanisms and proteins in Archaea. Conclusions In all eukaryotes, chromatin is organized for DNA replication as multiple replicons. In each replicon, replication is initiated at an origin. With the exception of those in budding yeast, replication origins, including the only one to be isolated so far from a plant, do not appear to embody a specific sequence; rather, they are AT-rich, with short tracts of locally bent DNA. The proteins involved in initiation are remarkably similar across the range of eukaryotes. Nevertheless, their activity may be modified by plant-specific mechanisms, including regulation by plant hormones. The molecular features of initiation are seen in a much simpler form in the Archaea. In particular, where eukaryotes possess a number of closely related proteins that form ‘hetero-complexes’ (such as the origin recognition complex and the MCM complex), archaeans typically possess one type of protein (e.g. one MCM) that forms a homo-complex. This suggests that several eukaryotic initiation proteins have evolved from archaeal ancestors by gene duplication and divergence. PMID:21508040

Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).

PubMed Central

Ishiai, M; Wada, C; Kawasaki, Y; Yura, T

1994-01-01

Replication of mini-F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherichia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dimeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization of RepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication. Images PMID:8170998

Sequential self-assembly of DNA functionalized droplets

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

Zhang, Yin; McMullen, Angus; Pontani, Lea-Laetitia

Complex structures and devices, both natural and manmade, are often constructed sequentially. From crystallization to embryogenesis, a nucleus or seed is formed and built upon. Sequential assembly allows for initiation, signaling, and logical programming, which are necessary for making enclosed, hierarchical structures. Though biology relies on such schemes, they have not been available in materials science. We demonstrate programmed sequential self-assembly of DNA functionalized emulsions. The droplets are initially inert because the grafted DNA strands are pre-hybridized in pairs. Active strands on initiator droplets then displace one of the paired strands and thus release its complement, which in turn activatesmore » the next droplet in the sequence, akin to living polymerization. This strategy provides time and logic control during the self-assembly process, and offers a new perspective on the synthesis of materials.« less

Sequential self-assembly of DNA functionalized droplets

DOE PAGES

Zhang, Yin; McMullen, Angus; Pontani, Lea-Laetitia; ...

2017-06-16

Complex structures and devices, both natural and manmade, are often constructed sequentially. From crystallization to embryogenesis, a nucleus or seed is formed and built upon. Sequential assembly allows for initiation, signaling, and logical programming, which are necessary for making enclosed, hierarchical structures. Though biology relies on such schemes, they have not been available in materials science. We demonstrate programmed sequential self-assembly of DNA functionalized emulsions. The droplets are initially inert because the grafted DNA strands are pre-hybridized in pairs. Active strands on initiator droplets then displace one of the paired strands and thus release its complement, which in turn activatesmore » the next droplet in the sequence, akin to living polymerization. This strategy provides time and logic control during the self-assembly process, and offers a new perspective on the synthesis of materials.« less

Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin, inducing specific immune responses and protection in cotton rats.

PubMed

Pasetti, Marcela F; Barry, Eileen M; Losonsky, Genevieve; Singh, Mahender; Medina-Moreno, Sandra M; Polo, John M; Ulmer, Jeffrey; Robinson, Harriet; Sztein, Marcelo B; Levine, Myron M

2003-05-01

Measles remains a leading cause of child mortality in developing countries. Residual maternal measles antibodies and immunologic immaturity dampen immunogenicity of the current vaccine in young infants. Because cotton rat respiratory tract is susceptible to measles virus (MV) replication after intranasal (i.n.) challenge, this model can be used to assess the efficacy of MV vaccines. Pursuing a new measles vaccine strategy that might be effective in young infants, we used attenuated Salmonella enterica serovar Typhi CVD 908-htrA and Shigella flexneri 2a CVD 1208 vaccines to deliver mucosally to cotton rats eukaryotic expression plasmid pGA3-mH and Sindbis virus-based DNA replicon pMSIN-H encoding MV hemagglutinin (H). The initial i.n. dose-response with bacterial vectors alone identified a well-tolerated dosage (1 x 10(9) to 7 x 10(9) CFU) and a volume (20 micro l) that elicited strong antivector immune responses. Animals immunized i.n. on days 0, 28, and 76 with bacterial vectors carrying DNA plasmids encoding MV H or immunized parenterally with these naked DNA vaccine plasmids developed MV plaque reduction neutralizing antibodies and proliferative responses against MV antigens. In a subsequent experiment of identical design, cotton rats were challenged with wild-type MV 1 month after the third dose of vaccine or placebo. MV titers were significantly reduced in lung tissue of animals immunized with MV DNA vaccines delivered either via bacterial live vectors or parenterally. Since attenuated serovar Typhi and S. flexneri can deliver measles DNA vaccines mucosally in cotton rats, inducing measles immune responses (including neutralizing antibodies) and protection, boosting strategies can now be evaluated in animals primed with MV DNA vaccines.

Interactions of the C-terminal Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Huff, Janice; Pluth, Janice M.; Cucinotta, Francis A.

2007-01-01

NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku(sub 70/80) complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The C-terminal domain of Ku70 (Ku70c, residues 559-609), contains an helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the Ku70c/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the subunit Ku70c and a 14 base pairs DNA duplex, whose starting structures are designed to be variable so as to mimic their different binding modes. By analyzing conformational changes and energetic properties of the complex during MD simulations, we found that interactions are preferred at DNA ends, and within the major groove, which is consistent with previous experimental investigations. In addition, the results indicate that cooperation of Ku70c with other subunits of Ku(sub 70/80) is necessary to explain the high affinity of binding as observed in experiments.

Application of environmental DNA analysis to inform invasive fish eradication operations

NASA Astrophysics Data System (ADS)

Davison, Phillip I.; Copp, Gordon H.; Créach, Véronique; Vilizzi, Lorenzo; Britton, J. R.

2017-04-01

Environmental DNA (eDNA) detection of non-native species has considerable potential to inform management decisions, including identifying the need for population control and/or eradication. An invasive species of European concern is the Asian cyprinid fish, topmouth gudgeon ( Pseudorasbora parva) . Here, eDNA analyses were applied at a commercial angling venue in southern England to inform operations aiming to eradicate P. parva, which had only ever been observed in one of the venue's seven unconnected angling ponds. Eradication of P. parva was initially attempted by repeated depletion of the population using fish traps (crayfish traps fitted with 5 mm mesh netting) and the introduction of native predators over a 4-year period. The very low number of P. parva captured following these eradication efforts suggested a possible population crash. Conventional PCR analysis of water samples using species-specific primers was applied to all seven ponds to confirm that P. parva was present in only one pond, that the eradication attempt had indeed failed and that the species' distribution in the pond appeared to be restricted to three bankside locations. The continued presence of P. parva at these locations was confirmed by subsequent trapping. Water samples from an adjacent, unconnected stream were also analysed using the eDNA methodology, but no DNA of P. parva was detected. The results suggest that further management action to eradicate P. parva be focused on the pond shown to contain the isolated P. parva population and thereby eliminate the risk of further dispersal. This study is the first to apply eDNA analysis to assess the efficacy of an eradication attempt and to provide evidence that the species was unlikely to be present in the other ponds, thus reducing the resources needed to control the species.

Neuronal DNA Methylation Profiling of Blast-Related Traumatic Brain Injury.

PubMed

Haghighi, Fatemeh; Ge, Yongchao; Chen, Sean; Xin, Yurong; Umali, Michelle U; De Gasperi, Rita; Gama Sosa, Miguel A; Ahlers, Stephen T; Elder, Gregory A

2015-08-15

Long-term molecular changes in the brain resulting from blast exposure may be mediated by epigenetic changes, such as deoxyribonucleic acid (DNA) methylation, that regulate gene expression. Aberrant regulation of gene expression is associated with behavioral abnormalities, where DNA methylation bridges environmental signals to sustained changes in gene expression. We assessed DNA methylation changes in the brains of rats exposed to three 74.5 kPa blast overpressure events, conditions that have been associated with long-term anxiogenic manifestations weeks or months following the initial exposures. Rat frontal cortex eight months post-exposure was used for cell sorting of whole brain tissue into neurons and glia. We interrogated DNA methylation profiles in these cells using Expanded Reduced Representation Bisulfite Sequencing. We obtained data for millions of cytosines, showing distinct methylation profiles for neurons and glia and an increase in global methylation in neuronal versus glial cells (p<10(-7)). We detected DNA methylation perturbations in blast overpressure-exposed animals, compared with sham blast controls, within 458 and 379 genes in neurons and glia, respectively. Differentially methylated neuronal genes showed enrichment in cell death and survival and nervous system development and function, including genes involved in transforming growth factor β and nitric oxide signaling. Functional validation via gene expression analysis of 30 differentially methylated neuronal and glial genes showed a 1.2 fold change in gene expression of the serotonin N-acetyltransferase gene (Aanat) in blast animals (p<0.05). These data provide the first genome-based evidence for changes in DNA methylation induced in response to multiple blast overpressure exposures. In particular, increased methylation and decreased gene expression were observed in the Aanat gene, which is involved in converting serotonin to the circadian hormone melatonin and is implicated in sleep disturbance and depression associated with traumatic brain injury.

Application of environmental DNA analysis to inform invasive fish eradication operations.

PubMed

Davison, Phillip I; Copp, Gordon H; Créach, Véronique; Vilizzi, Lorenzo; Britton, J R

2017-04-01

Environmental DNA (eDNA) detection of non-native species has considerable potential to inform management decisions, including identifying the need for population control and/or eradication. An invasive species of European concern is the Asian cyprinid fish, topmouth gudgeon (Pseudorasbora parva). Here, eDNA analyses were applied at a commercial angling venue in southern England to inform operations aiming to eradicate P. parva, which had only ever been observed in one of the venue's seven unconnected angling ponds. Eradication of P. parva was initially attempted by repeated depletion of the population using fish traps (crayfish traps fitted with 5 mm mesh netting) and the introduction of native predators over a 4-year period. The very low number of P. parva captured following these eradication efforts suggested a possible population crash. Conventional PCR analysis of water samples using species-specific primers was applied to all seven ponds to confirm that P. parva was present in only one pond, that the eradication attempt had indeed failed and that the species' distribution in the pond appeared to be restricted to three bankside locations. The continued presence of P. parva at these locations was confirmed by subsequent trapping. Water samples from an adjacent, unconnected stream were also analysed using the eDNA methodology, but no DNA of P. parva was detected. The results suggest that further management action to eradicate P. parva be focused on the pond shown to contain the isolated P. parva population and thereby eliminate the risk of further dispersal. This study is the first to apply eDNA analysis to assess the efficacy of an eradication attempt and to provide evidence that the species was unlikely to be present in the other ponds, thus reducing the resources needed to control the species.

Methods of introducing nucleic acids into cellular DNA

DOEpatents

Lajoie, Marc J.; Gregg, Christopher J.; Mosberg, Joshua A.; Church, George M.

2017-06-27

A method of introducing a nucleic acid sequence into a cell is provided where the cell has impaired or inhibited or disrupted DnaG primase activity or impaired or inhibited or disrupted DnaB helicase activity, or larger or increased gaps or distance between Okazaki fragments or lowered or reduced frequency of Okazaki fragment initiation, or the cell has increased single stranded DNA (ssDNA) on the lagging strand of the replication fork including transforming the cell through recombination with a nucleic acid oligomer.

Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified.

PubMed

Hammond, Elizabeth R; McGillivray, Brent C; Wicker, Sophie M; Peek, John C; Shelling, Andrew N; Stone, Peter; Chamley, Larry W; Cree, Lynsey M

2017-01-01

To characterize nuclear and mitochondrial DNA (mtDNA) in spent culture media from normally developing blastocysts to determine whether it could be used for noninvasive genetic assessment. Prospective embryo cohort study. Academic center and private in vitro fertilization (IVF) clinic. Seventy patients undergoing intracytoplasmic sperm injection (ICSI) and 227 blastocysts. Culture media assessment, artificial blastocoele fluid collapse and DNA analysis using digital polymerase chain reaction (dPCR), long-range PCR, quantitative PCR (qPCR), and DNA fingerprinting. Presence of nuclear and mtDNA in three different commercial culture media from Vitrolife and Irvine Scientific, spent embryo media assessment at the cleavage and blastocyst stages of development, and analysis of the internal media controls for each patient that had been exposed to identical conditions as embryo media but did not come into contact with embryos. Higher levels of nuclear and mtDNA were observed in the culture media that had been exposed to embryos compared with the internal media controls. Nuclear DNA (∼4 copies) and mtDNA (∼600 copies) could be detected in spent media, and the levels increased at the blastocyst stage. No increase in DNA was detected after artificial blastocoele fluid collapse. Mixed sex chromosome DNA was detected. This originated from contamination in the culture media and from maternal (cumulus) cells. Due to the limited amount of template, the presence of embryonic nuclear DNA could not be confirmed by DNA fingerprinting analysis. Currently DNA from culture media cannot be used for genetic assessment because embryo-associated structures release DNA into the culture medium and the DNA is of mixed origin. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes

PubMed Central

Taylor, Benjamin JM; Wu, Yee Ling; Rada, Cristina

2014-01-01

Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. DOI: http://dx.doi.org/10.7554/eLife.03553.001 PMID:25237741

The σ70 region 1.2 regulates promoter escape by unwinding DNA downstream of the transcription start site

PubMed Central

Bochkareva, Aleksandra; Zenkin, Nikolay

2013-01-01

The mechanisms of abortive synthesis and promoter escape during initiation of transcription are poorly understood. Here, we show that, after initiation of RNA synthesis, non-specific interaction of σ70 region 1.2, present in all σ70 family factors, with the non-template strand around position −4 relative to the transcription start site facilitates unwinding of the DNA duplex downstream of the transcription start site. This leads to stabilization of short RNA products and allows their extension, i.e. promoter escape. We show that this activity of σ70 region 1.2 is assisted by the β-lobe domain, but does not involve the β′-rudder or the β′-switch-2, earlier proposed to participate in promoter escape. DNA sequence independence of this function of σ70 region 1.2 suggests that it may be conserved in all σ70 family factors. Our results indicate that the abortive nature of initial synthesis is caused, at least in part, by failure to open the downstream DNA by the β-lobe and σ region 1.2. PMID:23430153

Structures of transcription pre-initiation complex with TFIIH and Mediator.

PubMed

Schilbach, S; Hantsche, M; Tegunov, D; Dienemann, C; Wigge, C; Urlaub, H; Cramer, P

2017-11-09

For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC-core Mediator complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the 'E-bridge' helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC-core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II.

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

PubMed Central

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

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

Chaotic Image Encryption Algorithm Based on Bit Permutation and Dynamic DNA Encoding.

PubMed

Zhang, Xuncai; Han, Feng; Niu, Ying

2017-01-01

With the help of the fact that chaos is sensitive to initial conditions and pseudorandomness, combined with the spatial configurations in the DNA molecule's inherent and unique information processing ability, a novel image encryption algorithm based on bit permutation and dynamic DNA encoding is proposed here. The algorithm first uses Keccak to calculate the hash value for a given DNA sequence as the initial value of a chaotic map; second, it uses a chaotic sequence to scramble the image pixel locations, and the butterfly network is used to implement the bit permutation. Then, the image is coded into a DNA matrix dynamic, and an algebraic operation is performed with the DNA sequence to realize the substitution of the pixels, which further improves the security of the encryption. Finally, the confusion and diffusion properties of the algorithm are further enhanced by the operation of the DNA sequence and the ciphertext feedback. The results of the experiment and security analysis show that the algorithm not only has a large key space and strong sensitivity to the key but can also effectively resist attack operations such as statistical analysis and exhaustive analysis.

Chaotic Image Encryption Algorithm Based on Bit Permutation and Dynamic DNA Encoding

PubMed Central

2017-01-01

With the help of the fact that chaos is sensitive to initial conditions and pseudorandomness, combined with the spatial configurations in the DNA molecule's inherent and unique information processing ability, a novel image encryption algorithm based on bit permutation and dynamic DNA encoding is proposed here. The algorithm first uses Keccak to calculate the hash value for a given DNA sequence as the initial value of a chaotic map; second, it uses a chaotic sequence to scramble the image pixel locations, and the butterfly network is used to implement the bit permutation. Then, the image is coded into a DNA matrix dynamic, and an algebraic operation is performed with the DNA sequence to realize the substitution of the pixels, which further improves the security of the encryption. Finally, the confusion and diffusion properties of the algorithm are further enhanced by the operation of the DNA sequence and the ciphertext feedback. The results of the experiment and security analysis show that the algorithm not only has a large key space and strong sensitivity to the key but can also effectively resist attack operations such as statistical analysis and exhaustive analysis. PMID:28912802

Intragenic origins due to short G1 phases underlie oncogene-induced DNA replication stress.

PubMed

Macheret, Morgane; Halazonetis, Thanos D

2018-03-01

Oncogene-induced DNA replication stress contributes critically to the genomic instability that is present in cancer. However, elucidating how oncogenes deregulate DNA replication has been impeded by difficulty in mapping replication initiation sites on the human genome. Here, using a sensitive assay to monitor nascent DNA synthesis in early S phase, we identified thousands of replication initiation sites in cells before and after induction of the oncogenes CCNE1 and MYC. Remarkably, both oncogenes induced firing of a novel set of DNA replication origins that mapped within highly transcribed genes. These ectopic origins were normally suppressed by transcription during G1, but precocious entry into S phase, before all genic regions had been transcribed, allowed firing of origins within genes in cells with activated oncogenes. Forks from oncogene-induced origins were prone to collapse, as a result of conflicts between replication and transcription, and were associated with DNA double-stranded break formation and chromosomal rearrangement breakpoints both in our experimental system and in a large cohort of human cancers. Thus, firing of intragenic origins caused by premature S phase entry represents a mechanism of oncogene-induced DNA replication stress that is relevant for genomic instability in human cancer.

A novel chaos-based image encryption algorithm using DNA sequence operations

NASA Astrophysics Data System (ADS)

Chai, Xiuli; Chen, Yiran; Broyde, Lucie

2017-01-01

An image encryption algorithm based on chaotic system and deoxyribonucleic acid (DNA) sequence operations is proposed in this paper. First, the plain image is encoded into a DNA matrix, and then a new wave-based permutation scheme is performed on it. The chaotic sequences produced by 2D Logistic chaotic map are employed for row circular permutation (RCP) and column circular permutation (CCP). Initial values and parameters of the chaotic system are calculated by the SHA 256 hash of the plain image and the given values. Then, a row-by-row image diffusion method at DNA level is applied. A key matrix generated from the chaotic map is used to fuse the confused DNA matrix; also the initial values and system parameters of the chaotic system are renewed by the hamming distance of the plain image. Finally, after decoding the diffused DNA matrix, we obtain the cipher image. The DNA encoding/decoding rules of the plain image and the key matrix are determined by the plain image. Experimental results and security analyses both confirm that the proposed algorithm has not only an excellent encryption result but also resists various typical attacks.

Reduction of nitric oxide and DNA/RNA oxidation products are associated with active disease in systemic lupus erythematosus patients.

