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Sample records for coordinate dna repair

  1. Coordination of Steps in Single-nucleotide Base Excision Repair Mediated by Apurinic/Apyrimidinic Endonuclease 1 and DNA Polymerase β*

    PubMed Central

    Liu, Yuan; Prasad, Rajendra; Beard, William A.; Kedar, Padmini S.; Hou, Esther W.; Shock, David D.; Wilson, Samuel H.

    2008-01-01

    The individual steps in single-nucleotide base excision repair (SN-BER) are coordinated to enable efficient repair without accumulation of cytotoxic DNA intermediates. The DNA transactions and various proteins involved in SN-BER of abasic sites are well known in mammalian systems. Yet, despite a wealth of information on SN-BER, the mechanism of step-by-step coordination is poorly understood. In this study we conducted experiments toward understanding step-by-step coordination during BER by comparing DNA binding specificities of two major human SN-BER enzymes, apurinic/aprymidinic endonuclease 1 (APE) and DNA polymerase β (Pol β). It is known that these enzymes do not form a stable complex in solution. For each enzyme, we found that DNA binding specificity appeared sufficient to explain the sequential processing of BER intermediates. In addition, however, we identified at higher enzyme concentrations a ternary complex of APE·Pol β·DNA that formed specifically at BER intermediates containing a 5′-deoxyribose phosphate group. Formation of this ternary complex was associated with slightly stronger Pol β gap-filling and much stronger 5′-deoxyribose phosphate lyase activities than was observed with the Pol β·DNA binary complex. These results indicate that step-by-step coordination in SN-BER can rely on DNA binding specificity inherent in APE and Pol β, although coordination also may be facilitated by APE·Pol β·DNA ternary complex formation with appropriate enzyme expression levels or enzyme recruitment to sites of repair. PMID:17355977

  2. Replication protein A and proliferating cell nuclear antigen coordinate DNA polymerase selection in 8-oxo-guanine repair.

    PubMed

    Maga, Giovanni; Crespan, Emmanuele; Wimmer, Ursula; van Loon, Barbara; Amoroso, Alessandra; Mondello, Chiara; Belgiovine, Cristina; Ferrari, Elena; Locatelli, Giada; Villani, Giuseppe; Hübscher, Ulrich

    2008-12-30

    The adenine misincorporated by replicative DNA polymerases (pols) opposite 7,8-dihydro-8-oxoguanine (8-oxo-G) is removed by a specific glycosylase, leaving the lesion on the DNA. Subsequent incorporation of C opposite 8-oxo-G on the resulting 1-nt gapped DNA is essential for the removal of the 8-oxo-G to prevent G-C to T-A transversion mutations. By using model DNA templates, purified DNA pols beta and lambda and knockout cell extracts, we show here that the auxiliary proteins replication protein A and proliferating cell nuclear antigen act as molecular switches to activate the DNA pol lambda- dependent highly efficient and faithful repair of A:8-oxo-G mismatches in human cells and to repress DNA pol beta activity. By using an immortalized human fibroblast cell line that has the potential to induce cancer in mice, we show that the development of a tumoral phenotype in these cells correlated with a differential expression of DNA pols lambda and beta.

  3. p53 coordinates DNA repair with nucleotide synthesis by suppressing PFKFB3 expression and promoting the pentose phosphate pathway

    PubMed Central

    Franklin, Derek A.; He, Yizhou; Leslie, Patrick L.; Tikunov, Andrey P.; Fenger, Nick; Macdonald, Jeffrey M.; Zhang, Yanping

    2016-01-01

    Activation of p53 in response to DNA damage is essential for tumor suppression. Although previous studies have emphasized the importance of p53-dependent cell cycle arrest and apoptosis for tumor suppression, recent studies have suggested that other areas of p53 regulation, such as metabolism and DNA damage repair (DDR), are also essential for p53-dependent tumor suppression. However, the intrinsic connections between p53-mediated DDR and metabolic regulation remain incompletely understood. Here, we present data suggesting that p53 promotes nucleotide biosynthesis in response to DNA damage by repressing the expression of the phosphofructokinase-2 (PFK2) isoform 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a rate-limiting enzyme that promotes glycolysis. PFKFB3 suppression increases the flux of glucose through the pentose phosphate pathway (PPP) to increase nucleotide production, which results in more efficient DNA damage repair and increased cell survival. Interestingly, although p53-mediated suppression of PFKFB3 could increase the two major PPP products, NADPH and nucleotides, only nucleotide production was essential to promote DDR. By identifying the novel p53 target PFKFB3, we report an important mechanistic connection between p53-regulated metabolism and DDR, both of which play crucial roles in tumor suppression. PMID:27901115

  4. DNA Repair by Reversal of DNA Damage

    PubMed Central

    Yi, Chengqi; He, Chuan

    2013-01-01

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

  5. DNA excision repair at telomeres.

    PubMed

    Jia, Pingping; Her, Chengtao; Chai, Weihang

    2015-12-01

    DNA damage is caused by either endogenous cellular metabolic processes such as hydrolysis, oxidation, alkylation, and DNA base mismatches, or exogenous sources including ultraviolet (UV) light, ionizing radiation, and chemical agents. Damaged DNA that is not properly repaired can lead to genomic instability, driving tumorigenesis. To protect genomic stability, mammalian cells have evolved highly conserved DNA repair mechanisms to remove and repair DNA lesions. Telomeres are composed of long tandem TTAGGG repeats located at the ends of chromosomes. Maintenance of functional telomeres is critical for preventing genome instability. The telomeric sequence possesses unique features that predispose telomeres to a variety of DNA damage induced by environmental genotoxins. This review briefly describes the relevance of excision repair pathways in telomere maintenance, with the focus on base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). By summarizing current knowledge on excision repair of telomere damage and outlining many unanswered questions, it is our hope to stimulate further interest in a better understanding of excision repair processes at telomeres and in how these processes contribute to telomere maintenance.