PubMed

Iriyoda, T M V; Stadtlober, N; Lozovoy, M A B; Delongui, F; Costa, N T; Reiche, E M V; Dichi, I; Simão, A N C

2017-09-01

The aims of the present study were to evaluate biomarkers of oxidative and nitrosative stress in systemic lupus erythematosus (SLE) patients, in particular products of DNA/RNA oxidative damage and their correlation with disease activity. This study included 188 controls and 203 patients; 153 with inactive SLE (SLEDAI < 6) and 50 with active SLE (SLEDAI ≥ 6) without renal impairment. Oxidative stress was assessed by tert-butyl hydroperoxide-initiated by chemiluminescence, advanced oxidation protein products (AOPP), total radical-trapping antioxidant parameter (TRAP), nitric oxide metabolites (NOx), and DNA/RNA oxidation products. Patients with SLE showed increased oxidative stress, as demonstrated by the augmentation of lipid hydroperoxides ( p < 0.0001) and AOPP ( p < 0.001) and reduced total antioxidant capacity ( p < 0.0001), without differences between patients with active disease and in remission. NOx levels and DNA/RNA oxidation products were inversely and independently associated with disease activity ( p < 0.0001 and p = 0.021, respectively), regardless of BMI and prednisone use. The linear regression analysis showed that about 5% of the SLEDAI score can be explained by the levels of DNA/RNA oxidation products ( r 2 :0.051; p = 0.002) and about 9% of this score by the levels of NOx ( r 2 :0.091; p < 0.0001). This study provides evidence for an inverse association between serum NOx levels and DNA/RNA oxidation products and SLE disease activity, suggesting that oxidative/nitrosative stress markers may be useful in evaluating SLE disease activity and progression of the disease.

DNA Damage Response Resulting from Replication Stress Induced by Synchronization of Cells by Inhibitors of DNA Replication: Analysis by Flow Cytometry.

PubMed

Halicka, Dorota; Zhao, Hong; Li, Jiangwei; Garcia, Jorge; Podhorecka, Monika; Darzynkiewicz, Zbigniew

2017-01-01

Cell synchronization is often achieved by transient inhibition of DNA replication. When cultured in the presence of such inhibitors as hydroxyurea, aphidicolin or excess of thymidine the cells that become arrested at the entrance to S-phase upon release from the block initiate progression through S then G 2 and M. However, exposure to these inhibitors at concentrations commonly used to synchronize cells leads to activation of ATR and ATM protein kinases as well as phosphorylation of Ser139 of histone H2AX. This observation of DNA damage signaling implies that synchronization of cells by these inhibitors is inducing replication stress. Thus, a caution should be exercised while interpreting data obtained with use of cells synchronized this way since they do not represent unperturbed cell populations in a natural metabolic state. This chapter critically outlines virtues and vices of most cell synchronization methods. It also presents the protocol describing an assessment of phosphorylation of Ser139 on H2AX and activation of ATM in cells treated with aphidicolin, as a demonstrative of one of several DNA replication inhibitors that are being used for cell synchronization. Phosphorylation of Ser139H2AX and Ser1981ATM in individual cells is detected immunocytochemically with phospho-specific Abs and intensity of immunofluorescence is measured by flow cytometry. Concurrent measurement of cellular DNA content followed by multiparameter analysis allows one to correlate the extent of phosphorylation of these proteins in response to aphidicolin with the cell cycle phase.

Emerging commercial molecular tests for the diagnosis of bloodstream infection.

PubMed

Mwaigwisya, Solomon; Assiri, Rasha Assad M; O'Grady, Justin

2015-05-01

Bloodstream infection (BSI) by microorganisms can lead to sepsis. This condition has a high mortality rate, which rises significantly with delays in initiation of appropriate antimicrobial treatment. Current culture methods for diagnosing BSI have long turnaround times and poor clinical sensitivity. While clinicians wait for culture diagnosis, patients are treated empirically, which can result in inappropriate treatment, undesirable side effects and contribute to drug resistance development. Molecular diagnostics assays that target pathogen DNA can identify pathogens and resistance markers within hours. Early diagnosis improves antibiotic stewardship and is associated with favorable clinical outcomes. Nonetheless, limitations of current molecular diagnostic methods are substantial. This article reviews recent commercially available molecular methods that use pathogen DNA to diagnose BSI, either by testing positive blood cultures or directly testing patient blood. We critically assess these tests and their application in clinical microbiology. A view of future directions in BSI diagnosis is also provided.

The potential of vitamin K3 as an anticancer agent against breast cancer that acts via the mitochondria-related apoptotic pathway.

PubMed

Akiyoshi, Takeshi; Matzno, Sumio; Sakai, Mika; Okamura, Noboru; Matsuyama, Kenji

2009-12-01

We tried to clarify the cytotoxic mechanism of VK(3) using the breast cancer cell line MCF-7. Cytotoxicity was measured via intracellular esterase activity. DNA fragmentation was assessed by agarose gel electrophoresis. JC-1 staining was applied to measure mitochondrial dysfunction. Caspase activation and reactive oxidative species (ROS) generation were also measured. VK(3) exhibited cytotoxicity that caused DNA fragmentation in MCF-7 cells with an IC(50) of 14.2 microM. JC-1 staining revealed that VK(3) caused mitochondrial dysfunction including a disappearance of mitochondrial membrane potential. Additional investigation showed that the mitochondrial damage was induced by the generation of ROS and the subsequent activation of caspase-7 and -9. Our findings demonstrate that VK(3)-induced apoptosis is selectively initiated by the mitochondria-related pathway and might be useful in breast cancer chemotherapy.

Radiation-induced transmethylation and transsulfuration in the system DNA-methionine

NASA Astrophysics Data System (ADS)

Köhnlein, W.; Merwitz, O.; Ohneseit, P.

Evidence is presented for the radiation-induced transmethylation and transsulfuration in a DNA-methionine model system. The extent of such alkylation of DNA is found to be comparable with that of alkylating agents. Therefore, both processes could be initial steps in radiation carcinogenesis. The protective effect of methionine on DNA strand breaks, due to scavenging of OH radicals, causes the formation of methyl and thiyl radicals.

A dual promoter system regulating λ DNA replication initiation

PubMed Central

Olszewski, Paweł; Szambowska, Anna; Barańska, Sylwia; Narajczyk, Magdalena; Węgrzyn, Grzegorz; Glinkowska, Monika

2014-01-01

Transcription and DNA replication are tightly regulated to ensure coordination of gene expression with growth conditions and faithful transmission of genetic material to progeny. A large body of evidence has accumulated, indicating that encounters between protein machineries carrying out DNA and RNA synthesis occur in vivo and may have important regulatory consequences. This feature may be exacerbated in the case of compact genomes, like the one of bacteriophage λ, used in our study. Transcription that starts at the rightward pR promoter and proceeds through the λ origin of replication and downstream of it was proven to stimulate the initiation of λ DNA replication. Here, we demonstrate that the activity of a convergently oriented pO promoter decreases the efficiency of transcription starting from pR. Our results show, however, that a lack of the functional pO promoter negatively influences λ phage and λ-derived plasmid replication. We present data, suggesting that this effect is evoked by the enhanced level of the pR-driven transcription, occurring in the presence of the defective pO, which may result in the impeded formation of the replication initiation complex. Our data suggest that the cross talk between the two promoters regulates λ DNA replication and coordinates transcription and replication processes. PMID:24500197

U.S. initiatives to strengthen forensic science & international standards in forensic DNA.

PubMed

Butler, John M

2015-09-01

A number of initiatives are underway in the United States in response to the 2009 critique of forensic science by a National Academy of Sciences committee. This article provides a broad review of activities including efforts of the White House National Science and Technology Council Subcommittee on Forensic Science and a partnership between the Department of Justice (DOJ) and the National Institute of Standards and Technology (NIST) to create the National Commission on Forensic Science and the Organization of Scientific Area Committees. These initiatives are seeking to improve policies and practices of forensic science. Efforts to fund research activities and aid technology transition and training in forensic science are also covered. The second portion of the article reviews standards in place or in development around the world for forensic DNA. Documentary standards are used to help define written procedures to perform testing. Physical standards serve as reference materials for calibration and traceability purposes when testing is performed. Both documentary and physical standards enable reliable data comparison, and standard data formats and common markers or testing regions are crucial for effective data sharing. Core DNA markers provide a common framework and currency for constructing DNA databases with compatible data. Recent developments in expanding core DNA markers in Europe and the United States are discussed. Published by Elsevier Ireland Ltd.

U.S. initiatives to strengthen forensic science & international standards in forensic DNA

PubMed Central

Butler, John M.

2015-01-01

A number of initiatives are underway in the United States in response to the 2009 critique of forensic science by a National Academy of Sciences committee. This article provides a broad review of activities including efforts of the White House National Science and Technology Council Subcommittee on Forensic Science and a partnership between the Department of Justice (DOJ) and the National Institute of Standards and Technology (NIST) to create the National Commission on Forensic Science and the Organization of Scientific Area Committees. These initiatives are seeking to improve policies and practices of forensic science. Efforts to fund research activities and aid technology transition and training in forensic science are also covered. The second portion of the article reviews standards in place or in development around the world for forensic DNA. Documentary standards are used to help define written procedures to perform testing. Physical standards serve as reference materials for calibration and traceability purposes when testing is performed. Both documentary and physical standards enable reliable data comparison, and standard data formats and common markers or testing regions are crucial for effective data sharing. Core DNA markers provide a common framework and currency for constructing DNA databases with compatible data. Recent developments in expanding core DNA markers in Europe and the United States are discussed. PMID:26164236

A leukocyte activation test identifies food items which induce release of DNA by innate immune peripheral blood leucocytes.

PubMed

Garcia-Martinez, Irma; Weiss, Theresa R; Yousaf, Muhammad N; Ali, Ather; Mehal, Wajahat Z

2018-01-01

Leukocyte activation (LA) testing identifies food items that induce a patient specific cellular response in the immune system, and has recently been shown in a randomized double blinded prospective study to reduce symptoms in patients with irritable bowel syndrome (IBS). We hypothesized that test reactivity to particular food items, and the systemic immune response initiated by these food items, is due to the release of cellular DNA from blood immune cells. We tested this by quantifying total DNA concentration in the cellular supernatant of immune cells exposed to positive and negative foods from 20 healthy volunteers. To establish if the DNA release by positive samples is a specific phenomenon, we quantified myeloperoxidase (MPO) in cellular supernatants. We further assessed if a particular immune cell population (neutrophils, eosinophils, and basophils) was activated by the positive food items by flow cytometry analysis. To identify the signaling pathways that are required for DNA release we tested if specific inhibitors of key signaling pathways could block DNA release. Foods with a positive LA test result gave a higher supernatant DNA content when compared to foods with a negative result. This was specific as MPO levels were not increased by foods with a positive LA test. Protein kinase C (PKC) inhibitors resulted in inhibition of positive food stimulated DNA release. Positive foods resulted in CD63 levels greater than negative foods in eosinophils in 76.5% of tests. LA test identifies food items that result in release of DNA and activation of peripheral blood innate immune cells in a PKC dependent manner, suggesting that this LA test identifies food items that result in release of inflammatory markers and activation of innate immune cells. This may be the basis for the improvement in symptoms in IBS patients who followed an LA test guided diet.

Sorption of DNA by diatomite-Zn(II) embedded supermacroporous monolithic p(HEMA) cryogels.

PubMed

Tozak, Kabil Özcan; Erzengin, Mahmut; Sargin, Idris; Ünlü, Nuri

2013-01-01

In this study, the DNA sorption performance of diatomite-Zn(II) embedded supermacroporous monolithic p(HEMA) cryogels were investigated for the purpose of designing a novel adsorbent that can be utilized for DNA purification, separation and immunoadsorption studies such as removal of anti-dsDNA antibodies from systemic lupus erythematosus (SLE) patient plasma. Poly(2-hydroxyethyl methacrylate) [p(HEMA)]-based monolithic cryogel column embedded with Zn(2+)-diatomite particles was prepared by free radical cryo-copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N,N'-methylene-bis-acrylamide (MBAAm). The polymerization reaction was initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. After thawing, the monolithic composite cryogels were used for affinity sorption and then subsequent desorption of DNA molecules from aqueous solutions. Diatomite (DA) particles were characterized by XRF and BET method. The characterization of composite cryogel was done through SEM imaging. The effects of pH of the solution, initial DNA concentration, ionic strength, temperature and flow rates on adsorption were investigated to determine the optimum conditions for adsorption/desorption experiments. The particle embedding procedure was shown to yield significantly enhanced adsorption of DNA on the adsorbent. Furthermore, considering its excellent bio-compatibility, p(HEMA) cryogels are promising a candidate for further DNA sorption studies.

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.

PubMed

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-09

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Sorption of DNA by diatomite-Zn(II) embedded supermacroporous monolithic p(HEMA) cryogels

PubMed Central

Tozak, Kabil Özcan; Erzengin, Mahmut; Sargin, Idris; Ünlü, Nuri

2013-01-01

In this study, the DNA sorption performance of diatomite-Zn(II) embedded supermacroporous monolithic p(HEMA) cryogels were investigated for the purpose of designing a novel adsorbent that can be utilized for DNA purification, separation and immunoadsorption studies such as removal of anti-dsDNA antibodies from systemic lupus erythematosus (SLE) patient plasma. Poly(2-hydroxyethyl methacrylate) [p(HEMA)]-based monolithic cryogel column embedded with Zn2+-diatomite particles was prepared by free radical cryo-copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N,N'-methylene-bis-acrylamide (MBAAm). The polymerization reaction was initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. After thawing, the monolithic composite cryogels were used for affinity sorption and then subsequent desorption of DNA molecules from aqueous solutions. Diatomite (DA) particles were characterized by XRF and BET method. The characterization of composite cryogel was done through SEM imaging. The effects of pH of the solution, initial DNA concentration, ionic strength, temperature and flow rates on adsorption were investigated to determine the optimum conditions for adsorption/desorption experiments. The particle embedding procedure was shown to yield significantly enhanced adsorption of DNA on the adsorbent. Furthermore, considering its excellent bio-compatibility, p(HEMA) cryogels are promising a candidate for further DNA sorption studies. PMID:26600734

Dynamic changes to survivin subcellular localization are initiated by DNA damage

PubMed Central

Asumen, Maritess Gay; Ifeacho, Tochukwu V; Cockerham, Luke; Pfandl, Christina; Wall, Nathan R

2010-01-01

Subcellular distribution of the apoptosis inhibitor survivin and its ability to relocalize as a result of cell cycle phase or therapeutic insult has led to the hypothesis that these subcellular pools may coincide with different survivin functions. The PIK kinases (ATM, ATR and DNA-PK) phosphorylate a variety of effector substrates that propagate DNA damage signals, resulting in various biological outputs. Here we demonstrate that subcellular repartitioning of survivin in MCF-7 cells as a result of UV light-mediated DNA damage is dependent upon DNA damage-sensing proteins as treatment with the pan PIK kinase inhibitor wortmannin repartitioned survivin in the mitochondria and diminished it from the cytosol and nucleus. Mitochondrial redistribution of survivin, such as was recorded after wortmannin treatment, occurred in cells lacking any one of the three DNA damage sensing protein kinases: DNA-PK, ATM or ATR. However, failed survivin redistribution from the mitochondria in response to low-dose UV occurred only in the cells lacking ATM, implying that ATM may be the primary kinase involved in this process. Taken together, this data implicates survivian’s subcellular distribution is a dynamic physiological process that appears responsive to UV light-initiated DNA damage and that its distribution may be responsible for its multifunctionality. PMID:20856848

DNA Recognition by a σ 54 Transcriptional Activator from Aquifex aeolicus

DOE PAGES

Vidangos, Natasha K.; Heideker, Johanna; Lyubimov, Artem; ...

2014-08-23

Transcription initiation by bacterial σ 54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain. The structurally characterized DNA-binding domains from activators all belong to the Factor for Inversion Stimulation (Fis) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DNA-binding domain of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ 54 activators. Two NtrC4 binding sites were identified upstream (-145 and -85 base pairs) from the start of the lpxC gene, which is responsiblemore » for the first committed step in Lipid A biosynthesis. This is the first experimental evidence for σ 54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the -145 binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homologue, Fis. Ultimately, the greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base specific contacts contributing to affinity than for Fis.« less

Double-probe signal enhancing strategy for toxin aptasensing based on rolling circle amplification.

PubMed

Tong, Ping; Zhao, Wei-Wei; Zhang, Lan; Xu, Jing-Juan; Chen, Hong-Yuan

2012-03-15

On the basis of aptamer-based rolling circle amplification (RCA) and magnetic beads (MBs), a highly sensitive electrochemical method was developed for the determination of Ochratoxin A (OTA). Initially, an amino-modified capture DNA was immobilized onto MBs for the following hybridization with an OTA aptamer and a phosphate labeled padlock DNA. In the presence of OTA, the aptamer would dissociate from the bioconjugate, and the padlock DNA would subsequently hybridize with the capture DNA to form a circular template with the aid of the T4 ligase. Next, capture DNA would act as primer to initiate a linear RCA reaction and hence generate a long tandem repeated sequences by phi29 DNA polymerase and dNTPs. Then, two quantum dots (QDs) labeled DNA probes were tagged on the resulted RCA product to indicate the OTA recognition event by electrochemical readout. This strategy, based on the novel design of OTA-mediated DNA circularization, the combination of RCA and double signal probes introduction, could detect OTA down to the level of 0.2 pg mL(-1) with a dynamic range spanning more than 4 orders of magnitude. The proposed approach is tested to determine OTA in red wines and shows good application potential in real samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Identification of Proteins Required for Repair of Double-Strand Chromosome Breaks, a Predisposing Factor in Breast Cancer

DTIC Science & Technology

2001-06-01

enzymatic apparatus needed to initiate DNA replication on recombination intermediates. Escherichia coli PriA protein was found to play a critical function in...the transition from recombination to DNA replication . PriA specifically binds to forked DNA structures created by recombination or replication fork

The DNA Inflammasome in Human Myeloid Cells Is Initiated by a STING-Cell Death Program Upstream of NLRP3

PubMed Central

Gaidt, Moritz M.; Ebert, Thomas S.; Chauhan, Dhruv; Ramshorn, Katharina; Pinci, Francesca; Zuber, Sarah; O’Duill, Fionan; Schmid-Burgk, Jonathan L.; Hoss, Florian; Buhmann, Raymund; Wittmann, Georg; Latz, Eicke; Subklewe, Marion; Hornung, Veit

2018-01-01

Summary Detection of cytosolic DNA constitutes a central event in the context of numerous infectious and sterile inflammatory conditions. Recent studies have uncovered a bipartite mode of cytosolic DNA recognition, in which the cGAS-STING axis triggers antiviral immunity, whereas AIM2 triggers inflammasome activation. Here, we show that AIM2 is dispensable for DNA-mediated inflammasome activation in human myeloid cells. Instead, detection of cytosolic DNA by the cGAS-STING axis induces a cell death program initiating potassium efflux upstream of NLRP3. Forward genetics identified regulators of lysosomal trafficking to modulate this cell death program, and subsequent studies revealed that activated STING traffics to the lysosome, where it triggers membrane permeabilization and thus lysosomal cell death (LCD). Importantly, the cGAS-STING-NLRP3 pathway constitutes the default inflammasome response during viral and bacterial infections in human myeloid cells. We conclude that targeting the cGAS-STING-LCD-NLRP3 pathway will ameliorate pathology in inflammatory conditions that are associated with cytosolic DNA sensing. PMID:29033128

Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway.

PubMed

Llanos, Susana; Serrano, Manuel

2010-10-01

Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 by a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely, UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 constitutes a mechanistic link between DNA damage and the ribosomal stress pathway, and is a relevant contributing signaling pathway for the activation of p53 in response to DNA damage.

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

PubMed Central

Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

2017-01-01

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA–DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA–DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs. PMID:28484024

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

PubMed

Borgogno, María V; Monti, Mariela R; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E; Pezza, Roberto J

2016-03-04

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange*

PubMed Central

Borgogno, María V.; Monti, Mariela R.; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E.; Pezza, Roberto J.

2016-01-01

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. PMID:26709229

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation.

PubMed

Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

2017-05-23

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

The DNA damage response during mitosis.

PubMed

Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

2013-10-01

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Nuclear sensing of viral DNA, epigenetic regulation of herpes simplex virus infection, and innate immunity

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

Knipe, David M., E-mail: david_knipe@hms.harvard.edu

Herpes simplex virus (HSV) undergoes a lytic infection in epithelial cells and a latent infection in neuronal cells, and epigenetic mechanisms play a major role in the differential gene expression under the two conditions. HSV viron DNA is not associated with histones but is rapidly loaded with heterochromatin upon entry into the cell. Viral proteins promote reversal of the epigenetic silencing in epithelial cells while the viral latency-associated transcript promotes additional heterochromatin in neuronal cells. The cellular sensors that initiate the chromatinization of foreign DNA have not been fully defined. IFI16 and cGAS are both essential for innate sensing ofmore » HSV DNA, and new evidence shows how they work together to initiate innate signaling. IFI16 also plays a role in the heterochromatinization of HSV DNA, and this review will examine how IFI16 integrates epigenetic regulation and innate sensing of foreign viral DNA to show how these two responses are related. - Highlights: • HSV lytic and latent gene expression is regulated differentially by epigenetic processes. • The sensors of foreign DNA have not been defined fully. • IFI16 and cGAS cooperate to sense viral DNA in HSV-infected cells. • IFI16 plays a role in both innate sensing of HSV DNA and in restricting its expression.« less

Boron neutron capture therapy for newly diagnosed glioblastoma multiforme: an assessment of clinical potential

PubMed Central

Sköld, K; Gorlia, T; Pellettieri, L; Giusti, V; H-Stenstam, B; Hopewell, J W

2010-01-01

The purpose of this study was to assess the potential of boron neutron capture therapy (BNCT), with a 6-h infusion of the boron carrier l-boronophenylalanine as a fructose preparation (BPA-f), as first-line radiotherapy for newly diagnosed glioblastoma multiforme (GBM). Patient survival data from a Phase II study using BNCT were compared with retrospective data from the two arms of a Phase III study using conventional radiotherapy (RT) in the reference arm and using RT plus concomitant and adjuvant medication with temozolomide (TMZ) in the experimental arm, and were also compared with small subgroups of these patients for whom the methylation status of the MGMT (O6-methylguanine–DNA methyltransferase) DNA repair gene was known. Differences in the baseline characteristics, salvage therapy after recurrence and levels of severe adverse events were also considered. The results indicate that BNCT offers a treatment that is at least as effective as conventional RT alone. For patients with an unmethylated MGMT DNA repair gene, a possible clinical advantage of BNCT over RT/TMZ was suggested. BNCT is a single-day treatment, which is of convenience to patients, with mild side effects, which would offer an initial 6 weeks of good-quality life during the time when patients would otherwise be undergoing daily treatments with RT and TMZ. It is suggested that the use of BNCT with a 6-h infusion of BPA-f should be explored in a stratified randomised Phase II trial in which patients with the unmethylated MGMT DNA repair gene are offered BNCT in the experimental arm and RT plus TMZ in the reference arm. PMID:20603410

Role and evolution of viral tropism in patients with advanced HIV disease receiving intensified initial regimen in the ANRS 130 APOLLO trial.

PubMed

Charpentier, Charlotte; Joly, Véronique; Larrouy, Lucile; Fagard, Catherine; Visseaux, Benoit; de Verdière, Nathalie Colin; Raffi, François; Yeni, Patrick; Descamps, Diane

2013-03-01

The aims of the study were to assess in patients with advanced HIV disease receiving antiretroviral therapy (ART) intensification with enfuvirtide (i) resistance at virological failure (VF), (ii) impact of baseline tropism on immunovirological response, and (iii) HIV-1 DNA tropism evolution during ART. The ANRS 130 APOLLO randomized trial evaluated in naive patients the immunovirological impact of standard ART without (control arm) or with enfuvirtide. Tropism was determined on RNA and DNA by V3-loop sequencing interpreted using the Geno2Pheno algorithm. At baseline the median CD4 cell count was 30 cells/mm(3). Among the 170 patients assessable in this virological substudy, HIV-1 RNA tropism was as follows: 60% of viruses were R5 and 40% were R5X4/X4. HIV-1 DNA tropism was as follows: 54% were R5 and 46% were R5X4/X4. At week 24, 39% and 49% of patients experienced VF in the enfuvirtide and control arms, respectively. In the enfuvirtide arm, only resistance-associated mutations to enfuvirtide were detected. In the control arm, two patients displayed drug-resistant viruses at the time of VF. No impact of baseline tropism was observed on immunovirological response, regardless of the study arm. Among the 25 patients experiencing DNA tropism switch between baseline and week 24, 16 (64%) switched from R5 to R5X4/X4. These latter were mostly successfully suppressed patients receiving enfuvirtide and exhibiting poorer immunological response. Baseline RNA tropism had no impact on the immunovirological response. Drug resistance mutations were only detected for the fusion inhibitor. Finally, the mechanism of replenishment of the viral cellular reservoir with X4 viruses observed needs to be further analysed.

PAX1 and SOX1 methylation as an initial screening method for cervical cancer: a meta-analysis of individual studies in Asians

PubMed Central

Cui, Zhaolei; Xiao, Zhenzhou; Hu, Minhua; Jiang, Chuanhui; Lin, Yingying; Chen, Yansong

2016-01-01

Background Epigenetic alterations of gene or DNA methylation have been highlighted as promising biomarkers for early cervical cancer screening. Herein, we evaluated the diagnostic performance of paired boxed gene 1 (PAX1) and sex determining region Y-box 1 (SOX1) methylation for cervical cancer detection. Methods Eligible studies were retrieved by searching the electronic databases. Study quality was assessed according to the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) checklist. The bivariate meta-analysis model was employed to plot the summary receiver operator characteristic (SROC) curve using Stata 12.0 software. Results The pooled sensitivity of PAX1 methylation was estimated to be 0.73 [95% confidence interval (CI): 0.70–0.75] in differentiating patients with HSIL (high-grade squamous intraepithelial lesion) or CIN3+ (cervical intraepithelial neoplasia type III/worse) or cervical cancer from normal individuals, corresponding to a specificity of 0.87 (95% CI: 0.85–0.89) and area under the curve (AUC) of 0.91. The SOX1 methylation test yielded an AUC of 0.82, under which, the pooled sensitivity was 0.71 (95% CI: 0.67–0.74) and specificity was 0.64 (95% CI: 0.61–0.67). Notably, the stratified analysis suggested that combing parallel testing of PAX1 methylation and human papillomavirus (HPV) DNA (AUC, sensitivity, and specificity of 0.89, 0.75, and 0.81, respectively) achieved higher accuracy than single HPV DNA testing (AUC, sensitivity, and specificity of 0.77, 0.81, and 0.70, respectively). Conclusions PAX1 or SOX1 methylation has a prospect to be an auxiliary biomarker for cervical cancer screening, and parallel testing of PAX1 methylation and HPV DNA in cervical swabs confers an improved diagnostic accuracy than single HPV DNA testing. PMID:27826568

From structure to mechanism-understanding initiation of DNA replication.

PubMed

Riera, Alberto; Barbon, Marta; Noguchi, Yasunori; Reuter, L Maximilian; Schneider, Sarah; Speck, Christian

2017-06-01

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability. © 2017 Riera et al.; Published by Cold Spring Harbor Laboratory Press.

Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq)

PubMed Central

Langley, Alexander R.; Gräf, Stefan; Smith, James C.; Krude, Torsten

2016-01-01

Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq. PMID:27587586

Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq).

PubMed

Langley, Alexander R; Gräf, Stefan; Smith, James C; Krude, Torsten

2016-12-01

Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Studies on DNA damage: discordant responses of rate of DNA disentanglement (viscosimetrically evaluated) and alkaline elution rate, obtained for several compounds. Possible explanations of the discrepancies.

PubMed

Parodi, S; Balbi, C; Abelmoschi, M L; Pala, M; Russo, P; Santi, L

1983-12-01

Alkaline elution is a well-known method for detecting DNA damage. Recently we have developed a viscosimetric method that is even more sensitive than alkaline elution. Here we report that the two methods, although apparently both revealing alkaline DNA fragmentation, can give dramatically different results for a significant series of compounds. We suspect that alkaline elution might reveal not only DNA fragmentation but also the extent of disentanglement of chromatin structure, whereas this DNA disentanglement rate, when evaluated viscosimetrically , is more strictly correlated with the initiation of DNA unwinding.

Four years of DNA barcoding: current advances and prospects.

PubMed

Frézal, Lise; Leblois, Raphael

2008-09-01

Research using cytochrome c oxidase barcoding techniques on zoological specimens was initiated by Hebert et al. [Hebert, P.D.N., Ratnasingham, S., deWaard, J.R., 2003. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. Soc. Lond. B 270, S96-S99]. By March 2004, the Consortium for the Barcode of Life started to promote the use of a standardized DNA barcoding approach, consisting of identifying a specimen as belonging to a certain animal species based on a single universal marker: the DNA barcode sequence. Over the last 4 years, this approach has become increasingly popular and advances as well as limitations have clearly emerged as increasing amounts of organisms have been studied. Our purpose is to briefly expose DNA Barcode of Life principles, pros and cons, relevance and universality. The initially proposed Barcode of life framework has greatly evolved, giving rise to a flexible description of DNA barcoding and a larger range of applications.

Both Chromosome Decondensation and Condensation Are Dependent on DNA Replication in C. elegans Embryos

PubMed Central

Sonneville, Remi; Craig, Gillian; Labib, Karim; Gartner, Anton; Blow, J. Julian

2015-01-01

Summary During cell division, chromatin alternates between a condensed state to facilitate chromosome segregation and a decondensed form when DNA replicates. In most tissues, S phase and mitosis are separated by defined G1 and G2 gap phases, but early embryogenesis involves rapid oscillations between replication and mitosis. Using Caenorhabditis elegans embryos as a model system, we show that chromosome condensation and condensin II concentration on chromosomal axes require replicated DNA. In addition, we found that, during late telophase, replication initiates on condensed chromosomes and promotes the rapid decondensation of the chromatin. Upon replication initiation, the CDC-45-MCM-GINS (CMG) DNA helicase drives the release of condensin I complexes from chromatin and the activation or displacement of inactive MCM-2–7 complexes, which together with the nucleoporin MEL-28/ELYS tethers condensed chromatin to the nuclear envelope, thereby promoting chromatin decondensation. Our results show how, in an early embryo, the chromosome-condensation cycle is functionally linked with DNA replication. PMID:26166571

DNA Replication Control During Drosophila Development: Insights into the Onset of S Phase, Replication Initiation, and Fork Progression

PubMed Central

Hua, Brian L.; Orr-Weaver, Terry L.

2017-01-01

Proper control of DNA replication is critical to ensure genomic integrity during cell proliferation. In addition, differential regulation of the DNA replication program during development can change gene copy number to influence cell size and gene expression. Drosophila melanogaster serves as a powerful organism to study the developmental control of DNA replication in various cell cycle contexts in a variety of differentiated cell and tissue types. Additionally, Drosophila has provided several developmentally regulated replication models to dissect the molecular mechanisms that underlie replication-based copy number changes in the genome, which include differential underreplication and gene amplification. Here, we review key findings and our current understanding of the developmental control of DNA replication in the contexts of the archetypal replication program as well as of underreplication and differential gene amplification. We focus on the use of these latter two replication systems to delineate many of the molecular mechanisms that underlie the developmental control of replication initiation and fork elongation. PMID:28874453

Reduced 3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy)-Initiated Oxidative DNA Damage and Neurodegeneration in Prostaglandin H Synthase-1 Knockout Mice

PubMed Central

2010-01-01

The neurodegenerative potential of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and underlying mechanisms are under debate. Here, we show that MDMA is a substrate for CNS prostaglandin H synthase (PHS)-catalyzed bioactivation to a free radical intermediate that causes reactive oxygen species (ROS) formation and neurodegenerative oxidative DNA damage. In vitro PHS-1-catalyzed bioactivation of MDMA stereoselectively produced free radical intermediate formation and oxidative DNA damage that was blocked by the PHS inhibitor eicosatetraynoic acid. In vivo, MDMA stereoselectively caused gender-independent DNA oxidation and dopaminergic nerve terminal degeneration in several brain regions, dependent on regional PHS-1 levels. Conversely, MDMA-initiated striatal DNA oxidation, nerve terminal degeneration, and motor coordination deficits were reduced in PHS-1 +/− and −/− knockout mice in a gene dose-dependent fashion. These results confirm the neurodegenerative potential of MDMA and provide the first direct evidence for a novel molecular mechanism involving PHS-catalyzed formation of a neurotoxic MDMA free radical intermediate. PMID:22778832

Enlightenment of Yeast Mitochondrial Homoplasmy: Diversified Roles of Gene Conversion

PubMed Central

Ling, Feng; Mikawa, Tsutomu; Shibata, Takehiko

2011-01-01

Mitochondria have their own genomic DNA. Unlike the nuclear genome, each cell contains hundreds to thousands of copies of mitochondrial DNA (mtDNA). The copies of mtDNA tend to have heterogeneous sequences, due to the high frequency of mutagenesis, but are quickly homogenized within a cell (“homoplasmy”) during vegetative cell growth or through a few sexual generations. Heteroplasmy is strongly associated with mitochondrial diseases, diabetes and aging. Recent studies revealed that the yeast cell has the machinery to homogenize mtDNA, using a common DNA processing pathway with gene conversion; i.e., both genetic events are initiated by a double-stranded break, which is processed into 3′ single-stranded tails. One of the tails is base-paired with the complementary sequence of the recipient double-stranded DNA to form a D-loop (homologous pairing), in which repair DNA synthesis is initiated to restore the sequence lost by the breakage. Gene conversion generates sequence diversity, depending on the divergence between the donor and recipient sequences, especially when it occurs among a number of copies of a DNA sequence family with some sequence variations, such as in immunoglobulin diversification in chicken. MtDNA can be regarded as a sequence family, in which the members tend to be diversified by a high frequency of spontaneous mutagenesis. Thus, it would be interesting to determine why and how double-stranded breakage and D-loop formation induce sequence homogenization in mitochondria and sequence diversification in nuclear DNA. We will review the mechanisms and roles of mtDNA homoplasmy, in contrast to nuclear gene conversion, which diversifies gene and genome sequences, to provide clues toward understanding how the common DNA processing pathway results in such divergent outcomes. PMID:24710143

Methodologic European external quality assurance for DNA sequencing: the EQUALseq program.

PubMed

Ahmad-Nejad, Parviz; Dorn-Beineke, Alexandra; Pfeiffer, Ulrike; Brade, Joachim; Geilenkeuser, Wolf-Jochen; Ramsden, Simon; Pazzagli, Mario; Neumaier, Michael

2006-04-01

DNA sequencing is a key technique in molecular diagnostics, but to date no comprehensive methodologic external quality assessment (EQA) programs have been instituted. Between 2003 and 2005, the European Union funded, as specific support actions, the EQUAL initiative to develop methodologic EQA schemes for genotyping (EQUALqual), quantitative PCR (EQUALquant), and sequencing (EQUALseq). Here we report on the results of the EQUALseq program. The participating laboratories received a 4-sample set comprising 2 DNA plasmids, a PCR product, and a finished sequencing reaction to be analyzed. Data and information from detailed questionnaires were uploaded online and evaluated by use of a scoring system for technical skills and proficiency of data interpretation. Sixty laboratories from 21 European countries registered, and 43 participants (72%) returned data and samples. Capillary electrophoresis was the predominant platform (n = 39; 91%). The median contiguous correct sequence stretch was 527 nucleotides with considerable variation in quality of both primary data and data evaluation. The association between laboratory performance and the number of sequencing assays/year was statistically significant (P <0.05). Interestingly, more than 30% of participants neither added comments to their data nor made efforts to identify the gene sequences or mutational positions. Considerable variations exist even in a highly standardized methodology such as DNA sequencing. Methodologic EQAs are appropriate tools to uncover strengths and weaknesses in both technique and proficiency, and our results emphasize the need for mandatory EQAs. The results of EQUALseq should help improve the overall quality of molecular genetics findings obtained by DNA sequencing.

Improving the Conservation of Mediterranean Chondrichthyans: The ELASMOMED DNA Barcode Reference Library

PubMed Central

Arculeo, Marco; Bonello, Juan J.; Bonnici, Leanne; Cannas, Rita; Carbonara, Pierluigi; Cau, Alessandro; Charilaou, Charis; El Ouamari, Najib; Fiorentino, Fabio; Follesa, Maria Cristina; Garofalo, Germana; Golani, Daniel; Guarniero, Ilaria; Hanner, Robert; Hemida, Farid; Kada, Omar; Lo Brutto, Sabrina; Mancusi, Cecilia; Morey, Gabriel; Schembri, Patrick J.; Serena, Fabrizio; Sion, Letizia; Stagioni, Marco; Tursi, Angelo; Vrgoc, Nedo; Steinke, Dirk; Tinti, Fausto

2017-01-01

Cartilaginous fish are particularly vulnerable to anthropogenic stressors and environmental change because of their K-selected reproductive strategy. Accurate data from scientific surveys and landings are essential to assess conservation status and to develop robust protection and management plans. Currently available data are often incomplete or incorrect as a result of inaccurate species identifications, due to a high level of morphological stasis, especially among closely related taxa. Moreover, several diagnostic characters clearly visible in adult specimens are less evident in juveniles. Here we present results generated by the ELASMOMED Consortium, a regional network aiming to sample and DNA-barcode the Mediterranean Chondrichthyans with the ultimate goal to provide a comprehensive DNA barcode reference library. This library will support and improve the molecular taxonomy of this group and the effectiveness of management and conservation measures. We successfully barcoded 882 individuals belonging to 42 species (17 sharks, 24 batoids and one chimaera), including four endemic and several threatened ones. Morphological misidentifications were found across most orders, further confirming the need for a comprehensive DNA barcoding library as a valuable tool for the reliable identification of specimens in support of taxonomist who are reviewing current identification keys. Despite low intraspecific variation among their barcode sequences and reduced samples size, five species showed preliminary evidence of phylogeographic structure. Overall, the ELASMOMED initiative further emphasizes the key role accurate DNA barcoding libraries play in establishing reliable diagnostic species specific features in otherwise taxonomically problematic groups for biodiversity management and conservation actions. PMID:28107413

The structural changes of T7 RNA polymerase from transcription initiation to elongation

PubMed Central

Steitz, Thomas A

2010-01-01

Summary The structures of T7 RNA polymerase (T7 RNAP) captured in the initiation and elongation phases of transcription, as well as an intermediate stage provide insights into how this RNA polymerase protein can initiate RNA synthesis and synthesize 7 to 10 nucleotides of RNA while remaining bound to the DNA promoter site. Recently, the structures of T7 RNAP bound to it promoter DNA along with either a 7 nucleotide or 8 nucleotide transcript show an elongated product site resulting from a 40° or 45° rotation of the promoter and domain that binds it. The different functional properties of the initiation and elongation phases of transcription are illuminated from structures of the initiation and elongation complexes. Structural insights into the translocation of the product transcript of RNAP, its separation of the downstream duplex DNA and its removal of the transcript from the heteroduplex are provided by the structures of several states of nucleotide incorporation. A conformational change in the “fingers” domain that results from the binding or dissociation of incoming NTP or PPi appears to be associated with the state of translocation of T7 RNAP. PMID:19811903

Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1.