  6. Coordination and processing of DNA ends during double-strand break repair: the role of the bacteriophage T4 Mre11/Rad50 (MR) complex.

    PubMed

    Almond, Joshua R; Stohr, Bradley A; Panigrahi, Anil K; Albrecht, Dustin W; Nelson, Scott W; Kreuzer, Kenneth N

    2013-11-01

    The in vivo functions of the bacteriophage T4 Mre11/Rad50 (MR) complex (gp46/47) in double-strand-end processing, double-strand break repair, and recombination-dependent replication were investigated. The complex is essential for T4 growth, but we wanted to investigate the in vivo function during productive infections. We therefore generated a suppressed triple amber mutant in the Rad50 subunit to substantially reduce the level of complex and thereby reduce phage growth. Growth-limiting amounts of the complex caused a concordant decrease in phage genomic recombination-dependent replication. However, the efficiencies of double-strand break repair and of plasmid-based recombination-dependent replication remained relatively normal. Genetic analyses of linked markers indicated that double-strand ends were less protected from nuclease erosion in the depleted infection and also that end coordination during repair was compromised. We discuss models for why phage genomic recombination-dependent replication is more dependent on Mre11/Rad50 levels when compared to plasmid recombination-dependent replication. We also tested the importance of the conserved histidine residue in nuclease motif I of the T4 Mre11 protein. Substitution with multiple different amino acids (including serine) failed to support phage growth, completely blocked plasmid recombination-dependent replication, and led to the stabilization of double-strand ends. We also constructed and expressed an Mre11 mutant protein with the conserved histidine changed to serine. The mutant protein was found to be completely defective for nuclease activities, but retained the ability to bind the Rad50 subunit and double-stranded DNA. These results indicate that the nuclease activity of Mre11 is critical for phage growth and recombination-dependent replication during T4 infections.

  7. DNA repair in cultured keratinocytes

    SciTech Connect

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

    1983-07-01

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

  8. Rethinking transcription coupled DNA repair.

    PubMed

    Kamarthapu, Venu; Nudler, Evgeny

    2015-04-01

    Nucleotide excision repair (NER) is an evolutionarily conserved, multistep process that can detect a wide variety of DNA lesions. Transcription coupled repair (TCR) is a subpathway of NER that repairs the transcribed DNA strand faster than the rest of the genome. RNA polymerase (RNAP) stalled at DNA lesions mediates the recruitment of NER enzymes to the damage site. In this review we focus on a newly identified bacterial TCR pathway in which the NER enzyme UvrD, in conjunction with NusA, plays a major role in initiating the repair process. We discuss the tradeoff between the new and conventional models of TCR, how and when each pathway operates to repair DNA damage, and the necessity of pervasive transcription in maintaining genome integrity.

  9. DNA encoding a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  10. Mammalian DNA Repair. Final Report

    SciTech Connect

    2003-01-24

    The Gordon Research Conference (GRC) on Mammalian DNA Repair was held at Harbortown Resort, Ventura Beach, CA. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  11. Regulation of DNA repair by parkin

    SciTech Connect

    Kao, Shyan-Yuan

    2009-05-01

    Mutation of parkin is one of the most prevalent causes of autosomal recessive Parkinson's disease (PD). Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here we show that parkin is essential for optimal repair of DNA damage. Parkin-deficient cells exhibit reduced DNA excision repair that can be restored by transfection of wild-type parkin, but not by transfection of a pathological parkin mutant. Parkin also protects against DNA damage-induced cell death, an activity that is largely lost in the pathological mutant. Moreover, parkin interacts with the proliferating cell nuclear antigen (PCNA), a protein that coordinates DNA excision repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in PD.

  12. DNA Damage, DNA Repair, Aging, and Neurodegeneration.

    PubMed

    Maynard, Scott; Fang, Evandro Fei; Scheibye-Knudsen, Morten; Croteau, Deborah L; Bohr, Vilhelm A

    2015-09-18

    Aging in mammals is accompanied by a progressive atrophy of tissues and organs, and stochastic damage accumulation to the macromolecules DNA, RNA, proteins, and lipids. The sequence of the human genome represents our genetic blueprint, and accumulating evidence suggests that loss of genomic maintenance may causally contribute to aging. Distinct evidence for a role of imperfect DNA repair in aging is that several premature aging syndromes have underlying genetic DNA repair defects. Accumulation of DNA damage may be particularly prevalent in the central nervous system owing to the low DNA repair capacity in postmitotic brain tissue. It is generally believed that the cumulative effects of the deleterious changes that occur in aging, mostly after the reproductive phase, contribute to species-specific rates of aging. In addition to nuclear DNA damage contributions to aging, there is also abundant evidence for a causative link between mitochondrial DNA damage and the major phenotypes associated with aging. Understanding the mechanistic basis for the association of DNA damage and DNA repair with aging and age-related diseases, such as neurodegeneration, would give insight into contravening age-related diseases and promoting a healthy life span.

  13. Targeting DNA Repair in Cancer: Beyond PARP Inhibitors.

    PubMed

    Brown, Jessica S; O'Carrigan, Brent; Jackson, Stephen P; Yap, Timothy A

    2017-01-01

    Germline aberrations in critical DNA-repair and DNA damage-response (DDR) genes cause cancer predisposition, whereas various tumors harbor somatic mutations causing defective DDR/DNA repair. The concept of synthetic lethality can be exploited in such malignancies, as exemplified by approval of poly(ADP-ribose) polymerase inhibitors for treating BRCA1/2-mutated ovarian cancers. Herein, we detail how cellular DDR processes engage various proteins that sense DNA damage, initiate signaling pathways to promote cell-cycle checkpoint activation, trigger apoptosis, and coordinate DNA repair. We focus on novel therapeutic strategies targeting promising DDR targets and discuss challenges of patient selection and the development of rational drug combinations.

  14. Function of transcription factors at DNA lesions in DNA repair.

    PubMed

    Malewicz, Michal; Perlmann, Thomas

    2014-11-15

    Cellular systems for DNA repair ensure prompt removal of DNA lesions that threaten the genomic stability of the cell. Transcription factors (TFs) have long been known to facilitate DNA repair via transcriptional regulation of specific target genes encoding key DNA repair proteins. However, recent findings identified TFs as DNA repair components acting directly at the DNA lesions in a transcription-independent fashion. Together this recent progress is consistent with the hypothesis that TFs have acquired the ability to localize DNA lesions and function by facilitating chromatin remodeling at sites of damaged DNA. Here we review these recent findings and discuss how TFs may function in DNA repair.

  15. Final report [DNA Repair and Mutagenesis - 1999

    SciTech Connect

    Walker, Graham C.

    2001-05-30

    The meeting, titled ''DNA Repair and Mutagenesis: Mechanism, Control, and Biological Consequences'', was designed to bring together the various sub-disciplines that collectively comprise the field of DNA Repair and Mutagenesis. The keynote address was titled ''Mutability Doth Play Her Cruel Sports to Many Men's Decay: Variations on the Theme of Translesion Synthesis.'' Sessions were held on the following themes: Excision repair of DNA damage; Transcription and DNA excision repair; UmuC/DinB/Rev1/Rad30 superfamily of DNA polymerases; Cellular responses to DNA damage, checkpoints, and damage tolerance; Repair of mismatched bases, mutation; Genome-instability, and hypermutation; Repair of strand breaks; Replicational fidelity, and Late-breaking developments; Repair and mutation in challenging environments; and Defects in DNA repair: consequences for human disease and aging.

  16. Dynamics of DNA Mismatch Repair

    NASA Astrophysics Data System (ADS)

    Coats, Julie; Lin, Yuyen; Rasnik, Ivan

    2009-11-01

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

  17. DNA repair capacity of zebrafish.

    PubMed

    Sussman, Raquel

    2007-08-14

    Damage to the genome is unavoidable in living creatures, because of sunlight exposure as well as environmental chemicals present in food and drinking water. There is a need to monitor and purify the drinking water; therefore, several methods of detection have been developed. A very promising model system for this purpose is the zebrafish (Danio rerio), which is endowed with special qualities for detecting external as well as internal abnormalities. Grossman and Wei's assay [Grossman L, Wei Q (1995) Clin Chem 12:1854-1863], which measures the expression level of a nonreplicating recombinant plasmid DNA containing a UV-damaged luciferase reporter gene, shows that zebrafish can repair chromosomal lesions to a much greater extent than the human population. This vertebrate model is still very promising after possible down-regulation of the DNA repair enzymes.

  18. ATM prevents DSB formation by coordinating SSB repair and cell cycle progression.

    PubMed

    Khoronenkova, Svetlana V; Dianov, Grigory L

    2015-03-31

    DNA single-strand breaks (SSBs) arise as a consequence of spontaneous DNA instability and are also formed as DNA repair intermediates. Their repair is critical because they otherwise terminate gene transcription and generate toxic DNA double-strand breaks (DSBs) on replication. To prevent the formation of DSBs, SSB repair must be completed before DNA replication. To accomplish this, cells should be able to detect unrepaired SSBs, and then delay cell cycle progression to allow more time for repair; however, to date there is no evidence supporting the coordination of SSB repair and replication in human cells. Here we report that ataxia-telangiectasia mutated kinase (ATM) plays a major role in restricting the replication of SSB-containing DNA and thus prevents DSB formation. We show that ATM is activated by SSBs and coordinates their repair with DNA replication. SSB-mediated ATM activation is followed by a G1 cell cycle delay that allows more time for repair and thus prevents the replication of damaged DNA and DSB accrual. These findings establish an unanticipated role for ATM in the signaling of DNA SSBs and provide important insight into the molecular defects leading to genetic instability in patients with ataxia-telangiectasia.

  19. Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair

    PubMed Central

    Çağlayan, Melike; Wilson, Samuel H.

    2015-01-01

    DNA lesions arise from many endogenous and environmental agents, and they promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5'-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER. PMID:26596511

  20. Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair

    PubMed Central

    çağlayan, Melike; Wilson, Samuel H.

    2015-01-01

    DNA lesions arise from many endogenous and environmental agents, and they promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5′-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER. PMID:26466358

  1. Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair.

    PubMed

    Çağlayan, Melike; Wilson, Samuel H

    2015-11-01

    DNA lesions arise from many endogenous and environmental agents, and such lesions can promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5'-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER.