PubMed

Alver, Robert C; Chadha, Gaganmeet Singh; Gillespie, Peter J; Blow, J Julian

2017-03-07

Dbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Fractionation and reconstitution of factors required for accurate transcription of mammalian ribosomal RNA genes: identification of a species-dependent initiation factor.

PubMed Central

Mishima, Y; Financsek, I; Kominami, R; Muramatsu, M

1982-01-01

Mouse and human cell extracts (S100) can support an accurate and efficient transcription initiation on homologous ribosomal RNA gene (rDNA) templates. The cell extracts were fractionated with the aid of a phosphocellulose column into four fractions (termed A, B, C and D), including one containing a major part of the RNA polymerase I activity. Various reconstitution experiments indicate that fraction D is an absolute requirement for the correct and efficient transcription initiation by RNA polymerase I on both mouse and human genes. Fraction B effectively suppresses random initiation on these templates. Fraction A appears to further enhance the transcription which takes place with fractions C and D. Although fractions A, B and C are interchangeable between mouse and human extracts, fraction D is not; i.e. initiation of transcription required the presence of a homologous fraction D for both templates. The factor(s) in fraction D, however, is not literally species-specific, since mouse D fraction is capable of supporting accurate transcription initiation on a rat rDNA template in the presence of all the other fractions from human cell extract under the conditions where human D fraction is unable to support it. We conclude from these experiments that a species-dependent factor in fraction D plays an important role in the initiation of rDNA transcription in each animal species. Images PMID:7177852

Function of BRCA1 at a DNA Replication Origin

DTIC Science & Technology

2004-07-01

origin of Epstein-Barr Virus DNA replication (Ori P). OriP replicates once and only once per cell cycle in synchrony with the cellular genome, and is...modifications, and to investigate its function at OriP in DNA replication and plasmid maintenance. We propose that these studies will provide valuable...information concerning the function of OriP at replication origins and in the control of DNA replication initiation and genome stability.

Molecular Technique to Reduce PCR Bias for Deeper Understanding of Microbial Diversity

NASA Technical Reports Server (NTRS)

Vaishampayan, Parag A.; Venkateswaran, Kasthuri J.

2012-01-01

Current planetary protection policies require that spacecraft targeted to sensitive solar system bodies be assembled and readied for launch in controlled cleanroom environments. A better understanding of the distribution and frequency at which high-risk contaminant microbes are encountered on spacecraft surfaces would significantly aid in assessing the threat of forward contamination. However, despite a growing understanding of the diverse microbial populations present in cleanrooms, less abundant microbial populations are probably not adequately taken into account due to technological limitations. This novel approach encompasses a wide spectrum of microbial species and will represent the true picture of spacecraft cleanroom-associated microbial diversity. All of the current microbial diversity assessment techniques are based on an initial PCR amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of a minor template appears to be suppressed by the amplification of a more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck overlooks the presence of the less abundant minority population and may underestimate their role in the ecosystem maintenance. DNA intercalating agents such as propidium monoazide (PMA) covalently bind with DNA molecules upon photolysis using visible light, and make it unavailable for DNA polymerase enzyme during polymerase chain reaction (PCR). Environmental DNA samples will be treated with suboptimum PMA concentration, enough to intercalate with 90 99% of the total DNA. The probability of PMA binding with DNA from abundant bacterial species will be much higher than binding with DNA from less abundant species. This will increase the relative DNA concentration of previously "shadowed" less abundant species available for PCR amplification. These PCR products obtained with and without PMA treatment will then be subjected to downstream diversity analyses such as sequencing and DNA microarray. It is expected that PMA-coupled PCR will amplify the "minority population" and help in understanding microbial diversity spectrum of an environmental sample at a much deeper level. This new protocol aims to overcome the major potential biases faced when analyzing microbial 16S rRNA gene diversity. This study will lead to a technological advancement and a commercial product that will aid microbial ecologists in understanding microbial diversity from various environmental niches. Implementation of this technique may lead to discoveries of novel microbes and their functions in sustenance of the ecosystem.

Role of mtDNA haplogroups in the prevalence of osteoarthritis in different geographic populations: a meta-analysis.

PubMed

Shen, Jin-Ming; Feng, Lei; Feng, Chun

2014-01-01

Osteoarthritis (OA) is the most common form of arthritis and has become an increasingly important public-health problem. However, the pathogenesis of OA is still unclear. In recent years, its correlation with mtDNA haplogroups attracts much attention. We aimed to perform a meta-analysis to investigate the association between mtDNA haplogroups and OA. Published English or Chinese literature from PubMed, Web of Science, SDOS, and CNKI was retrieved up until April 15, 2014. Case-control or cohort studies that detected the frequency of mtDNA haplogroups in OA patients and controls were included. The quality of the included studies was evaluated by the Newcastle-Ottawa Scale (NOS) assessment. A meta-analysis was conducted to calculate pooled odds ratio (OR) with 95% confidence interval (CI) through the random or fixed effect model, which was selected based on the between-study heterogeneity assessed by Q test and I2 test. Subgroup analysis was performed to explore the origin of heterogeneity. A total of 6 case-control studies (10590 cases and 7161 controls) with an average NOS score of 6.9 were involved. For the analysis between mtDNA haplogroup J and OA, random model was selected due to high heterogeneity. No significant association was found initially (OR = 0.73, 95%CI: 0.52-1.03), however, once any study from UK population was removed the association emerged. Further subgroup analysis demonstrated that there was a significant association in Spain population (OR = 0.57, 95%CI: 0.46-0.71), but not in UK population. Also, subgroup analysis revealed that there was a significant correlation between cluster TJ and OA in Spain population (OR = 0.70, 95%CI: 0.58-0.84), although not in UK population. No significant correlation was found between haplogroup T/cluster HV/cluster KU and OA. Our current meta-analysis suggests that mtDNA haplogroup J and cluster TJ correlate with the risk of OA in Spanish population, but the associations in other populations require further investigation.

Mitochondrial DNA Damage Initiates Acute Lung Injury and Multi-Organ System Failure Evoked in Rats by Intra-Tracheal Pseudomonas Aeruginosa.

PubMed

Lee, Yann-Leei; Obiako, Boniface; Gorodnya, Olena M; Ruchko, Mykhaylo V; Kuck, Jamie L; Pastukh, Viktor M; Wilson, Glenn L; Simmons, Jon D; Gillespie, Mark N

2017-07-01

Although studies in rat cultured pulmonary artery endothelial cells, perfused lungs, and intact mice support the concept that oxidative mitochondrial (mt) DNA damage triggers acute lung injury (ALI), it has not yet been determined whether enhanced mtDNA repair forestalls development of ALI and its progression to multiple organ system failure (MOSF). Accordingly, here we examined the effect of a fusion protein construct targeting the DNA glycosylase, Ogg1, to mitochondria in a rat model intra-tracheal Pseudomonas aeruginosa (strain 103; PA103)-induced ALI and MOSF. Relative to controls, animals given PA103 displayed increases in lung vascular filtration coefficient accompanied by transient lung tissue oxidative mtDNA damage and variable changes in mtDNA copy number without evidence of nuclear DNA damage. The approximate 40% of animals surviving 24 h after bacterial administration exhibited multiple organ dysfunction, manifest as increased serum and tissue-specific indices of kidney and liver failure, along with depressed heart rate and blood pressure. While administration of mt-targeted Ogg1 to control animals was innocuous, the active fusion protein, but not a DNA repair-deficient mutant, prevented bacteria-induced increases in lung tissue oxidative mtDNA damage, failed to alter mtDNA copy number, and attenuated lung endothelial barrier degradation. These changes were associated with suppression of liver, kidney, and cardiovascular dysfunction and with decreased 24 h mortality. Collectively, the present findings indicate that oxidative mtDNA damage to lung tissue initiates PA103-induced ALI and MOSF in rats.

DNA methylation analysis from saliva samples for epidemiological studies.

PubMed

Nishitani, Shota; Parets, Sasha E; Haas, Brian W; Smith, Alicia K

2018-06-18

Saliva is a non-invasive, easily accessible tissue, which is regularly collected in large epidemiological studies to examine genetic questions. Recently, it is becoming more common to use saliva to assess DNA methylation. However, DNA extracted from saliva is a mixture of both bacterial and human DNA derived from epithelial and immune cells in the mouth. Thus, there are unique challenges to using salivary DNA in methylation studies that can influence data quality. This study assesses: (1) quantification of human DNA after extraction; (2) delineation of human and bacterial DNA; (3) bisulfite conversion (BSC); (4) quantification of BSC DNA; (5) PCR amplification of BSC DNA from saliva and; (6) quantitation of DNA methylation with a targeted assay. The framework proposed will allow saliva samples to be more widely used in targeted epigenetic studies.

Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System.

PubMed

Cooper, Lauren A; Stringer, Anne M; Wade, Joseph T

2018-04-17

In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo , for the type I-E system of Escherichia coli Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5' end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing. IMPORTANCE Many bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61-nucleotide (nt) CRISPR RNAs (crRNAs). The Cascade complex is directed to invading DNA molecules through base pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease, which destroys the invading DNA molecule and promotes acquisition of new immunity elements. We made the first in vivo measurements of Cascade binding to DNA targets. Thus, we show that Cascade binding to DNA is highly promiscuous; endogenous E. coli crRNAs can direct Cascade binding to >100 chromosomal locations. In contrast, we show that targeted degradation and acquisition of new immunity elements require highly specific association of Cascade with DNA, limiting CRISPR-Cas function to the appropriate targets. Copyright © 2018 Cooper et al.

Seasonal variation in environmental DNA in relation to population size and environmental factors

PubMed Central

Buxton, Andrew S.; Groombridge, Jim J.; Zakaria, Nurulhuda B.; Griffiths, Richard A.

2017-01-01

Analysing DNA that organisms release into the environment (environmental DNA, or eDNA) has enormous potential for assessing rare and cryptic species. At present the method is only reliably used to assess the presence-absence of species in natural environments, as seasonal influences on eDNA in relation to presence, abundance, life stages and seasonal behaviours are poorly understood. A naturally colonised, replicated pond system was used to show how seasonal changes in eDNA were influenced by abundance of adults and larvae of great crested newts (Triturus cristatus). Peaks in eDNA were observed in early June when adult breeding was coming to an end, and between mid-July and mid-August corresponding to a peak in newt larval abundance. Changes in adult body condition associated with reproduction also influenced eDNA concentrations, as did temperature (but not rainfall or UV). eDNA concentration fell rapidly as larvae metamorphosed and left the ponds. eDNA concentration may therefore reflect relative abundance in different ponds, although environmental factors can affect the concentrations observed. Nevertheless, eDNA surveys may still represent an improvement over unadjusted counts which are widely used in population assessments but have unreliable relationships with population size. PMID:28393885

Implementation options for DNA-based identification into ecological status assessment under the European Water Framework Directive.

PubMed

Hering, Daniel; Borja, Angel; Jones, J Iwan; Pont, Didier; Boets, Pieter; Bouchez, Agnes; Bruce, Kat; Drakare, Stina; Hänfling, Bernd; Kahlert, Maria; Leese, Florian; Meissner, Kristian; Mergen, Patricia; Reyjol, Yorick; Segurado, Pedro; Vogler, Alfried; Kelly, Martyn

2018-07-01

Assessment of ecological status for the European Water Framework Directive (WFD) is based on "Biological Quality Elements" (BQEs), namely phytoplankton, benthic flora, benthic invertebrates and fish. Morphological identification of these organisms is a time-consuming and expensive procedure. Here, we assess the options for complementing and, perhaps, replacing morphological identification with procedures using eDNA, metabarcoding or similar approaches. We rate the applicability of DNA-based identification for the individual BQEs and water categories (rivers, lakes, transitional and coastal waters) against eleven criteria, summarised under the headlines representativeness (for example suitability of current sampling methods for DNA-based identification, errors from DNA-based species detection), sensitivity (for example capability to detect sensitive taxa, unassigned reads), precision of DNA-based identification (knowledge about uncertainty), comparability with conventional approaches (for example sensitivity of metrics to differences in DNA-based identification), cost effectiveness and environmental impact. Overall, suitability of DNA-based identification is particularly high for fish, as eDNA is a well-suited sampling approach which can replace expensive and potentially harmful methods such as gill-netting, trawling or electrofishing. Furthermore, there are attempts to replace absolute by relative abundance in metric calculations. For invertebrates and phytobenthos, the main challenges include the modification of indices and completing barcode libraries. For phytoplankton, the barcode libraries are even more problematic, due to the high taxonomic diversity in plankton samples. If current assessment concepts are kept, DNA-based identification is least appropriate for macrophytes (rivers, lakes) and angiosperms/macroalgae (transitional and coastal waters), which are surveyed rather than sampled. We discuss general implications of implementing DNA-based identification into standard ecological assessment, in particular considering any adaptations to the WFD that may be required to facilitate the transition to molecular data. Copyright © 2018 Elsevier Ltd. All rights reserved.

Early diagnosis and monitoring of mucormycosis by detection of circulating DNA in serum: retrospective analysis of 44 cases collected through the French Surveillance Network of Invasive Fungal Infections (RESSIF).

PubMed

Millon, L; Herbrecht, R; Grenouillet, F; Morio, F; Alanio, A; Letscher-Bru, V; Cassaing, S; Chouaki, T; Kauffmann-Lacroix, C; Poirier, P; Toubas, D; Augereau, O; Rocchi, S; Garcia-Hermoso, D; Bretagne, S

2016-09-01

The main objective of this study was to assess the diagnostic performance of a set of three Mucorales quantitative PCR assays in a retrospective multicentre study. Mucormycosis cases were recorded thanks to the French prospective surveillance programme (RESSIF network). The day of sampling of the first histological or mycological positive specimen was defined as day 0 (D0). Detection of circulating DNA was performed on frozen serum samples collected from D-30 to D30, using quantitative PCR assays targeting Rhizomucor, Lichtheimia, Mucor/Rhizopus. Forty-four patients diagnosed with probable (n = 19) or proven (n = 25) mucormycosis were included. Thirty-six of the 44 patients (81%) had at least one PCR-positive serum. The first PCR-positive sample was observed 9 days (range 0-28 days) before diagnosis was made using mycological criteria and at least 2 days (range 0-24 days) before imaging. The identifications provided with the quantitative PCR assays were all concordant with culture and/or PCR-based identification of the causal species. Survival rate at D84 was significantly higher for patients with an initially positive PCR that became negative after treatment initiation than for patients whose PCR remained positive (48% and 4%, respectively; p <10 -6 ). The median time for complete negativity of PCR was 7 days (range 3-19 days) after initiation of l-AmB treatment. Despite some limitations due to the retrospective design of the study, we showed that Mucorales quantitative PCR could not only confirm the mucormycosis diagnosis when other mycological arguments were present but could also anticipate this diagnosis. Quantification of DNA loads may also be a useful adjunct to treatment monitoring. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Combined quantification of pulmonary Pneumocystis jirovecii DNA and serum (1->3)-β-D-glucan for differential diagnosis of pneumocystis pneumonia and Pneumocystis colonization.

PubMed

Damiani, Céline; Le Gal, Solène; Da Costa, Cécilia; Virmaux, Michèle; Nevez, Gilles; Totet, Anne

2013-10-01

This study assessed a quantitative PCR (qPCR) assay for Pneumocystis jirovecii quantification in bronchoalveolar lavage (BAL) fluid samples combined with serum (1→3)-β-d-glucan (BG) level detection to distinguish Pneumocystis pneumonia (PCP) from pulmonary colonization with P. jirovecii. Forty-six patients for whom P. jirovecii was initially detected in BAL fluid samples were retrospectively enrolled. Based on clinical data and results of P. jirovecii detection, 17 and 29 patients were diagnosed with PCP and colonization, respectively. BAL fluid samples were reassayed using a qPCR assay targeting the mitochondrial large subunit rRNA gene. qPCR results and serum BG levels (from a Fungitell kit) were analyzed conjointly. P. jirovecii DNA copy numbers were significantly higher in the PCP group than in the colonization group (1.3 × 10(7) versus 3.4 × 10(3) copies/μl, P < 0.05). A lower cutoff value (1.6 × 10(3) copies/μl) achieving 100% sensitivity for PCP diagnosis and an upper cutoff value (2 × 10(4) copies/μl) achieving 100% specificity were determined. Applying these two values, 13/17 PCP patients and 19/29 colonized patients were correctly assigned to their patient groups. For the remaining 14 patients with P. jirovecii DNA copy numbers between the cutoff values, PCP and colonization could not be distinguished on the basis of qPCR results. Four of these patients who were initially assigned to the PCP group presented BG levels of ≥100 pg/ml. The other 10 patients, who were initially assigned to the colonization group, presented BG levels of <100 pg/ml. These results suggest that the combination of the qPCR assay, applying cutoff values of 1.6 × 10(3) and 2 × 10(4) copies/μl, and serum BG detection, applying a 100 pg/ml threshold, can differentiate PCP and colonization diagnoses.