  2. An alternative eukaryotic DNA excision repair pathway.

    PubMed Central

    Freyer, G A; Davey, S; Ferrer, J V; Martin, A M; Beach, D; Doetsch, P W

    1995-01-01

    DNA lesions induced by UV light, cyclobutane pyrimidine dimers, and (6-4)pyrimidine pyrimidones are known to be repaired by the process of nucleotide excision repair (NER). However, in the fission yeast Schizosaccharomyces pombe, studies have demonstrated that at least two mechanisms for excising UV photo-products exist; NER and a second, previously unidentified process. Recently we reported that S. pombe contains a DNA endonuclease, SPDE, which recognizes and cleaves at a position immediately adjacent to cyclobutane pyrimidine dimers and (6-4)pyrimidine pyrimidones. Here we report that the UV-sensitive S. pombe rad12-502 mutant lacks SPDE activity. In addition, extracts prepared from the rad12-502 mutant are deficient in DNA excision repair, as demonstrated in an in vitro excision repair assay. DNA repair activity was restored to wild-type levels in extracts prepared from rad12-502 cells by the addition of partially purified SPDE to in vitro repair reaction mixtures. When the rad12-502 mutant was crossed with the NER rad13-A mutant, the resulting double mutant was much more sensitive to UV radiation than either single mutant, demonstrating that the rad12 gene product functions in a DNA repair pathway distinct from NER. These data directly link SPDE to this alternative excision repair process. We propose that the SPDE-dependent DNA repair pathway is the second DNA excision repair process present in S. pombe. PMID:7623848

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

    PubMed Central

    Sorrell, Melanie; Berman, Zachary

    2014-01-01

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

  4. DNA Damage and Repair in Vascular Disease.

    PubMed

    Uryga, Anna; Gray, Kelly; Bennett, Martin

    2016-01-01

    DNA damage affecting both genomic and mitochondrial DNA is present in a variety of both inherited and acquired vascular diseases. Multiple cell types show persistent DNA damage and a range of lesions. In turn, DNA damage activates a variety of DNA repair mechanisms, many of which are activated in vascular disease. Such DNA repair mechanisms either stall the cell cycle to allow repair to occur or trigger apoptosis or cell senescence to prevent propagation of damaged DNA. Recent evidence has indicated that DNA damage occurs early, is progressive, and is sufficient to impair function of cells composing the vascular wall. The consequences of persistent genomic and mitochondrial DNA damage, including inflammation, cell senescence, and apoptosis, are present in vascular disease. DNA damage can thus directly cause vascular disease, opening up new possibilities for both prevention and treatment. We review the evidence for and the causes, types, and consequences of DNA damage in vascular disease.

  5. Effect of acrylamide on hepatocellular DNA repair

    SciTech Connect

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

    1986-01-01

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

  6. Role of Deubiquitinating Enzymes in DNA Repair

    PubMed Central

    2015-01-01

    Both proteolytic and nonproteolytic functions of ubiquitination are essential regulatory mechanisms for promoting DNA repair and the DNA damage response in mammalian cells. Deubiquitinating enzymes (DUBs) have emerged as key players in the maintenance of genome stability. In this minireview, we discuss the recent findings on human DUBs that participate in genome maintenance, with a focus on the role of DUBs in the modulation of DNA repair and DNA damage signaling. PMID:26644404

  7. Flavonoids and DNA Repair in Prostate Cancer

    DTIC Science & Technology

    2005-12-01

    hours of naringenin treatment. The tea flavonoid EGC and apigenin from parsley did not show any DNA repair-stimulatory activity. 15. SUBJECT TERMS No...flavonoids to continue the investigations on the stimulatory effect on DNA repair: -naringenin from citrus -apigenin from parsley -epicatechin

  8. DNA excision repair in permeable human fibroblasts

    SciTech Connect

    Kaufmann, W.K.; Bodell, W.J.; Cleaver, J.E.

    1983-01-01

    U.v. irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized. Incubation of intact cells for 20 min between irradiation and harvesting was necessary to obtain a maximum rate of reparative DNA synthesis. Cells harvested immediately after irradiation before repair was initiated displayed only a small stimulation of DNA synthesis, indicating that permeable cells have a reduced capacity to recognize pyrimidine dimers and activate repair. The distribution of sizes of DNA strands labeled during 10 min of reparative DNA synthesis resembled that of parental DNA. However, during a 60-min incubation of permeable cells at 37 degrees C, parental DNA and DNA labeled by reparative DNA synthesis were both cleaved to smaller sizes. Cleavage also occurred in unirradiated cells, indicating that endogenous nuclease was active during incubation. Repair patches synthesized in permeable cells displayed increased sensitivity to digestion by micrococcal nuclease. However, the change in sensitivity during a chase with unlabeled DNA precursors was small, suggesting that reassembly of nucleosome structure at sites of repair was impaired. To examine whether this deficiency was due to a preponderance of incomplete or unligated repair patches, 3H-labeled (repaired) DNA was purified, then digested with exonuclease III and nuclease S1 to probe for free 3' ends and single-stranded regions. About 85% of the (3H)DNA synthesized during a 10-min pulse resisted digestion, suggesting that a major fraction of the repair patches that were filled were also ligated. U.v. light-activated DNA synthesis in permeable cells, therefore, appears to represent the continuation of reparative gap-filling at sites of excision repair activated within intact cells. Gap-filling and ligation were comparatively efficient processes in permeable cells.

  9. DNA Triplet Repeat Expansion and Mismatch Repair

    PubMed Central

    Iyer, Ravi R.; Pluciennik, Anna; Napierala, Marek; Wells, Robert D.

    2016-01-01

    DNA mismatch repair is a conserved antimutagenic pathway that maintains genomic stability through rectification of DNA replication errors and attenuation of chromosomal rearrangements. Paradoxically, mutagenic action of mismatch repair has been implicated as a cause of triplet repeat expansions that cause neurological diseases such as Huntington disease and myotonic dystrophy. This mutagenic process requires the mismatch recognition factor MutSβ and the MutLα (and/or possibly MutLγ) endonuclease, and is thought to be triggered by the transient formation of unusual DNA structures within the expanded triplet repeat element. This review summarizes the current knowledge of DNA mismatch repair involvement in triplet repeat expansion, which encompasses in vitro biochemical findings, cellular studies, and various in vivo transgenic animal model experiments. We present current mechanistic hypotheses regarding mismatch repair protein function in mediating triplet repeat expansions and discuss potential therapeutic approaches targeting the mismatch repair pathway. PMID:25580529

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

    PubMed Central

    Uphoff, Stephan; Kapanidis, Achillefs N.