Combined Quantification of Pulmonary Pneumocystis jirovecii DNA and Serum (1→3)-β-d-Glucan for Differential Diagnosis of Pneumocystis Pneumonia and Pneumocystis Colonization

PubMed Central

Le Gal, Solène; Da Costa, Cécilia; Virmaux, Michèle; Nevez, Gilles; Totet, Anne

2013-01-01

This study assessed a quantitative PCR (qPCR) assay for Pneumocystis jirovecii quantification in bronchoalveolar lavage (BAL) fluid samples combined with serum (1→3)-β-d-glucan (BG) level detection to distinguish Pneumocystis pneumonia (PCP) from pulmonary colonization with P. jirovecii. Forty-six patients for whom P. jirovecii was initially detected in BAL fluid samples were retrospectively enrolled. Based on clinical data and results of P. jirovecii detection, 17 and 29 patients were diagnosed with PCP and colonization, respectively. BAL fluid samples were reassayed using a qPCR assay targeting the mitochondrial large subunit rRNA gene. qPCR results and serum BG levels (from a Fungitell kit) were analyzed conjointly. P. jirovecii DNA copy numbers were significantly higher in the PCP group than in the colonization group (1.3 × 107 versus 3.4 × 103 copies/μl, P < 0.05). A lower cutoff value (1.6 × 103 copies/μl) achieving 100% sensitivity for PCP diagnosis and an upper cutoff value (2 × 104 copies/μl) achieving 100% specificity were determined. Applying these two values, 13/17 PCP patients and 19/29 colonized patients were correctly assigned to their patient groups. For the remaining 14 patients with P. jirovecii DNA copy numbers between the cutoff values, PCP and colonization could not be distinguished on the basis of qPCR results. Four of these patients who were initially assigned to the PCP group presented BG levels of ≥100 pg/ml. The other 10 patients, who were initially assigned to the colonization group, presented BG levels of <100 pg/ml. These results suggest that the combination of the qPCR assay, applying cutoff values of 1.6 × 103 and 2 × 104 copies/μl, and serum BG detection, applying a 100 pg/ml threshold, can differentiate PCP and colonization diagnoses. PMID:23903553

Non-parenchymal liver cells support the growth advantage in the first stages of hepatocarcinogenesis.

PubMed

Drucker, Claudia; Parzefall, Wolfram; Teufelhofer, Olga; Grusch, Michael; Ellinger, Adolf; Schulte-Hermann, Rolf; Grasl-Kraupp, Bettina

2006-01-01

Hepatocellular carcinoma almost always arises in chronically inflamed livers. We developed a culture model to study the role of non-parenchymal cells (NPCs) for inflammation-driven hepatocarcinogenesis. Rats were treated with the carcinogen N-nitrosomorpholine, which induced initiated hepatocytes expressing the marker placental glutathione-S-transferase (GSTp). After 21 days two preparations of hepatocytes were made: (i) conventional ones (Hep-conv) containing NPCs and (ii) hepatocytes purified of NPCs (Hep-pur). Initiated hepatocytes, being positive for GSTp (GSTp-pos) were present in both preparations and were cultured along with normal hepatocytes, being negative for GSTp (GSTp-neg). Under any culture condition DNA synthesis was approximately 4-fold higher in GSTp-pos than in GSTp-neg hepatocytes demonstrating the inherent growth advantage of the first stages of hepatocarcinogenesis. Hepatocytes showed approximately 3-fold lower rates of DNA synthesis in Hep-pur than in Hep-conv, which was elevated above Hep-conv levels by addition of NPC or NPC-supernatant. Pretreatment of NPCs with proinflammatory lipopolysaccharide (LPS) further increased DNA synthesis. Thus, NPCs release soluble growth stimulators. Next we investigated the effect of specific cytokines produced by NPCs. Tumour necrosis factor alpha and interleukin 6 barely altered DNA synthesis, whereas hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and the heparin-binding epidermal growth factor-like growth factor (HB-EGF) were potent inducers of DNA replication in both, GSTp-neg and GSTp-pos cells. In conclusion, DNA synthesis of hepatocytes is increased by factors released from NPCs, an effect augmented by LPS-stimulation. NPC-derived cytokines, such as KGF, HGF and HB-EGF, stimulate DNA synthesis preferentially in initiated hepatocytes, presumably resulting in tumour promotion. Similar mechanisms may contribute to carcinogenesis in human inflammatory liver diseases.

An Mcm10 Mutant Defective in ssDNA Binding Shows Defects in DNA Replication Initiation.

PubMed

Perez-Arnaiz, Patricia; Kaplan, Daniel L

2016-11-20

Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and a substantially decreased DNA replication. We also observed a substantially reduced replication protein A- chromatin immunoprecipitation signal at origins of replication, reduced levels of DDK-phosphorylated Mcm2, and diminished Go, Ichi, Ni, and San (GINS) association with Mcm2-7 in vivo. mcm5-bob1 bypasses the growth defect conferred by DDK-phosphodead Mcm2 in budding yeast. However, the growth defect observed by expressing mcm10-m2,3,4 is not bypassed by the mcm5-bob1 mutation. Furthermore, origin melting and GINS association with Mcm2-7 are substantially decreased for cells expressing mcm10-m2,3,4 in the mcm5-bob1 background. Thus, the origin melting and GINS-Mcm2-7 interaction defects we observed for mcm10-m2,3,4 are not explained by decreased Mcm2 phosphorylation by DDK, since the defects persist in an mcm5-bob1 background. These data suggest that DNA binding by Mcm10 is essential for the initiation of DNA replication. Copyright © 2016 Elsevier Ltd. All rights reserved.

N-methylpurine DNA glycosylase and DNA polymerase β modulate BER inhibitor potentiation of glioma cells to temozolomide

PubMed Central

Tang, Jiang-bo; Svilar, David; Trivedi, Ram N.; Wang, Xiao-hong; Goellner, Eva M.; Moore, Briana; Hamilton, Ronald L.; Banze, Lauren A.; Brown, Ashley R.; Sobol, Robert W.

2011-01-01

Temozolomide (TMZ) is the preferred chemotherapeutic agent in the treatment of glioma following surgical resection and/or radiation. Resistance to TMZ is attributed to efficient repair and/or tolerance of TMZ-induced DNA lesions. The majority of the TMZ-induced DNA base adducts are repaired by the base excision repair (BER) pathway and therefore modulation of this pathway can enhance drug sensitivity. N-methylpurine DNA glycosylase (MPG) initiates BER by removing TMZ-induced N3-methyladenine and N7-methylguanine base lesions, leaving abasic sites (AP sites) in DNA for further processing by BER. Using the human glioma cell lines LN428 and T98G, we report here that potentiation of TMZ via BER inhibition [methoxyamine (MX), the PARP inhibitors PJ34 and ABT-888 or depletion (knockdown) of PARG] is greatly enhanced by over-expression of the BER initiating enzyme MPG. We also show that methoxyamine-induced potentiation of TMZ in MPG expressing glioma cells is abrogated by elevated-expression of the rate-limiting BER enzyme DNA polymerase β (Polβ), suggesting that cells proficient for BER readily repair AP sites in the presence of MX. Further, depletion of Polβ increases PARP inhibitor-induced potentiation in the MPG over-expressing glioma cells, suggesting that expression of Polβ modulates the cytotoxic effect of combining increased repair initiation and BER inhibition. This study demonstrates that MPG overexpression, together with inhibition of BER, sensitizes glioma cells to the alkylating agent TMZ in a Polβ-dependent manner, suggesting that the expression level of both MPG and Polβ might be used to predict the effectiveness of MX and PARP-mediated potentiation of TMZ in cancer treatment. PMID:21377995

Role of non-equilibrium conformations on driven polymer translocation

NASA Astrophysics Data System (ADS)

Katkar, H. H.; Muthukumar, M.

2018-01-01

One of the major theoretical methods in understanding polymer translocation through a nanopore is the Fokker-Planck formalism based on the assumption of quasi-equilibrium of polymer conformations. The criterion for applicability of the quasi-equilibrium approximation for polymer translocation is that the average translocation time per Kuhn segment, ⟨τ⟩/NK, is longer than the relaxation time τ0 of the polymer. Toward an understanding of conditions that would satisfy this criterion, we have performed coarse-grained three dimensional Langevin dynamics and multi-particle collision dynamics simulations. We have studied the role of initial conformations of a polyelectrolyte chain (which were artificially generated with a flow field) on the kinetics of its translocation across a nanopore under the action of an externally applied transmembrane voltage V (in the absence of the initial flow field). Stretched (out-of-equilibrium) polyelectrolyte chain conformations are deliberately and systematically generated and used as initial conformations in translocation simulations. Independent simulations are performed to study the relaxation behavior of these stretched chains, and a comparison is made between the relaxation time scale and the mean translocation time (⟨τ⟩). For such artificially stretched initial states, ⟨τ⟩/NK < τ0, demonstrating the inapplicability of the quasi-equilibrium approximation. Nevertheless, we observe a scaling of ⟨τ⟩ ˜ 1/V over the entire range of chain stretching studied, in agreement with the predictions of the Fokker-Planck model. On the other hand, for realistic situations where the initial artificially imposed flow field is absent, a comparison of experimental data reported in the literature with the theory of polyelectrolyte dynamics reveals that the Zimm relaxation time (τZimm) is shorter than the mean translocation time for several polymers including single stranded DNA (ssDNA), double stranded DNA (dsDNA), and synthetic polymers. Even when these data are rescaled assuming a constant effective velocity of translocation, it is found that for flexible (ssDNA and synthetic) polymers with NK Kuhn segments, the condition ⟨τ⟩/NK < τZimm is satisfied. We predict that for flexible polymers such as ssDNA, a crossover from quasi-equilibrium to non-equilibrium behavior would occur at NK ˜ O(1000).

The Human Cytomegalovirus IE2 and UL112-113 Proteins Accumulate in Viral DNA Replication Compartments That Initiate from the Periphery of Promyelocytic Leukemia Protein-Associated Nuclear Bodies (PODs or ND10)

PubMed Central

Ahn, Jin-Hyun; Jang, Won-Jong; Hayward, Gary S.

1999-01-01

During human cytomegalovirus (HCMV) infection, the periphery of promyelocytic leukemia protein (PML)-associated nuclear bodies (also known as PML oncogenic domains [PODs] or ND10) are sites for both input viral genome deposition and immediate-early (IE) gene transcription. At very early times after infection, the IE1 protein localizes to and subsequently disrupts PODs, whereas the IE2 protein localizes within or adjacent to PODs. This process appears to be required for efficient viral gene expression and DNA replication. We have investigated the initiation of viral DNA replication compartment formation by studying the localization of viral IE proteins, DNA replication proteins, and the PML protein during productive infection. Localization of IE2 adjacent to PODs between 2 and 6 h after infection was confirmed by confocal microscopy of human fibroblasts (HF cells) infected with both wild-type HCMV(Towne) and with an IE1-deletion mutant HCMV(CR208) that fails to disrupt PODs. In HCMV(Towne)-infected HF cells at 24 to 48 h, IE2 also accumulated in newly formed viral DNA replication compartments containing the polymerase processivity factor (UL44), the single-stranded DNA binding protein (SSB; UL57), the UL112-113 accessory protein, and newly incorporated bromodeoxyuridine (BrdU). Double labeling of the HCMV(CR208)-infected HF cells demonstrated that formation of viral DNA replication compartments initiates within granular structures that bud from the periphery of some of the PODs and subsequently coalesce into larger structures that are flanked by PODs. In transient DNA transfection assays, both the N terminus (codons 136 to 290) and the C terminus (codons 379 to 579) of IE2 exon 5, but not the central region between them, were found to be necessary for both the punctate distribution of IE2 and its association with PODs. Like IE2, the UL112-113 accessory replication protein was also distributed in a POD-associated pattern in both DNA-transfected and virus-infected cells beginning at 6 h. Furthermore, when all six replication core machinery proteins (polymerase complex, SSB, and helicase-primase complex) were expressed together in the presence of UL112-113, they also accumulated at POD-associated sites, suggesting that the UL112-113 protein (but not IE2) may play a role in recruitment of viral replication fork proteins into the periphery of PODs. These results show that (i) subsequent to accumulating at the periphery of PODs, IE2 is incorporated together with the core proteins into viral DNA replication compartments that initiate from the periphery of PODs and then grow to fill the space between groups of PODs, and (ii) the UL112-113 protein appears to have a key role in assembling and recruiting the core replication machinery proteins in the initial stages of viral replication compartment formation. PMID:10559364

A Demonstration Marine Biodiversity Observation Network (MBON): Understanding Marine Life and its Role in Maintaining Ecosystem Services

NASA Astrophysics Data System (ADS)

Muller-Karger, F. E.; Iken, K.; Miller, R. J.; Duffy, J. E.; Chavez, F.; Montes, E.

2016-02-01

The U.S. Federal government (NOAA, NASA, BOEM, and the Smithsonian Institution), academic researchers, and private partners are laying the foundation for a Marine Biodiversity Observation Network (MBON). The goals of the network are to: 1) Observe and understand life, from microbes to whales, in different coastal and continental shelf habitats; 2) Define an efficient set of observations required for implementing a useful MBON; 3) Develop technology for biodiversity assessments including emerging environmental DNA (eDNA), remote sensing, and image analysis methods to coordinate with classical sampling; 4) Integrate and synthesize information in coordination with the Integrated Ocean Observing System (IOOS), the international Group on Earth Observations Biodiversity Observation Network(GEO BON), and the Ocean Biogeographic Information System (OBIS) sponsored by UNESCO's Intergovernmental Oceanographic Commission (IOC); and 5) Understand the linkages between marine biodiversity, ecosystem processes, and the social-economic context of a region. Pilot projects have been implemented within three NOAA National Marine Sanctuaries (Florida Keys, Monterey Bay, and Channel Islands), the wider Santa Barbara Channel, in the Chukchi Sea, and through the Smithsonian's Tennenbaum Marine Observatories Network (TMON) at several sites in the U.S. and collaborating countries. Together, these MBON sites encompass a wide range of marine environments, including deep sea, continental shelves, and coastal habitats including estuaries, wetlands, and coral reefs. The present MBON partners are open to growth of the MBON through additional collaborations. Given these initiatives, GEO BON is proposing an MBON effort that spans from pole to pole, with a pathfinder effort among countries in the Americas. By specializing in coastal ecosystems—where marine biodiversity and people are concentrated and interact most—the MBON and TMON initiatives aim to provide policymakers with the science to support innovative solutions and advance management and protection of our oceans. The initiative contributes to addressing U.N. Sustainable Development Goal 14 to conserve and sustainably use marine resources. The MBON will facilitate and enable regional biodiversity assessments.

Toward DNA-based Security Circuitry: First Step - Random Number Generation.

PubMed

Bogard, Christy M; Arazi, Benjamin; Rouchka, Eric C

2008-08-10

DNA-based circuit design is an area of research in which traditional silicon-based technologies are replaced by naturally occurring phenomena taken from biochemistry and molecular biology. Our team investigates the implications of DNA-based circuit design in serving security applications. As an initial step we develop a random number generation circuitry. A novel prototype schema employs solid-phase synthesis of oligonucleotides for random construction of DNA sequences. Temporary storage and retrieval is achieved through plasmid vectors.

Integration of single-molecule detection with magnetic separation for multiplexed detection of DNA glycosylases.

PubMed

Li, Chen-Chen; Zhang, Yan; Tang, Bo; Zhang, Chun-Yang

2018-06-05

We combine single-molecule detection with magnetic separation for simultaneous measurement of human 8-oxoG DNA glycosylase 1 (hOGG1) and uracil DNA glycosylase (UDG) based on excision repair-initiated endonuclease IV (Endo IV)-assisted signal amplification. This method can sensitively detect multiple DNA glycosylases, and it can be further applied for the simultaneous measurement of enzyme kinetic parameters and screening of both hOGG1 and UDG inhibitors.

Ectopic Integration of Transforming DNA Is Rare among Neurospora Transformants Selected for Gene Replacement

PubMed Central

Miao, VPW.; Rountree, M. R.; Selker, E. U.

1995-01-01

In a variety of organisms, DNA-mediated transformation experiments commonly produce transformants with multiple copies of the transforming DNA, including both selected and unselected molecules. Such ``cotransformants'' are much more common than expected from the individual transformation frequencies, suggesting that subpopulations of cells, or nuclei, are particularly competent for transformation. We found that Neurospora crassa transformants selected for gene replacement at the am gene had not efficiently incorporated additional DNA, suggesting that nuclei that undergo transformation by homologous recombination are not highly competent at integration of DNA by illegitimate recombination. Spheroplasts were treated with DNA fragments homologous to am and with an Escherichia coli hph plasmid. Transformants were initially selected for hph (hygromycin(R)), allowed to conidiate to generate homokaryons and then selected for either Am(-) (gene replacements) or hph. Surprisingly, most am replacement strains were hygromycin(S) (124/140) and carried no extraneous DNA (116/140). Most transformants selected for hph also had ectopic copies of am DNA and/or multiple copies of hph sequences (32/35), generally at multiple sites, confirming that efficient cotransformation could occur. To test the implication that cotransformation involving gene replacement and ectopic integration is rare, we compared the yields of am replacement strains with or without prior selection for hph. The initial selection did not appreciably help (or hinder) recovery of strains with replacements. PMID:7789758

Human ISWI complexes are targeted by SMARCA5 ATPase and SLIDE domains to help resolve lesion-stalled transcription

PubMed Central

Aydin, Özge Z.; Marteijn, Jurgen A.; Ribeiro-Silva, Cristina; Rodríguez López, Aida; Wijgers, Nils; Smeenk, Godelieve; van Attikum, Haico; Poot, Raymond A.; Vermeulen, Wim; Lans, Hannes

2014-01-01

Chromatin compaction of deoxyribonucleic acid (DNA) presents a major challenge to the detection and removal of DNA damage. Helix-distorting DNA lesions that block transcription are specifically repaired by transcription-coupled nucleotide excision repair, which is initiated by binding of the CSB protein to lesion-stalled RNA polymerase II. Using live cell imaging, we identify a novel function for two distinct mammalian ISWI adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in resolving lesion-stalled transcription. Human ISWI isoform SMARCA5/SNF2H and its binding partners ACF1 and WSTF are rapidly recruited to UV-C induced DNA damage to specifically facilitate CSB binding and to promote transcription recovery. SMARCA5 targeting to UV-C damage depends on transcription and histone modifications and requires functional SWI2/SNF2-ATPase and SLIDE domains. After initial recruitment to UV damage, SMARCA5 re-localizes away from the center of DNA damage, requiring its HAND domain. Our studies support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis-dependent scanning and proofreading mechanism, highlighting how SWI2/SNF2 chromatin remodelers identify and bind nucleosomes containing damaged DNA. PMID:24990377

Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC

PubMed Central

Kong, Daochun; Coleman, Thomas R.; DePamphilis, Melvin L.

2003-01-01

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S.pombe. PMID:12840006

The rationale for liquid biopsy in colorectal cancer: a focus on circulating tumor cells.

PubMed

Gazzaniga, Paola; Raimondi, Cristina; Nicolazzo, Chiara; Carletti, Raffaella; di Gioia, Cira; Gradilone, Angela; Cortesi, Enrico

2015-01-01

Capturing circulating tumor cells (CTCs) and/or circulating tumor DNA from blood, which represents a precious source of biological material derived from both primary and metastatic tumors, has been named a 'liquid biopsy'. While the circulating tumor DNA might be more representative of the bulk of the metastatic tumor, CTCs are thought to reflect more of the metastases-initiating cells. Consequently, a liquid biopsy made of tumor cells and tumor DNA that is able to track cancer evolution, as a fingerprint of the patient's individual tumor, and is easy to perform at every stage of the disease course, sounds attractive. This article mainly focuses on the applications of CTCs to track tumor dynamics in real time using colorectal cancer as a model system. The analysis of viable CTCs at DNA, RNA and protein levels, as well as their expansion in vitro, may allow deep investigation of the features of metastases-initiating cells.

Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

NASA Astrophysics Data System (ADS)

Senavirathne, Gayan; Bertram, Jeffrey G.; Jaszczur, Malgorzata; Chaurasiya, Kathy R.; Pham, Phuong; Mak, Chi H.; Goodman, Myron F.; Rueda, David

2015-12-01

Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ~5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.

Two subunits of human ORC are dispensable for DNA replication and proliferation.

PubMed

Shibata, Etsuko; Kiran, Manjari; Shibata, Yoshiyuki; Singh, Samarendra; Kiran, Shashi; Dutta, Anindya

2016-12-01

The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1 . The ORC1 or ORC2 -depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication.

Correlation of Particle Traversals with Clonogenic Survival Using Cell-Fluorescent Ion Track Hybrid Detector.

PubMed

Dokic, Ivana; Niklas, Martin; Zimmermann, Ferdinand; Mairani, Andrea; Seidel, Philipp; Krunic, Damir; Jäkel, Oliver; Debus, Jürgen; Greilich, Steffen; Abdollahi, Amir

2015-01-01

Development of novel approaches linking the physical characteristics of particles with biological responses are of high relevance for the field of particle therapy. In radiobiology, the clonogenic survival of cells is considered the gold standard assay for the assessment of cellular sensitivity to ionizing radiation. Toward further development of next generation biodosimeters in particle therapy, cell-fluorescent ion track hybrid detector (Cell-FIT-HD) was recently engineered by our group and successfully employed to study physical particle track information in correlation with irradiation-induced DNA damage in cell nuclei. In this work, we investigated the feasibility of Cell-FIT-HD as a tool to study the effects of clinical beams on cellular clonogenic survival. Tumor cells were grown on the fluorescent nuclear track detector as cell culture, mimicking the standard procedures for clonogenic assay. Cell-FIT-HD was used to detect the spatial distribution of particle tracks within colony-initiating cells. The physical data were associated with radiation-induced foci as surrogates for DNA double-strand breaks, the hallmark of radiation-induced cell lethality. Long-term cell fate was monitored to determine the ability of cells to form colonies. We report the first successful detection of particle traversal within colony-initiating cells at subcellular resolution using Cell-FIT-HD.

Interplay between CedA, rpoB and double stranded DNA: A step towards understanding CedA mediated cell division in E. coli.

PubMed

Sharma, Pankaj; Tomar, Anil Kumar; Kundu, Bishwajit

2018-02-01

Cell division is compromised in DnaAcos mutant E. coli cells due to chromosome over-replication. In these cells, CedA acts as a regulatory protein and initiates cell division by a hitherto unknown mechanism. CedA, a double stranded DNA binding protein, interacts with various subunits of RNA polymerase complex, including rpoB. To reveal how this concert between CedA, rpoB and DNA brings about cell division in E. coli, we performed biophysical and in silico analysis and obtained mechanistic insights. Interaction between CedA and rpoB was shown by circular dichroism spectrometry and in silico docking experiments. Further, CedA and rpoB were allowed to interact individually to a selected DNA and their binding was monitored by fluorescence spectroscopy. The binding constants of these interactions as determined by BioLayer Interferometry clearly show that rpoB binds to DNA with higher affinity (K D2 =<1.0E-12M) as compared to CedA (K D2 =9.58E-09M). These findings were supported by docking analysis where 12 intermolecular H-bonds were formed in rpoB-DNA complex as compared to 4 in CedA-DNA complex. Based on our data we propose that in E. coli cells chromosome over-replication signals CedA to recruit rpoB to specific DNA site(s), which initiates transcription of cell division regulatory elements. Copyright © 2017 Elsevier B.V. All rights reserved.

Promoter-proximal rDNA terminator augments initiation by preventing disruption of the stable transcription complex caused by polymerase read-in

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

Henderson, S.L.; Ryan, K.; Sollner-Webb, B.

1989-02-01

We have examined the mechanism by which transcriptional initiation at the mouse rDNA promoter is augmented by the RNA polymerase I terminator element that resides just upstream of it. Using templates in which terminator elements are instead positioned at the opposite side of the plasmid rather than proximal to the promoter, or conditions where transcription is terminated elsewhere in the plasmid by UV-induced lesions, we show that the terminator's stimulatory effect is not position dependent. Mouse terminator elements therefore do not stimulate via the previously postulated 'read-through enhancement' model in which terminated polymerases are handed off to an adjacent promotermore » in a concerted reaction. The position independence and orientation dependence of the terminator also makes it unlikely that the terminator functions as a promoter element or as an enhancer. Instead, terminators serve to augment initiation by preventing polymerases from reading completely around the plasmid and through the promoter from upstream, an event which we show interferes with subsequent rounds of initiation. Notably, this transcriptional interference arises because polymerase passage across a promoter disrupts the otherwise stable transcription complex, specifically releasing the bound transcription factor D. These liberated D molecules can then bind to other templates and activate their expression. The rDNA transcriptional interference is not due to a steric impediment to the binding of new polymerase molecules, and it does not similarly liberate the initiation-competent polymerase (factor C). These studies have also convincingly demonstrated that multiple rounds of transcription are obtained from rDNA template molecules in vitro.« less

DNA hybridization kinetics: zippering, internal displacement and sequence dependence.

PubMed

Ouldridge, Thomas E; Sulc, Petr; Romano, Flavio; Doye, Jonathan P K; Louis, Ard A

2013-10-01

Although the thermodynamics of DNA hybridization is generally well established, the kinetics of this classic transition is less well understood. Providing such understanding has new urgency because DNA nanotechnology often depends critically on binding rates. Here, we explore DNA oligomer hybridization kinetics using a coarse-grained model. Strand association proceeds through a complex set of intermediate states, with successful binding events initiated by a few metastable base-pairing interactions, followed by zippering of the remaining bonds. But despite reasonably strong interstrand interactions, initial contacts frequently dissociate because typical configurations in which they form differ from typical states of similar enthalpy in the double-stranded equilibrium ensemble. Initial contacts must be stabilized by two or three base pairs before full zippering is likely, resulting in negative effective activation enthalpies. Non-Arrhenius behavior arises because the number of base pairs required for nucleation increases with temperature. In addition, we observe two alternative pathways-pseudoknot and inchworm internal displacement-through which misaligned duplexes can rearrange to form duplexes. These pathways accelerate hybridization. Our results explain why experimentally observed association rates of GC-rich oligomers are higher than rates of AT- rich equivalents, and more generally demonstrate how association rates can be modulated by sequence choice.

DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle.

PubMed

Demarre, Gaëlle; Chattoraj, Dhruba K

2010-05-06

DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated) sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII) of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.

Strong minor groove base conservation in sequence logos implies DNA distortion or base flipping during replication and transcription initiation | Center for Cancer Research

Cancer.gov

Dubbed "Tom's T" by Dhruba Chattoraj, the unusually conserved thymine at position +7 in bacteriophage P1 plasmid RepA DNA binding sites rises above repressor and acceptor sequence logos. The T appears to represent base flipping prior to helix opening in this DNA replication initation protein.

Oxidation of DNA bases, deoxyribonucleosides and homopolymers by peroxyl radicals.

PubMed Central

Simandan, T; Sun, J; Dix, T A

1998-01-01

DNA base oxidation is considered to be a key event associated with disease initiation and progression in humans. Peroxyl radicals (ROO. ) are important oxidants found in cells whose ability to react with the DNA bases has not been characterized extensively. In this paper, the products resulting from ROO. oxidation of the DNA bases are determined by gas chromatography/MS in comparison with authentic standards. ROO. radicals oxidize adenine and guanine to their 8-hydroxy derivatives, which are considered biomarkers of hydroxyl radical (HO.) oxidations in cells. ROO. radicals also oxidize adenine to its hydroxylamine, a previously unidentified product. ROO. radicals oxidize cytosine and thymine to the monohydroxy and dihydroxy derivatives that are formed by oxidative damage in cells. Identical ROO. oxidation profiles are observed for each base when exposed as deoxyribonucleosides, monohomopolymers and base-paired dihomopolymers. These results have significance for the development, utilization and interpretation of DNA base-derived biomarkers of oxidative damage associated with disease initiation and propagation, and support the idea that the mutagenic potential of N-oxidized bases, when generated in cellular DNA, will require careful evaluation. Adenine hydroxylamine is proposed as a specific molecular probe for the activity of ROO. in cellular systems. PMID:9761719

Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway

PubMed Central

Llanos, Susana; Serrano, Manuel

2013-01-01

Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 by a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 constitutes a mechanistic link between DNA damage and the ribosomal stress pathway, and is a relevant contributing signaling pathway for the activation of p53 in response to DNA damage. PMID:20935493

The Specificity and Flexibility of L1 Reverse Transcription Priming at Imperfect T-Tracts

PubMed Central

Viollet, Sébastien; Mir, Ashfaq Ali; Gabus, Caroline; Darlix, Jean-Luc; Cristofari, Gaël

2013-01-01

L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5′-TTTT/A-3′ sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether—and to which degree—the liberated 3′-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3′ end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3′ overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3′ end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome. PMID:23675310

Most of the extant mtDNA boundaries in South and Southwest Asia were likely shaped during the initial settlement of Eurasia by anatomically modern humans

PubMed Central

Metspalu, Mait; Kivisild, Toomas; Metspalu, Ene; Parik, Jüri; Hudjashov, Georgi; Kaldma, Katrin; Serk, Piia; Karmin, Monika; Behar, Doron M; Gilbert, M Thomas P; Endicott, Phillip; Mastana, Sarabjit; Papiha, Surinder S; Skorecki, Karl; Torroni, Antonio; Villems, Richard

2004-01-01

Background Recent advances in the understanding of the maternal and paternal heritage of south and southwest Asian populations have highlighted their role in the colonization of Eurasia by anatomically modern humans. Further understanding requires a deeper insight into the topology of the branches of the Indian mtDNA phylogenetic tree, which should be contextualized within the phylogeography of the neighboring regional mtDNA variation. Accordingly, we have analyzed mtDNA control and coding region variation in 796 Indian (including both tribal and caste populations from different parts of India) and 436 Iranian mtDNAs. The results were integrated and analyzed together with published data from South, Southeast Asia and West Eurasia. Results Four new Indian-specific haplogroup M sub-clades were defined. These, in combination with two previously described haplogroups, encompass approximately one third of the haplogroup M mtDNAs in India. Their phylogeography and spread among different linguistic phyla and social strata was investigated in detail. Furthermore, the analysis of the Iranian mtDNA pool revealed patterns of limited reciprocal gene flow between Iran and the Indian sub-continent and allowed the identification of different assemblies of shared mtDNA sub-clades. Conclusions Since the initial peopling of South and West Asia by anatomically modern humans, when this region may well have provided the initial settlers who colonized much of the rest of Eurasia, the gene flow in and out of India of the maternally transmitted mtDNA has been surprisingly limited. Specifically, our analysis of the mtDNA haplogroups, which are shared between Indian and Iranian populations and exhibit coalescence ages corresponding to around the early Upper Paleolithic, indicates that they are present in India largely as Indian-specific sub-lineages. In contrast, other ancient Indian-specific variants of M and R are very rare outside the sub-continent. PMID:15339343

Push back to respond better: regulatory inhibition of the DNA double-strand break response.

PubMed

Panier, Stephanie; Durocher, Daniel

2013-10-01

Single DNA lesions such as DNA double-strand breaks (DSBs) can cause cell death or trigger genome rearrangements that have oncogenic potential, and so the pathways that mend and signal DNA damage must be highly sensitive but, at the same time, selective and reversible. When initiated, boundaries must be set to restrict the DSB response to the site of the lesion. The integration of positive and, crucially, negative control points involving post-translational modifications such as phosphorylation, ubiquitylation and acetylation is key for building fast, effective responses to DNA damage and for mitigating the impact of DNA lesions on genome integrity.

A critical role for topoisomerase IIb and DNA double strand breaks in transcription

PubMed Central

Calderwood, Stuart K.

2016-01-01

ABSTRACT Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb. PMID:27100743

A critical role for topoisomerase IIb and DNA double strand breaks in transcription.

PubMed

Calderwood, Stuart K

2016-05-26

Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb.

SU-E-T-565: RAdiation Resistance of Cancer CElls Using GEANT4 DNA: RACE

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

Perrot, Y; Payno, H; Delage, E

2014-06-01

Purpose: The objective of the RACE project is to develop a comparison between Monte Carlo simulation using the Geant4-DNA toolkit and measurements of radiation damage on 3D melanoma and chondrosarcoma culture cells coupled with gadolinium nanoparticles. We currently expose the status of the developments regarding simulations. Methods: Monte Carlo studies are driven using the Geant4 toolkit and the Geant4-DNA extension. In order to model the geometry of a cell population, the opensource CPOP++ program is being developed for the geometrical representation of 3D cell populations including a specific cell mesh coupled with a multi-agent system. Each cell includes cytoplasm andmore » nucleus. The correct modeling of the cell population has been validated with confocal microscopy images of spheroids. The Geant4 Livermore physics models are used to simulate the interactions of a 250 keV X-ray beam and the production of secondaries from gadolinium nanoparticles supposed to be fixed on the cell membranes. Geant4-DNA processes are used to simulate the interactions of charged particles with the cells. An atomistic description of the DNA molecule, from PDB (Protein Data Bank) files, is provided by the so-called PDB4DNA Geant4 user application we developed to score energy depositions in DNA base pairs and sugar-phosphate groups. Results: At the microscopic level, our simulations enable assessing microscopic energy distribution in each cell compartment of a realistic 3D cell population. Dose enhancement factors due to the presence of gadolinium nanoparticles can be estimated. At the nanometer scale, direct damages on nuclear DNA are also estimated. Conclusion: We successfully simulated the impact of direct radiations on a realistic 3D cell population model compatible with microdosimetry calculations using the Geant4-DNA toolkit. Upcoming validation and the future integration of the radiochemistry module of Geant4-DNA will propose to correlate clusters of ionizations with in vitro experiments. All those developments will be released publicly. This work was supported by grants from Plan Cancer 2009-2013 French national initiative managed by INSERM (Institut National de la Sante et de la Recherche Medicale)« less

Inclusion probability for DNA mixtures is a subjective one-sided match statistic unrelated to identification information

PubMed Central

Perlin, Mark William

2015-01-01

Background: DNA mixtures of two or more people are a common type of forensic crime scene evidence. A match statistic that connects the evidence to a criminal defendant is usually needed for court. Jurors rely on this strength of match to help decide guilt or innocence. However, the reliability of unsophisticated match statistics for DNA mixtures has been questioned. Materials and Methods: The most prevalent match statistic for DNA mixtures is the combined probability of inclusion (CPI), used by crime labs for over 15 years. When testing 13 short tandem repeat (STR) genetic loci, the CPI-1 value is typically around a million, regardless of DNA mixture composition. However, actual identification information, as measured by a likelihood ratio (LR), spans a much broader range. This study examined probability of inclusion (PI) mixture statistics for 517 locus experiments drawn from 16 reported cases and compared them with LR locus information calculated independently on the same data. The log(PI-1) values were examined and compared with corresponding log(LR) values. Results: The LR and CPI methods were compared in case examples of false inclusion, false exclusion, a homicide, and criminal justice outcomes. Statistical analysis of crime laboratory STR data shows that inclusion match statistics exhibit a truncated normal distribution having zero center, with little correlation to actual identification information. By the law of large numbers (LLN), CPI-1 increases with the number of tested genetic loci, regardless of DNA mixture composition or match information. These statistical findings explain why CPI is relatively constant, with implications for DNA policy, criminal justice, cost of crime, and crime prevention. Conclusions: Forensic crime laboratories have generated CPI statistics on hundreds of thousands of DNA mixture evidence items. However, this commonly used match statistic behaves like a random generator of inclusionary values, following the LLN rather than measuring identification information. A quantitative CPI number adds little meaningful information beyond the analyst's initial qualitative assessment that a person's DNA is included in a mixture. Statistical methods for reporting on DNA mixture evidence should be scientifically validated before they are relied upon by criminal justice. PMID:26605124

Inclusion probability for DNA mixtures is a subjective one-sided match statistic unrelated to identification information.

PubMed

Perlin, Mark William

2015-01-01

DNA mixtures of two or more people are a common type of forensic crime scene evidence. A match statistic that connects the evidence to a criminal defendant is usually needed for court. Jurors rely on this strength of match to help decide guilt or innocence. However, the reliability of unsophisticated match statistics for DNA mixtures has been questioned. The most prevalent match statistic for DNA mixtures is the combined probability of inclusion (CPI), used by crime labs for over 15 years. When testing 13 short tandem repeat (STR) genetic loci, the CPI(-1) value is typically around a million, regardless of DNA mixture composition. However, actual identification information, as measured by a likelihood ratio (LR), spans a much broader range. This study examined probability of inclusion (PI) mixture statistics for 517 locus experiments drawn from 16 reported cases and compared them with LR locus information calculated independently on the same data. The log(PI(-1)) values were examined and compared with corresponding log(LR) values. The LR and CPI methods were compared in case examples of false inclusion, false exclusion, a homicide, and criminal justice outcomes. Statistical analysis of crime laboratory STR data shows that inclusion match statistics exhibit a truncated normal distribution having zero center, with little correlation to actual identification information. By the law of large numbers (LLN), CPI(-1) increases with the number of tested genetic loci, regardless of DNA mixture composition or match information. These statistical findings explain why CPI is relatively constant, with implications for DNA policy, criminal justice, cost of crime, and crime prevention. Forensic crime laboratories have generated CPI statistics on hundreds of thousands of DNA mixture evidence items. However, this commonly used match statistic behaves like a random generator of inclusionary values, following the LLN rather than measuring identification information. A quantitative CPI number adds little meaningful information beyond the analyst's initial qualitative assessment that a person's DNA is included in a mixture. Statistical methods for reporting on DNA mixture evidence should be scientifically validated before they are relied upon by criminal justice.

Cigarette smoke-induced DNA damage and repair detected by the comet assay in HPV-transformed cervical cells.