    2014-01-01

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

  11. DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

    PubMed Central

    Genois, Marie-Michelle; Paquet, Eric R.; Laffitte, Marie-Claude N.; Maity, Ranjan; Rodrigue, Amélie

    2014-01-01

    SUMMARY All living organisms are continuously faced with endogenous or exogenous stress conditions affecting genome stability. DNA repair pathways act as a defense mechanism, which is essential to maintain DNA integrity. There is much to learn about the regulation and functions of these mechanisms, not only in human cells but also equally in divergent organisms. In trypanosomatids, DNA repair pathways protect the genome against mutations but also act as an adaptive mechanism to promote drug resistance. In this review, we scrutinize the molecular mechanisms and DNA repair pathways which are conserved in trypanosomatids. The recent advances made by the genome consortiums reveal the complete genomic sequences of several pathogens. Therefore, using bioinformatics and genomic sequences, we analyze the conservation of DNA repair proteins and their key protein motifs in trypanosomatids. We thus present a comprehensive view of DNA repair processes in trypanosomatids at the crossroads of DNA repair and drug resistance. PMID:24600040

  12. Antibody specific for a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-07-11

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  13. DNA-PKcs and ATM Co-Regulate DNA Double-Strand Break Repair

    PubMed Central

    Shrivastav, Meena; Miller, Cheryl A.; De Haro, Leyma P.; Durant, Stephen T.; Chen, Benjamin P.C.; Chen, David J.; Nickoloff, Jac A.

    2009-01-01

    DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ/HR decision is under complex regulation and involves DNA-dependent protein kinase (DNA-PKcs). HR is elevated in DNA-PKcs null cells, but suppressed by DNA-PKcs kinase inhibitors, suggesting that kinase-inactive DNA-PKcs (DNA-PKcs-KR) would suppress HR. Here we use a direct repeat assay to monitor HR repair of DSBs induced by I-SceI nuclease. Surprisingly, DSB-induced HR in DNA-PKcs-KR cells was 2- to 3-fold above the elevated HR level of DNA-PKcs null cells, and ∼4- to 7-fold above cells expressing wild-type DNA-PKcs. The hyperrecombination in DNA-PKcs-KR cells compared to DNA-PKcs null cells was also apparent as increased resistance to DNA crosslinks induced by mitomycin C. ATM phosphorylates many HR proteins, and ATM is expressed at a low level in cells lacking DNA-PKcs, but restored to wild-type level in cells expressing DNA-PKcs-KR. Several clusters of phosphorylation sites in DNA-PKcs, including the T2609 cluster, which is phosphorylated by DNA-PKcs and ATM, regulate access of repair factors to broken ends. Our results indicate that ATM-dependent phosphorylation of DNA-PKcs-KR contributes to the hyperrecombination phenotype. Interestingly, DNA-PKcs null cells showed more persistent ionizing radiation-induced RAD51 foci (but lower HR levels) compared to DNA-PKcs-KR cells, consistent with HR completion requiring RAD51 turnover. ATM may promote RAD51 turnover, suggesting a second (not mutually exclusive) mechanism by which restored ATM contributes to hyperrecombination in DNA-PKcs-KR cells. We propose a model in which DNA-PKcs and ATM coordinately regulate DSB repair by NHEJ and HR. PMID:19535303

  14. Nuclear position dictates DNA repair pathway choice

    PubMed Central

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

    2014-01-01

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

  15. DNA repair in ischemic acute kidney injury.

    PubMed

    Pressly, Jeffrey D; Park, Frank

    2017-04-01

    Ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury leading to an induction of oxidative stress, cellular dysfunction, and loss of renal function. DNA damage, including oxidative base modifications and physical DNA strand breaks, is a consequence of renal IRI. Like many other organs in the body, a redundant and highly conserved set of endogenous repair pathways have evolved to selectively recognize the various types of cellular DNA damage and combat its negative effects on cell viability. Severe damage to the DNA, however, can trigger cell death and elimination of the injured tubular epithelial cells. In this minireview, we summarize the state of the current field of DNA damage and repair in the kidney and provide some expected and, in some cases, unexpected effects of IRI on DNA damage and repair in the kidney. These findings may be applicable to other forms of acute kidney injury and could provide new opportunities for renal research.

  16. International congress on DNA damage and repair: Book of abstracts

    SciTech Connect

    Not Available

    1987-01-01

    This document contains the abstracts of 105 papers presented at the Congress. Topics covered include the Escherichia coli nucleotide excision repair system, DNA repair in malignant transformations, defective DNA repair, and gene regulation. (TEM)

  17. DNA repair variants and breast cancer risk.

    PubMed

    Grundy, Anne; Richardson, Harriet; Schuetz, Johanna M; Burstyn, Igor; Spinelli, John J; Brooks-Wilson, Angela; Aronson, Kristan J

    2016-05-01

    A functional DNA repair system has been identified as important in the prevention of tumour development. Previous studies have hypothesized that common polymorphisms in DNA repair genes could play a role in breast cancer risk and also identified the potential for interactions between these polymorphisms and established breast cancer risk factors such as physical activity. Associations with breast cancer risk for 99 single nucleotide polymorphisms (SNPs) from genes in ten DNA repair pathways were examined in a case-control study including both Europeans (644 cases, 809 controls) and East Asians (299 cases, 160 controls). Odds ratios in both additive and dominant genetic models were calculated separately for participants of European and East Asian ancestry using multivariate logistic regression. The impact of multiple comparisons was assessed by correcting for the false discovery rate within each DNA repair pathway. Interactions between several breast cancer risk factors and DNA repair SNPs were also evaluated. One SNP (rs3213282) in the gene XRCC1 was associated with an increased risk of breast cancer in the dominant model of inheritance following adjustment for the false discovery rate (P < 0.05), although no associations were observed for other DNA repair SNPs. Interactions of six SNPs in multiple DNA repair pathways with physical activity were evident prior to correction for FDR, following which there was support for only one of the interaction terms (P < 0.05). No consistent associations between variants in DNA repair genes and breast cancer risk or their modification by breast cancer risk factors were observed.

  18. Repair of DNA Double-Strand Breaks

    NASA Astrophysics Data System (ADS)

    Falk, Martin; Lukasova, Emilie; Kozubek, Stanislav

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

  19. Human DNA repair and recombination genes

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs.

  20. Chromatin Remodeling, DNA Damage Repair and Aging

    PubMed Central

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

    2012-01-01

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

  1. DNA repair genes in the Megavirales pangenome.

    PubMed

    Blanc-Mathieu, Romain; Ogata, Hiroyuki

    2016-06-01

    The order 'Megavirales' represents a group of eukaryotic viruses with a large genome encoding a few hundred up to two thousand five hundred genes. Several members of Megavirales possess genes involved in major DNA repair pathways. Some of these genes were likely inherited from an ancient virus world and some others were derived from the genomes of their hosts. Here we examine molecular phylogenies of key DNA repair enzymes in light of recent hypotheses on the origin of Megavirales, and propose that the last common ancestors of the individual families of the order Megavirales already possessed DNA repair functions to achieve and maintain a moderately large genome and that this repair capacity gradually increased, in a family-dependent manner, during their recent evolution.

  2. The awakening of DNA repair at Yale.

    PubMed

    Hanawalt, Philip C

    2013-12-13

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

  3. The Awakening of DNA Repair at Yale

    PubMed Central

    Hanawalt, Philip C.

    2013-01-01

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

  4. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a) Purpose. Bacterial DNA damage or repair tests measure DNA damage which is expressed as differential...

  5. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a) Purpose. Bacterial DNA damage or repair tests measure DNA damage which is expressed as differential...

  6. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a) Purpose. Bacterial DNA damage or repair tests measure DNA damage which is expressed as differential...

  7. Mechanisms of Post-Replication DNA Repair

    PubMed Central

    Gao, Yanzhe; Mutter-Rottmayer, Elizabeth; Zlatanou, Anastasia; Vaziri, Cyrus; Yang, Yang

    2017-01-01

    Accurate DNA replication is crucial for cell survival and the maintenance of genome stability. Cells have developed mechanisms to cope with the frequent genotoxic injuries that arise from both endogenous and environmental sources. Lesions encountered during DNA replication are often tolerated by post-replication repair mechanisms that prevent replication fork collapse and avert the formation of DNA double strand breaks. There are two predominant post-replication repair pathways, trans-lesion synthesis (TLS) and template switching (TS). TLS is a DNA damage-tolerant and low-fidelity mode of DNA synthesis that utilizes specialized ‘Y-family’ DNA polymerases to replicate damaged templates. TS, however, is an error-free ‘DNA damage avoidance’ mode of DNA synthesis that uses a newly synthesized sister chromatid as a template in lieu of the damaged parent strand. Both TLS and TS pathways are tightly controlled signaling cascades that integrate DNA synthesis with the overall DNA damage response and are thus crucial for genome stability. This review will cover the current knowledge of the primary mediators of post-replication repair and how they are regulated in the cell. PMID:28208741

  8. Ribonucleotides in DNA: Origins, repair and consequences

    PubMed Central

    Williams, Jessica S.; Kunkel, Thomas A.

    2014-01-01

    While primordial life is thought to have been RNA-based (Cech, Cold Spring Harbor Perspect. Biol. 4 (2012) a006742), all living organisms store genetic information in DNA, which is chemically more stable. Distinctions between the RNA and DNA worlds and our views of “DNA” synthesis continue to evolve as new details emerge on the incorporation, repair and biological effects of ribonucleotides in DNA genomes of organisms from bacteria through humans. PMID:24794402

  9. DNA Repair and Personalized Breast Cancer Therapy

    PubMed Central

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

    2010-01-01

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

  10. DNA repair responses in human skin cells

    SciTech Connect

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

    1981-07-01

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

  11. The SMX DNA Repair Tri-nuclease.

    PubMed

    Wyatt, Haley D M; Laister, Rob C; Martin, Stephen R; Arrowsmith, Cheryl H; West, Stephen C

    2017-03-02

    The efficient removal of replication and recombination intermediates is essential for the maintenance of genome stability. Resolution of these potentially toxic structures requires the MUS81-EME1 endonuclease, which is activated at prometaphase by formation of the SMX tri-nuclease containing three DNA repair structure-selective endonucleases: SLX1-SLX4, MUS81-EME1, and XPF-ERCC1. Here we show that SMX tri-nuclease is more active than the three individual nucleases, efficiently cleaving replication forks and recombination intermediates. Within SMX, SLX4 co-ordinates the SLX1 and MUS81-EME1 nucleases for Holliday junction resolution, in a reaction stimulated by XPF-ERCC1. SMX formation activates MUS81-EME1 for replication fork and flap structure cleavage by relaxing substrate specificity. Activation involves MUS81's conserved N-terminal HhH domain, which mediates incision site selection and SLX4 binding. Cell cycle-dependent formation and activation of this tri-nuclease complex provides a unique mechanism by which cells ensure chromosome segregation and preserve genome integrity.