PubMed

Moktar, Afsoon; Ravoori, Srivani; Vadhanam, Manicka V; Gairola, C Gary; Gupta, Ramesh C

2009-12-01

Human papillomavirus (HPV) is the causative factor in the development and progression of cervical cancers in >97% of the cases, although insufficient. Epidemiological studies suggest an elevated risk of cervical cancer for cigarette smokers; therefore, we examined cigarette smoke-induced DNA damage and repair in HPV16-transformed human ectocervical cells (ECT1/E6 E7). Cells were treated with cigarette smoke condensate (CSC) for 72 h to assess the formation of single- and double-strand DNA breaks, measured by alkaline and neutral single cell gel electrophoresis assays, respectively. The mean tail length of cells with single-strand breaks was increased by 1.8-, 2.7- and 3.7-fold (p<0.001) after treatment with 4, 8 and 12 microg/ml CSC, respectively. The tail length with double-strand breaks was also increased dose-dependently. These results were further supported by measurement of the mean tail moment: the increase in both single- and double-strand breaks were much more pronounced with increasing concentration of CSC, by up to 23.5-fold (p<0.0001 for both assays). To examine the DNA repair, cells were treated with CSC for 72 h, followed by CSC withdrawal and re-incubation of the cells with fresh medium for 24, 48, or 72 h. Both single- and double-strand DNA breaks were removed during the initial 24 h but no further removal of the damage was observed. Up to 80% of residual single- and double-strand DNA breaks (p<0.05) were found to persist at all CSC concentrations examined. Ellagic acid, a known antioxidant and free-radical scavenger, was found to significantly inhibit DNA breaks induced by CSC. Thus, free radicals may be a plausible source of CSC-induced DNA damage. These data show that CSC-mediated DNA strand breaks are highly persistent, and suggest that persistence of cigarette smoke-associated DNA damage in the presence of HPV infection may lead to increased mutations in cervical cells and ultimately higher cancer risk.

Demonstration of GTG as an endogenous initiation codon for a human mRNA transcript revealed by molecular cloning of the serpin endopin 2B.

PubMed

Hwang, Shin-Rong; Garza, Christina Z; Wegrzyn, Jill; Hook, Vivian Y H

2004-08-16

This study demonstrates utilization of the novel GTG initiation codon for translation of a human mRNA transcript that encodes the serpin endopin 2B, a protease inhibitor. Molecular cloning revealed the nucleotide sequence of the human endopin 2B cDNA. Its deduced primary sequence shows high homology to bovine endopin 2A that possesses cross-class protease inhibition of elastase and papain. Notably, the human endopin 2B cDNA sequence revealed GTG as the predicted translation initiation codon; the predicted translation product of 46 kDa endopin 2B was produced by in vitro translation of 35S-endopin 2B with mammalian (rabbit) protein translation components. Importantly, bioinformatic studies demonstrated the presence of the entire human endopin 2B cDNA sequence with GTG as initiation codon within the human genome on chromosome 14. Further evidence for GTG as a functional initiation codon was illustrated by GTG-mediated in vitro translation of the heterologous protein EGFP, and by GTG-mediated expression of EGFP in mammalian PC12 cells. Mutagenesis of GTG to GTC resulted in the absence of EGFP expression in PC12 cells, indicating the function of GTG as an initiation codon. In addition, it was apparent that the GTG initiation codon produces lower levels of translated protein compared to ATG as initiation codon. Significantly, GTG-mediated translation of endopin 2B demonstrates a functional human gene product not previously predicted from initial analyses of the human genome. Further analyses based on GTG as an alternative initiation codon may predict new candidate genes of the human genome.

Role of indirect readout mechanism in TATA box binding protein-DNA interaction.

PubMed

Mondal, Manas; Choudhury, Devapriya; Chakrabarti, Jaydeb; Bhattacharyya, Dhananjay

2015-03-01

Gene expression generally initiates from recognition of TATA-box binding protein (TBP) to the minor groove of DNA of TATA box sequence where the DNA structure is significantly different from B-DNA. We have carried out molecular dynamics simulation studies of TBP-DNA system to understand how the DNA structure alters for efficient binding. We observed rigid nature of the protein while the DNA of TATA box sequence has an inherent flexibility in terms of bending and minor groove widening. The bending analysis of the free DNA and the TBP bound DNA systems indicate presence of some similar structures. Principal coordinate ordination analysis also indicates some structural features of the protein bound and free DNA are similar. Thus we suggest that the DNA of TATA box sequence regularly oscillates between several alternate structures and the one suitable for TBP binding is induced further by the protein for proper complex formation.

Assessment of DNA replication in central nervous system by Laser Scanning Cytometry

NASA Astrophysics Data System (ADS)

Lenz, Dominik; Mosch, Birgit; Bocsi, Jozsef; Arendt, Thomas; Tárnok, Attila

2004-07-01

μIn neurons of patients with Alzheimers's disease (AD) signs of cell cycle re-entry as well as polyploidy have been reported1, 2, indicating that the entire or a part of the genome of the neurons is duplicated before its death but mitosis is not initiated so that the cellular DNA content remains tetraploid. It was concluded, that this imbalance is the direct cause of the neuronal loss in AD3. Manual counting of polyploidal cells is possible but time consuming and possibly statistically insufficient. The aim of this study was to develop an automated method that detects the neuronal DNA content abnormalities with Laser Scanning Cytometry (LSC).Frozen sections of formalin-fixed brain tissue of AD patients and control subjects were labelled with anti-cyclin B and anti-NeuN antibodies. Immunolabelling was performed using Cy5- and Cy2-conjugated secondary antibodies and biotin streptavidin or tyramid signal amplification. In the end sections of 20m thickness were incubated with propidium iodide (PI) (50μg/ml) and covered on slides. For analysis by the LSC PI was used as trigger. Cells identified as neurons by NeuN expression were analyzed for cyclin B expression. Per specimen data of at least 10,000 neurons were acquired. In the frozen brain sections an automated quantification of the amount of nuclear DNA is possible with LSC. The DNA ploidy as well as the cell cycle distribution can be analyzed. A high number of neurons can be scanned and the duration of measuring is shorter than a manual examination. The amount of DNA is sufficiently represented by the PI fluorescence to be able to distinguish between eu- and polyploid neurons.

Adverse Outcome Pathway (AOP) for a Mutagenic Mode of ...

EPA Pesticide Factsheets

AOPs provide a framework to describe a sequence of measureable key events (KEs), beginning with a molecular initiating event (MIE), followed by a series of identified KEs linked to one another by KE Relationships (KERs), all anchored by a specific adverse outcome (AO). Each KE/KER is supported by data and evaluated against criteria to assess biological plausibility, weight/strength of evidence, specificity, and confidence. AOPs offer an approach to using toxicological data and predictive modeling to actualize use of mode-of-action (MOA) for such purposes as read-across, integrated approaches to testing & assessment, and risk assessment. Different applications will depend partly on the scientific confidence underpinning each KE/KER and the overall AOP. An OECD program encourages development of AOPs, with a wiki that allows for public review & comment to foster collaborations and broaden understanding & application of AOPs. Developing an AOP for a mutagenic MOA for cancer as a case study in the OECD program lays a path towards determination of such an MOA and its use in chemical assessment programs. Aflatoxin B1 (AFB1), with ubiquitous exposure and a rich database, was selected for this case study. AFB1 has been determined to induce HCC via a DNA-reactive MOA in many species, including humans. The sequential KEs identified for AFB1 are as follows: pre-MIE: Hepatic metabolic activation; MIE: Formation of a pro-mutagenic DNA adduct (N7-AFB1-guanine or AFB1-FAPy); KE

Single Molecule Analysis of Replicated DNA Reveals the Usage of Multiple KSHV Genome Regions for Latent Replication

PubMed Central

Verma, Subhash C.; Lu, Jie; Cai, Qiliang; Kosiyatrakul, Settapong; McDowell, Maria E.; Schildkraut, Carl L.; Robertson, Erle S.

2011-01-01

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences. PMID:22072974

Genomic fragmentation and extrachromosomal telomeric repeats impact assessment of telomere length in human spermatozoa: quantitative experiments and systematic review.

PubMed

Kurjanowicz, P; Moskovtsev, S; Librach, C

2017-11-01

Can differences in DNA isolation alter assessment of sperm telomere length (spTL) and do they account for conflicting results in the literature on spTL and male fertility? DNA isolation methods preferentially include or exclude short, extrachromosomal (EC) telomere-specific sequences that alter spTL measurements, and are responsible for a proportion of the disparity observed between investigations. The relationship between spTL and male fertility has become an active area of research. The results across investigations, however, have been discordant, generating a need to critically evaluate the existing body of knowledge to guide future investigations. Quantitative experiments determined the effect of DNA isolation on the integrity of sperm DNA and measures of spTL, while a systematic analysis of the current literature evaluated the effect of DNA isolation and study design on experimental outcomes. Two DNA isolation methods were compared: Genomic Tips which isolate 'High Molecular Weight' (HMW) DNA exclusively, and QIAamp® DNA Mini which isolates 'Total' genomic DNA irrespective of size. DNA quality was assessed via field inversion gel electrophoresis (FIGE) and spTL was measured via terminal restriction fragment analysis. In addition, major databases in medicine, health and the life sciences were subject to a targeted search, and results were independently screened according to defined exclusion/inclusion criterion. Findings from primary articles were analyzed for concordance and study designs were compared across six moderator variables (sample size, participant age, fertility status, semen fraction, telomere population and type of analysis). HMW DNA spTL was significantly longer than spTL measured from total DNA (P < 0.01), indicating that Total DNA contained short, EC telomeric repeats that shifted downstream assessment towards shorter spTL. HMW DNA spTL reflected the length of intact, chromosomal telomeres. Major findings on spTL showed the greatest concordance amongst studies that implemented HMW DNA isolation prior to spTL assessment. Studies that utilized Total DNA varied in concordance, but outcomes were similar if (i) a comparative analysis was applied or (ii) a sample size threshold of 81 was achieved for correlative analysis. Chromosomal and EC telomeric DNA were distinguished based on outcomes of HMW DNA isolation and size. Further experiments are required to determine the nature and function of these two types of telomeric sequences. This study reveals a dramatic impact of upstream DNA processing and study design on measurements of spTL, which accounts for conflicting results in the literature. Future assessments of spTL should incorporate independent detection of chromosomal and EC telomeric DNA and specific experimental planning. This study was funded by CReATe Fertility Centre, Toronto, Ontario, Canada. The authors have declared no conflict of interest. N/A. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Field calibration of blowfly-derived DNA against traditional methods for assessing mammal diversity in tropical forests.

PubMed

Lee, Ping-Shin; Gan, Han Ming; Clements, Gopalasamy Reuben; Wilson, John-James

2016-11-01

Mammal diversity assessments based on DNA derived from invertebrates have been suggested as alternatives to assessments based on traditional methods; however, no study has field-tested both approaches simultaneously. In Peninsular Malaysia, we calibrated the performance of mammal DNA derived from blowflies (Diptera: Calliphoridae) against traditional methods used to detect species. We first compared five methods (cage trapping, mist netting, hair trapping, scat collection, and blowfly-derived DNA) in a forest reserve with no recent reports of megafauna. Blowfly-derived DNA and mist netting detected the joint highest number of species (n = 6). Only one species was detected by multiple methods. Compared to the other methods, blowfly-derived DNA detected both volant and non-volant species. In another forest reserve, rich in megafauna, we calibrated blowfly-derived DNA against camera traps. Blowfly-derived DNA detected more species (n = 11) than camera traps (n = 9), with only one species detected by both methods. The rarefaction curve indicated that blowfly-derived DNA would continue to detect more species with greater sampling effort. With further calibration, blowfly-derived DNA may join the list of traditional field methods. Areas for further investigation include blowfly feeding and dispersal biology, primer biases, and the assembly of a comprehensive and taxonomically-consistent DNA barcode reference library.

Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

PubMed

Zakian, V A; Brewer, B J; Fangman, W L

1979-08-01

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA.

Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model.

PubMed

Noguchi, Yasunori; Yuan, Zuanning; Bai, Lin; Schneider, Sarah; Zhao, Gongpu; Stillman, Bruce; Speck, Christian; Li, Huilin

2017-11-07

During replication initiation, the core component of the helicase-the Mcm2-7 hexamer-is loaded on origin DNA as a double hexamer (DH). The two ring-shaped hexamers are staggered, leading to a kinked axial channel. How the origin DNA interacts with the axial channel is not understood, but the interaction could provide key insights into Mcm2-7 function and regulation. Here, we report the cryo-EM structure of the Mcm2-7 DH on dsDNA and show that the DNA is zigzagged inside the central channel. Several of the Mcm subunit DNA-binding loops, such as the oligosaccharide-oligonucleotide loops, helix 2 insertion loops, and presensor 1 (PS1) loops, are well defined, and many of them interact extensively with the DNA. The PS1 loops of Mcm 3, 4, 6, and 7, but not 2 and 5, engage the lagging strand with an approximate step size of one base per subunit. Staggered coupling of the two opposing hexamers positions the DNA right in front of the two Mcm2-Mcm5 gates, with each strand being pressed against one gate. The architecture suggests that lagging-strand extrusion initiates in the middle of the DH that is composed of the zinc finger domains of both hexamers. To convert the Mcm2-7 DH structure into the Mcm2-7 hexamer structure found in the active helicase, the N-tier ring of the Mcm2-7 hexamer in the DH-dsDNA needs to tilt and shift laterally. We suggest that these N-tier ring movements cause the DNA strand separation and lagging-strand extrusion. Copyright © 2017 the Author(s). Published by PNAS.

A test of the transcription model for biased inheritance of yeast mitochondrial DNA.

PubMed

Lorimer, H E; Brewer, B J; Fangman, W L

1995-09-01

Two strand-specific origins of replication appear to be required for mammalian mitochondrial DNA (mtDNA) replication. Structural equivalents of these origins are found in the rep sequences of Saccharomyces cerevisiae mtDNA. These striking similarities have contributed to a universal model for the initiation of mtDNA replication in which a primer is created by cleavage of an origin region transcript. Consistent with this model are the properties of deletion mutants of yeast mtDNA ([rho-]) with a high density of reps (HS [rho-]). These mutant mtDNAs are preferentially inherited by the progeny resulting from the mating of HS [rho-] cells with cells containing wild-type mtDNA ([rho+]). This bias is presumed to result from a replication advantage conferred on HS [rho-] mtDNA by the high density of rep sequences acting as origins. To test whether transcription is indeed required for the preferential inheritance of HS [rho-] mtDNA, we deleted the nuclear gene (RPO41) for the mitochondrial RNA polymerase, reducing transcripts by at least 1000-fold. Since [rho-] genomes, but not [rho+] genomes, are stable when RPO41 is deleted, we examined matings between HS [rho-] and neutral [rho-] cells. Neutral [rho-] mtDNAs lack rep sequences and are not preferentially inherited in [rho-] x [rho+] crosses. In HS [rho-] x neutral [rho-] matings, the HS [rho-] mtDNA was preferentially inherited whether both parents were wild type or both were deleted for RPO41. Thus, transcription from the rep promoter does not appear to be necessary for biased inheritance. Our results, and analysis of the literature, suggest that priming by transcription is not a universal mechanism for mtDNA replication initiation.

DNA polymerase preference determines PCR priming efficiency.

PubMed

Pan, Wenjing; Byrne-Steele, Miranda; Wang, Chunlin; Lu, Stanley; Clemmons, Scott; Zahorchak, Robert J; Han, Jian

2014-01-30

Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3' hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3' end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially available DNA polymerases. The results suggest that the interaction of the DNA polymerase with the primer:template junction during the initiation of DNA polymerization is very important in terms of overall amplification bias and has broader implications for both the primer design process and multiplex PCR.

The hunt for origins of DNA replication in multicellular eukaryotes

PubMed Central

Urban, John M.; Foulk, Michael S.; Casella, Cinzia

2015-01-01

Origins of DNA replication (ORIs) occur at defined regions in the genome. Although DNA sequence defines the position of ORIs in budding yeast, the factors for ORI specification remain elusive in metazoa. Several methods have been used recently to map ORIs in metazoan genomes with the hope that features for ORI specification might emerge. These methods are reviewed here with analysis of their advantages and shortcomings. The various factors that may influence ORI selection for initiation of DNA replication are discussed. PMID:25926981

Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings.

PubMed

Jordan, Jeanne A; Ibe, Christine O; Moore, Miranda S; Host, Christel; Simon, Gary L

2012-05-01

In resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner. Assess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS. 60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells. The manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470 copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS. Using just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay. Copyright © 2012 Elsevier B.V. All rights reserved.

Defects in Mitochondrial DNA Replication and Human Disease

PubMed Central

Copeland, William C.

2011-01-01

Mitochondrial DNA (mtDNA) is replicated by the DNA polymerase γ in concert with accessory proteins such as the mitochondrial DNA helicase, single stranded DNA binding protein, topoisomerase, and initiating factors. Nucleotide precursors for mtDNA replication arise from the mitochondrial salvage pathway originating from transport of nucleosides, or alternatively from cytoplasmic reduction of ribonucleotides. Defects in mtDNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mtDNA deletions, point mutations, or depletion which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mtDNA depletion syndromes (MDS) such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders, such as progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). This review focuses on our current knowledge of genetic defects of mtDNA replication (POLG, POLG2, C10orf2) and nucleotide metabolism (TYMP, TK2, DGOUK, and RRM2B) that cause instability of mtDNA and mitochondrial disease. PMID:22176657

Mechanism for Clastogenic Activity of Naphthalene

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

Buchholz, Bruce A.

2016-06-24

Naphthalene incubations form DNA adducts in vitro in a dose dependent manner in both mouse and rat tissues. Rodent tissue incubations with naphthalene indicate that naphthalene forms as many DNA adducts as Benzo(a)pyrene, a known DNA binding carcinogen. The mouse airway has the greatest number of DNA adducts, corresponding to the higher metabolic activation of naphthalene in this location. Both rat tissues, the rat olfactory (tumor target) and the airways (non-tumor target), have similar levels of NA-DNA adducts, indicating that short term measures of initial adduct formation do not directly correlate with sites of tumor formation in the NTP bioassays.

A model for regulation of mammalian ribosomal DNA transcription. Co-ordination of initiation and termination.

PubMed Central

Nashimoto, M; Mishima, Y

1988-01-01

Based on recent experimental data about transcription initiation and termination, a model for regulation of mammalian ribosomal DNA transcription is developed using a simple kinetic scheme. In this model, the existence of the transition pathway from the terminator to the promoter increases the rate of ribosomal RNA precursor synthesis. In addition to this 'non-transcribed spacer' traverse of RNA polymerase I, the co-ordination of initiation and termination allows a rapid on/off switch transition from the minimum to the maximum rate of ribosomal RNA precursor synthesis. Furthermore, taking account of the participation of two factors in the termination event, we propose a plausible molecular mechanism for the co-ordination of initiation and termination. This co-ordination is emphasized by repetition of the terminator unit. PMID:3223915

Intrinsically bent DNA in replication origins and gene promoters.

PubMed

Gimenes, F; Takeda, K I; Fiorini, A; Gouveia, F S; Fernandez, M A

2008-06-24

Intrinsically bent DNA is an alternative conformation of the DNA molecule caused by the presence of dA/dT tracts, 2 to 6 bp long, in a helical turn phase DNA or with multiple intervals of 10 to 11 bp. Other than flexibility, intrinsic bending sites induce DNA curvature in particular chromosome regions such as replication origins and promoters. Intrinsically bent DNA sites are important in initiating DNA replication, and are sometimes found near to regions associated with the nuclear matrix. Many methods have been developed to localize bent sites, for example, circular permutation, computational analysis, and atomic force microscopy. This review discusses intrinsically bent DNA sites associated with replication origins and gene promoter regions in prokaryote and eukaryote cells. We also describe methods for identifying bent DNA sites for circular permutation and computational analysis.

The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.