  12. Nucleotide excision repair and homologous recombination systems commit differentially to the repair of DNA-protein crosslinks.

    PubMed

    Nakano, Toshiaki; Morishita, Soh; Katafuchi, Atsushi; Matsubara, Mayumi; Horikawa, Yusuke; Terato, Hiroaki; Salem, Amir M H; Izumi, Shunsuke; Pack, Seung Pil; Makino, Keisuke; Ide, Hiroshi

    2007-10-12

    DNA-protein crosslinks (DPCs)-where proteins are covalently trapped on the DNA strand-block the progression of replication and transcription machineries and hence hamper the faithful transfer of genetic information. However, the repair mechanism of DPCs remains largely elusive. Here we have analyzed the roles of nucleotide excision repair (NER) and homologous recombination (HR) in the repair of DPCs both in vitro and in vivo using a bacterial system. Several lines of biochemical and genetic evidence show that both NER and HR commit to the repair or tolerance of DPCs, but differentially. NER repairs DPCs with crosslinked proteins of sizes less than 12-14 kDa, whereas oversized DPCs are processed exclusively by RecBCD-dependent HR. These results highlight how NER and HR are coordinated when cells need to deal with unusually bulky DNA lesions such as DPCs.

  13. Epigenetic reduction of DNA repair in progression to gastrointestinal cancer

    PubMed Central

    Bernstein, Carol; Bernstein, Harris

    2015-01-01

    Deficiencies in DNA repair due to inherited germ-line mutations in DNA repair genes cause increased risk of gastrointestinal (GI) cancer. In sporadic GI cancers, mutations in DNA repair genes are relatively rare. However, epigenetic alterations that reduce expression of DNA repair genes are frequent in sporadic GI cancers. These epigenetic reductions are also found in field defects that give rise to cancers. Reduced DNA repair likely allows excessive DNA damages to accumulate in somatic cells. Then either inaccurate translesion synthesis past the un-repaired DNA damages or error-prone DNA repair can cause mutations. Erroneous DNA repair can also cause epigenetic alterations (i.e., epimutations, transmitted through multiple replication cycles). Some of these mutations and epimutations may cause progression to cancer. Thus, deficient or absent DNA repair is likely an important underlying cause of cancer. Whole genome sequencing of GI cancers show that between thousands to hundreds of thousands of mutations occur in these cancers. Epimutations that reduce DNA repair gene expression and occur early in progression to GI cancers are a likely source of this high genomic instability. Cancer cells deficient in DNA repair are more vulnerable than normal cells to inactivation by DNA damaging agents. Thus, some of the most clinically effective chemotherapeutic agents in cancer treatment are DNA damaging agents, and their effectiveness often depends on deficient DNA repair in cancer cells. Recently, at least 18 DNA repair proteins, each active in one of six DNA repair pathways, were found to be subject to epigenetic reduction of expression in GI cancers. Different DNA repair pathways repair different types of DNA damage. Evaluation of which DNA repair pathway(s) are deficient in particular types of GI cancer and/or particular patients may prove useful in guiding choice of therapeutic agents in cancer therapy. PMID:25987950

  14. Epigenetic reduction of DNA repair in progression to gastrointestinal cancer.

    PubMed

    Bernstein, Carol; Bernstein, Harris

    2015-05-15

    Deficiencies in DNA repair due to inherited germ-line mutations in DNA repair genes cause increased risk of gastrointestinal (GI) cancer. In sporadic GI cancers, mutations in DNA repair genes are relatively rare. However, epigenetic alterations that reduce expression of DNA repair genes are frequent in sporadic GI cancers. These epigenetic reductions are also found in field defects that give rise to cancers. Reduced DNA repair likely allows excessive DNA damages to accumulate in somatic cells. Then either inaccurate translesion synthesis past the un-repaired DNA damages or error-prone DNA repair can cause mutations. Erroneous DNA repair can also cause epigenetic alterations (i.e., epimutations, transmitted through multiple replication cycles). Some of these mutations and epimutations may cause progression to cancer. Thus, deficient or absent DNA repair is likely an important underlying cause of cancer. Whole genome sequencing of GI cancers show that between thousands to hundreds of thousands of mutations occur in these cancers. Epimutations that reduce DNA repair gene expression and occur early in progression to GI cancers are a likely source of this high genomic instability. Cancer cells deficient in DNA repair are more vulnerable than normal cells to inactivation by DNA damaging agents. Thus, some of the most clinically effective chemotherapeutic agents in cancer treatment are DNA damaging agents, and their effectiveness often depends on deficient DNA repair in cancer cells. Recently, at least 18 DNA repair proteins, each active in one of six DNA repair pathways, were found to be subject to epigenetic reduction of expression in GI cancers. Different DNA repair pathways repair different types of DNA damage. Evaluation of which DNA repair pathway(s) are deficient in particular types of GI cancer and/or particular patients may prove useful in guiding choice of therapeutic agents in cancer therapy.

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

    SciTech Connect

    Phoebe L. Stewart

    2005-06-27

    objective is to locate the kinase domain of DNA-PKcs by determining the structure of a kinase deletion mutant both as an isolated protein and as part of a DNA-PKcs/Ku/DNA complex. A third objective is to pursue higher resolution studies of DNA-PKcs and the DNA-PKcs/Ku/DNA complex. If the crystal structure determination of DNA-PKcs is completed during the project period, the atomic coordinates of DNA-PKcs will be modeled within the cryo-EM structure of the complex. In order to achieve these goals, a collaborative effort is proposed between Dr. Phoebe Stewart at UCLA, whose laboratory has expertise in cryo-EM reconstruction methods, and Dr. David Chen at the Lawrence Berkeley National Laboratory, who has a long-standing interest in DNA repair. Advantages of the cryo-EM structural method include the fact that the sample is imaged in a frozen-hydrated and unstained state, avoiding artifacts associated with drying and staining in other EM approaches. Also crystals of the sample are not needed for the single particle reconstruction method and only microgram quantities of sample are required. Cryo-EM structural information of macromolecular assemblies is complementary to both atomic structures of individual component molecules, as well as low resolution information obtained from x-ray and neutron scattering. Knowledge of the geometrical arrangement of the complex, and the position of the essential DNA-PKcs kinase domain, should lead to a greater understanding of the molecular events in DNA double-strand break repair following exposure to low doses of radiation.