PubMed

Daugaard, Iben; Kjeldsen, Tina E; Hager, Henrik; Hansen, Lise Lotte; Wojdacz, Tomasz K

2015-12-01

Readily accessible formalin-fixed paraffin embedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those samples is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE samples prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) samples was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR amplification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR amplification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE samples and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM. Copyright © 2015 Elsevier Inc. All rights reserved.

A new MCM modification cycle regulates DNA replication initiation

PubMed Central

Wei, Lei; Zhao, Xiaolan

2016-01-01

The MCM DNA helicase is a central regulatory target during genome replication. MCM is kept inactive during G1 and activated in S phase to initiate replication. During this transition, the only known chemical change on MCM is the gain of multi-site phosphorylation that promotes cofactor recruitment. As replication initiation is intimately linked to multiple biological cues, additional changes on MCM can provide further regulatory points. Here, we describe a yeast MCM sumoylation cycle that negatively regulates replication. MCM subunits undergo sumoylation upon loading at origins in G1 prior to MCM phosphorylation. MCM sumoylation levels then decline as MCM phosphorylation levels rise, suggesting an inhibitory role in replication. Indeed, increasing MCM sumoylation impairs replication initiation through promoting the recruitment of a phosphatase that reduces MCM phosphorylation and activation. MCM sumoylation thus counterbalances kinase-based regulation to ensure accurate control of replication initiation. PMID:26854664

A new MCM modification cycle regulates DNA replication initiation.

PubMed

Wei, Lei; Zhao, Xiaolan

2016-03-01

The MCM DNA helicase is a central regulatory target during genome replication. MCM is kept inactive during G1, and it initiates replication after being activated in S phase. During this transition, the only known chemical change to MCM is the gain of multisite phosphorylation that promotes cofactor recruitment. Because replication initiation is intimately linked to multiple biological cues, additional changes to MCM can provide further regulatory points. Here, we describe a yeast MCM SUMOylation cycle that regulates replication. MCM subunits undergo SUMOylation upon loading at origins in G1 before MCM phosphorylation. MCM SUMOylation levels then decline as MCM phosphorylation levels rise, thus suggesting an inhibitory role of MCM SUMOylation during replication. Indeed, increasing MCM SUMOylation impairs replication initiation, partly through promoting the recruitment of a phosphatase that decreases MCM phosphorylation and activation. We propose that MCM SUMOylation counterbalances kinase-based regulation, thus ensuring accurate control of replication initiation.

Structural basis of human PCNA sliding on DNA

NASA Astrophysics Data System (ADS)

de March, Matteo; Merino, Nekane; Barrera-Vilarmau, Susana; Crehuet, Ramon; Onesti, Silvia; Blanco, Francisco J.; de Biasio, Alfredo

2017-01-01

Sliding clamps encircle DNA and tether polymerases and other factors to the genomic template. However, the molecular mechanism of clamp sliding on DNA is unknown. Using crystallography, NMR and molecular dynamics simulations, here we show that the human clamp PCNA recognizes DNA through a double patch of basic residues within the ring channel, arranged in a right-hand spiral that matches the pitch of B-DNA. We propose that PCNA slides by tracking the DNA backbone via a `cogwheel' mechanism based on short-lived polar interactions, which keep the orientation of the clamp invariant relative to DNA. Mutation of residues at the PCNA-DNA interface has been shown to impair the initiation of DNA synthesis by polymerase δ (pol δ). Therefore, our findings suggest that a clamp correctly oriented on DNA is necessary for the assembly of a replication-competent PCNA-pol δ holoenzyme.

A Simple Laboratory Class Using a "Pseudomonas aeruginosa" Auxotroph to Illustrate UV-Mutagenic Killing, DNA Photorepair and Mutagenic DNA Repair

ERIC Educational Resources Information Center

Sobrero, Patricio; Valverde, Claudio

2013-01-01

A simple and cheap laboratory class is proposed to illustrate the lethal effect of UV radiation on bacteria and the operation of different DNA repair mechanisms. The class is divided into two sessions, an initial 3-hour experimental session and a second 2-hour analytical session. The experimental session involves two separate experiments: one…

Information analysis of sequences that bind the replication initiator RepA | Center for Cancer Research

Cancer.gov

The tall letters represent the highly conserved bases in DNA binding sites of several prokaryotic repressors and activators. Conservation is strongest where major grooves of the double helical DNA (represented by crests of a cosine wave) face the protein. This shows that conservation analysis alone can be used to predict the face of DNA that contacts the proteins.

DNA Damage Repair Factors have a Tumor Promoting Role in MLL-fusion Leukemia | Center for Cancer Research

Cancer.gov

Cancers develop when cells accumulate DNA mutations that allow them to grow and divide inappropriately. Thus, proteins involved in repairing DNA damage are generally suppressors of cancer formation, and their expression is often lost in the early stages of cancer initiation. In contrast, cancer stem cells, like their normal counterparts, must retain their ability to

Environmental DNA as a Tool for Inventory and Monitoring of Aquatic Vertebrates

DTIC Science & Technology

2017-07-01

geomorphic calculations and description of each reach. Methods Channel Surveys We initially selected reaches based on access and visual indicators...WA 99164 I-2 Environmental DNA lab protocol: designing species-specific qPCR assays Species-specific surveys should use quantitative polymerase...to traditional field sampling with respect to sensitivity, detection probabilities, and cost efficiency. Compared to field surveys , eDNA sampling

[Ubiquitin-proteasome system and sperm DNA repair: An update].

PubMed

Zhang, Guo-Wei; Cai, Hong-Cai; Shang, Xue-Jun

2016-09-01

The ubiquitin-proteasome system (UPS) is a proteasome system widely present in the human body, which is composed of ubiquitin (Ub), ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), ubiquitin protein ligases (E3), 26S proteasome, and deubiquitinating enzymes (DUBs) and involved in cell cycle regulation, immune response, signal transduction, DNA repair as well as protein degradation. Sperm DNA is vulnerable to interference or damage in the progression of chromosome association and homologous recombination. Recent studies show that UPS participates in DNA repair in spermatogenesis by modulating DNA repair enzymes via ubiquitination, assisting in the identification of DNA damage sites, raising damage repair-related proteins, initiating the DNA repair pathway, maintaining chromosome stability, and ensuring the normal process of spermatogenesis.

Emerging players in the initiation of eukaryotic DNA replication

PubMed Central

2012-01-01

Faithful duplication of the genome in eukaryotes requires ordered assembly of a multi-protein complex called the pre-replicative complex (pre-RC) prior to S phase; transition to the pre-initiation complex (pre-IC) at the beginning of DNA replication; coordinated progression of the replisome during S phase; and well-controlled regulation of replication licensing to prevent re-replication. These events are achieved by the formation of distinct protein complexes that form in a cell cycle-dependent manner. Several components of the pre-RC and pre-IC are highly conserved across all examined eukaryotic species. Many of these proteins, in addition to their bona fide roles in DNA replication are also required for other cell cycle events including heterochromatin organization, chromosome segregation and centrosome biology. As the complexity of the genome increases dramatically from yeast to human, additional proteins have been identified in higher eukaryotes that dictate replication initiation, progression and licensing. In this review, we discuss the newly discovered components and their roles in cell cycle progression. PMID:23075259

Expression of the Caulobacter heat shock gene dnaK is developmentally controlled during growth at normal temperatures.

PubMed Central

Gomes, S L; Gober, J W; Shapiro, L

1990-01-01

Caulobacter crescentus has a single dnaK gene that is highly homologous to the hsp70 family of heat shock genes. Analysis of the cloned and sequenced dnaK gene has shown that the deduced amino acid sequence could encode a protein of 67.6 kilodaltons that is 68% identical to the DnaK protein of Escherichia coli and 49% identical to the Drosophila and human hsp70 protein family. A partial open reading frame 165 base pairs 3' to the end of dnaK encodes a peptide of 190 amino acids that is 59% identical to DnaJ of E. coli. Northern blot analysis revealed a single 4.0-kilobase mRNA homologous to the cloned fragment. Since the dnaK coding region is 1.89 kilobases, dnaK and dnaJ may be transcribed as a polycistronic message. S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence. A single start site of transcription was identified during heat shock at 42 degrees C, and the predicted promoter sequence conformed to the consensus heat shock promoters of E. coli. At normal growth temperature (30 degrees C), a different start site was identified 3' to the heat shock start site that conformed to the E. coli sigma 70 promoter consensus sequence. S1 protection assays and analysis of expression of the dnaK gene fused to the lux transcription reporter gene showed that expression of dnaK is temporally controlled under normal physiological conditions and that transcription occurs just before the initiation of DNA replication. Thus, in both human cells (I. K. L. Milarski and R. I. Morimoto, Proc. Natl. Acad. Sci. USA 83:9517-9521, 1986) and in a simple bacterium, the transcription of a hsp70 gene is temporally controlled as a function of the cell cycle under normal growth conditions. Images PMID:2345134

Diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) is synthesized in response to DNA damage and inhibits the initiation of DNA replication.

PubMed

Marriott, Andrew S; Copeland, Nikki A; Cunningham, Ryan; Wilkinson, Mark C; McLennan, Alexander G; Jones, Nigel J

2015-09-01

The level of intracellular diadenosine 5', 5'''-P(1),P(4)-tetraphosphate (Ap4A) increases several fold in mammalian cells treated with non-cytotoxic doses of interstrand DNA-crosslinking agents such as mitomycin C. It is also increased in cells lacking DNA repair proteins including XRCC1, PARP1, APTX and FANCG, while >50-fold increases (up to around 25 μM) are achieved in repair mutants exposed to mitomycin C. Part of this induced Ap4A is converted into novel derivatives, identified as mono- and di-ADP-ribosylated Ap4A. Gene knockout experiments suggest that DNA ligase III is primarily responsible for the synthesis of damage-induced Ap4A and that PARP1 and PARP2 can both catalyze its ADP-ribosylation. Degradative proteins such as aprataxin may also contribute to the increase. Using a cell-free replication system, Ap4A was found to cause a marked inhibition of the initiation of DNA replicons, while elongation was unaffected. Maximum inhibition of 70-80% was achieved with 20 μM Ap4A. Ap3A, Ap5A, Gp4G and ADP-ribosylated Ap4A were without effect. It is proposed that Ap4A acts as an important inducible ligand in the DNA damage response to prevent the replication of damaged DNA. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of EhTIF-IA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IA.

PubMed

Srivastava, Ankita; Bhattacharya, Alok; Bhattacharya, Sudha; Jhingan, Gagan Deep

2016-03-01

Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at the rDNA promoter containing the RNA Pol I with auxiliary factors. One of these factors is known as Rrn3P in yeast and Transcription Initiation Factor IA (TIF-IA) in mammals. Rrn3p/TIF-IA serves as a bridge between RNA Pol I and the pre-initiation complex at the promoter. It is phosphorylated at multiple sites and is involved in regulation of rDNA transcription in a growth-dependent manner. In the early branching parasitic protist Entamoeba histolytica, the rRNA genes are present exclusively on circular extra chromosomal plasmids. The protein factors involved in regulation of rDNA transcription in E. histolytica are not known. We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within the database and was further cloned and expressed. Immuno-localization studies showed that EhTIF-IA co-localized partially with fibrillarin in the peripherally localized nucleolus. EhTIF-IA was shown to interact with the RNA Pol I-specific subunit RPA12 both in vivo and in vitro. Mass spectroscopy data identified RNA Pol I-specific subunits and other nucleolar proteins to be the interacting partners of EhTIF-IA. Our study demonstrates for the first time a conserved putative RNA Pol I transcription factor TIF-IA in E. histolytica.

Structural basis for the initiation of eukaryotic transcription-coupled DNA repair.

PubMed

Xu, Jun; Lahiri, Indrajit; Wang, Wei; Wier, Adam; Cianfrocco, Michael A; Chong, Jenny; Hare, Alissa A; Dervan, Peter B; DiMaio, Frank; Leschziner, Andres E; Wang, Dong

2017-11-30

Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II-CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II-Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation.

Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis.

PubMed Central

Winnacker, E L

1975-01-01

Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 DNA. PMID:1117487

Preservation and Significance of Extracellular DNA in Ferruginous Sediments from Lake Towuti, Indonesia

PubMed Central

Vuillemin, Aurèle; Horn, Fabian; Alawi, Mashal; Henny, Cynthia; Wagner, Dirk; Crowe, Sean A.; Kallmeyer, Jens

2017-01-01

Extracellular DNA is ubiquitous in soil and sediment and constitutes a dominant fraction of environmental DNA in aquatic systems. In theory, extracellular DNA is composed of genomic elements persisting at different degrees of preservation produced by processes occurring on land, in the water column and sediment. Extracellular DNA can be taken up as a nutrient source, excreted or degraded by microorganisms, or adsorbed onto mineral matrices, thus potentially preserving information from past environments. To test whether extracellular DNA records lacustrine conditions, we sequentially extracted extracellular and intracellular DNA from anoxic sediments of ferruginous Lake Towuti, Indonesia. We applied 16S rRNA gene Illumina sequencing on both fractions to discriminate exogenous from endogenous sources of extracellular DNA in the sediment. Environmental sequences exclusively found as extracellular DNA in the sediment originated from multiple sources. For instance, Actinobacteria, Verrucomicrobia, and Acidobacteria derived from soils in the catchment. Limited primary productivity in the water column resulted in few sequences of Cyanobacteria in the oxic photic zone, whereas stratification of the water body mainly led to secondary production by aerobic and anaerobic heterotrophs. Chloroflexi and Planctomycetes, the main degraders of sinking organic matter and planktonic sequences at the water-sediment interface, were preferentially preserved during the initial phase of burial. To trace endogenous sources of extracellular DNA, we used relative abundances of taxa in the intracellular DNA to define which microbial populations grow, decline or persist at low density with sediment depth. Cell lysis became an important additional source of extracellular DNA, gradually covering previous genetic assemblages as other microbial genera became more abundant with depth. The use of extracellular DNA as nutrient by active microorganisms led to selective removal of sequences with lowest GC contents. We conclude that extracellular DNA preserved in shallow lacustrine sediments reflects the initial environmental context, but is gradually modified and thereby shifts from its stratigraphic context. Discrimination of exogenous and endogenous sources of extracellular DNA allows simultaneously addressing in-lake and post-depositional processes. In deeper sediments, the accumulation of resting stages and sequences from cell lysis would require stringent extraction and specific primers if ancient DNA is targeted. PMID:28798742

The emerging role of nuclear viral DNA sensors.

PubMed

Diner, Benjamin A; Lum, Krystal K; Cristea, Ileana M

2015-10-30

Detecting pathogenic DNA by intracellular receptors termed "sensors" is critical toward galvanizing host immune responses and eliminating microbial infections. Emerging evidence has challenged the dogma that sensing of viral DNA occurs exclusively in sub-cellular compartments normally devoid of cellular DNA. The interferon-inducible protein IFI16 was shown to bind nuclear viral DNA and initiate immune signaling, culminating in antiviral cytokine secretion. Here, we review the newly characterized nucleus-originating immune signaling pathways, their links to other crucial host defenses, and unique mechanisms by which viruses suppress their functions. We frame these findings in the context of human pathologies associated with nuclear replicating DNA viruses. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Perspective on the pipeline of drugs being developed with modulation of DNA damage as a target.

PubMed

Plummer, Ruth

2010-09-15

Inhibitors of various elements of the DNA repair pathways have entered clinical development or are in late preclinical stages of drug development. It was initially considered that agents targeting DNA repair would act to overcome tumor resistance to chemotherapy and radiotherapy. More recent data have shown that targeting DNA repair pathways can be effective in selected tumors via a synthetically lethal route, with single agent activity having been shown with poly-ADP ribose polymerase (PARP) inhibitors. An increased understanding of the biology and interaction of the DNA repair pathways also means that rational combination of DNA repair inhibitors may also give great benefit in the clinic. ©2010 AACR.

Assessing macroinvertebrate biodiversity in freshwater ecosystems: Advances and challenges in dna-based approaches

USGS Publications Warehouse

Pfrender, M.E.; Ferrington, L.C.; Hawkins, C.P.; Hartzell, P.L.; Bagley, M.; Jackson, S.; Courtney, G.W.; Larsen, D.P.; Creutzburg, B.R.; Levesque, C.A.; Epler, J.H.; Morse, J.C.; Fend, S.; Petersen, M.J.; Ruiter, D.; Schindel, D.; Whiting, M.

2010-01-01

Assessing the biodiversity of macroinvertebrate fauna in freshwater ecosystems is an essential component of both basic ecological inquiry and applied ecological assessments. Aspects of taxonomic diversity and composition in freshwater communities are widely used to quantify water quality and measure the efficacy of remediation and restoration efforts. The accuracy and precision of biodiversity assessments based on standard morphological identifications are often limited by taxonomic resolution and sample size. Morphologically based identifications are laborious and costly, significantly constraining the sample sizes that can be processed. We suggest that the development of an assay platform based on DNA signatures will increase the precision and ease of quantifying biodiversity in freshwater ecosystems. Advances in this area will be particularly relevant for benthic and planktonic invertebrates, which are often monitored by regulatory agencies. Adopting a genetic assessment platform will alleviate some of the current limitations to biodiversity assessment strategies. We discuss the benefits and challenges associated with DNA-based assessments and the methods that are currently available. As recent advances in microarray and next-generation sequencing technologies will facilitate a transition to DNA-based assessment approaches, future research efforts should focus on methods for data collection, assay platform development, establishing linkages between DNA signatures and well-resolved taxonomies, and bioinformatics. ?? 2010 by The University of Chicago Press.

Novel adsorbent for DNA adsorption: Fe(3+)-attached sporopollenin particles embedded composite cryogels.

PubMed

Ceylan, Şeyda; Odabaşı, Mehmet

2013-12-01

The aim of this study is to prepare supermacroporous cryogels embedded with Fe(3+)-attached sporopollenin particles (Fe(3++)-ASPs) having large surface area for high DNA adsorption capacity. Supermacroporous poly(2-hydroxyethyl methacrylate) (PHEMA)-based monolithic cryogel column embedded with Fe3+(+)-ASPs was prepared by radical cryo-copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N,N´-methylene- bis-acrylamide (MBAAm) as cross-linker directly in a plastic syringe for DNA adsorption studies. Firstly, Fe3+(+) ions were attached to the sporopollenin particles (SPs), then the supermacroporous PHEMA cryogel with embedded Fe(3++)-ASPs was produced by free radical polymerization using N,N,N´, N´-Tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) as initiator/activator pair in an ice bath. Optimum conditions of adsorption experiments were performed at pH 6.0 (0.02 M Tris buffer containing 0.2 M NaCl), with flow rate of 0.5 mL/min, and at 5°C. The maximum amount of DNA adsorption from aqueous solution was very high (109 mg/g SPs) with initial concentration of 3 mg/mL. It was observed that DNA could be repeatedly adsorbed and desorbed with this composite cryogel without significant loss of adsorption capacity. As a result, higher amounts of DNA adsorbed these composite cryogels are expected to be good candidate for achieving higher removal of anti-DNA antibodies from systemic lupus erythematosus (SLE) patients plasma.