  16. Aging processes, DNA damage, and repair.

    PubMed

    Gilchrest, B A; Bohr, V A

    1997-04-01

    The second triennial FASEB Summer Research Conference on "Clonal Senescence and Differentiation" (August 17-22, 1996) focused on the interrelationships between aging processes and DNA damage and repair. The attendees represented a cross section of senior and junior investigators working in fields ranging from classic cellular gerontology to yeast and nematode models of aging to basic mechanisms of DNA damage and repair. The meeting opened with a keynote address by Dr. Bruce Ames that emphasized the documented relationships between oxidative damage, cancer, and aging. This was followed by eight platform sessions, one poster discussion, one featured presentation, and an after-dinner address. The following sections highlight the key points discussed.

  17. Oxidatively induced DNA damage and its repair in cancer.

    PubMed

    Dizdaroglu, Miral

    2015-01-01

    Oxidatively induced DNA damage is caused in living organisms by endogenous and exogenous reactive species. DNA lesions resulting from this type of damage are mutagenic and cytotoxic and, if not repaired, can cause genetic instability that may lead to disease processes including carcinogenesis. Living organisms possess DNA repair mechanisms that include a variety of pathways to repair multiple DNA lesions. Mutations and polymorphisms also occur in DNA repair genes adversely affecting DNA repair systems. Cancer tissues overexpress DNA repair proteins and thus develop greater DNA repair capacity than normal tissues. Increased DNA repair in tumors that removes DNA lesions before they become toxic is a major mechanism for development of resistance to therapy, affecting patient survival. Accumulated evidence suggests that DNA repair capacity may be a predictive biomarker for patient response to therapy. Thus, knowledge of DNA protein expressions in normal and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. DNA repair proteins constitute targets for inhibitors to overcome the resistance of tumors to therapy. Inhibitors of DNA repair for combination therapy or as single agents for monotherapy may help selectively kill tumors, potentially leading to personalized therapy. Numerous inhibitors have been developed and are being tested in clinical trials. The efficacy of some inhibitors in therapy has been demonstrated in patients. Further development of inhibitors of DNA repair proteins is globally underway to help eradicate cancer.

  18. How SUMOylation Fine-Tunes the Fanconi Anemia DNA Repair Pathway

    PubMed Central

    Coleman, Kate E.; Huang, Tony T.

    2016-01-01

    Fanconi anemia (FA) is a rare human genetic disorder characterized by developmental defects, bone marrow failure and cancer predisposition, primarily due to a deficiency in the repair of DNA interstrand crosslinks (ICLs). ICL repair through the FA DNA repair pathway is a complicated multi-step process, involving at least 19 FANC proteins and coordination of multiple DNA repair activities, including homologous recombination, nucleotide excision repair and translesion synthesis (TLS). SUMOylation is a critical regulator of several DNA repair pathways, however, the role of this modification in controlling the FA pathway is poorly understood. Here, we summarize recent advances in the fine-tuning of the FA pathway by small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligases (STUbLs) and other SUMO-related interactions, and discuss the implications of these findings in the design of novel therapeutics for alleviating FA-associated condition, including cancer. PMID:27148358

  19. The RecQ DNA helicases in DNA Repair

    PubMed Central

    Bernstein, Kara A.; Gangloff, Serge; Rothstein, Rodney

    2014-01-01

    The RecQ helicases are conserved from bacteria to humans and play a critical role in genome stability. In humans, loss of RecQ gene function is associated with cancer predisposition and/or premature aging. Recent data have shown that the RecQ helicases function during two distinct steps during DNA repair; DNA end resection and resolution of double Holliday junctions (dHJs). RecQ functions in these different processing steps has important implications for its role in repair of double-strand breaks (DSBs) that occur during DNA replication, meiosis and at specific genomic loci such as telomeres. PMID:21047263

  20. Early days of DNA repair: discovery of nucleotide excision repair and homology-dependent recombinational repair.

    PubMed

    Rupp, W Dean

    2013-12-13

    The discovery of nucleotide excision repair in 1964 showed that DNA could be repaired by a mechanism that removed the damaged section of a strand and replaced it accurately by using the remaining intact strand as the template. This result showed that DNA could be actively metabolized in a process that had no precedent. In 1968, experiments describing postreplication repair, a process dependent on homologous recombination, were reported. The authors of these papers were either at Yale University or had prior Yale connections. Here we recount some of the events leading to these discoveries and consider the impact on further research at Yale and elsewhere.

  1. Flavonoids and DNA Repair in Prostate Cancer

    DTIC Science & Technology

    2004-12-01

    responsible to fill the gap created by the excision of 8-OHdG. There is in vitro evidence that some flavonoids such as myricetin and baicalin will...myricetin. Methods Enzymol., 335, 308-316. 4. Chen,X., Nishida,H., and Konishi,T. (2003) Baicalin promoted the repair of DNA single strand breakage caused by

  2. Databases and Bioinformatics Tools for the Study of DNA Repair

    PubMed Central

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

    2011-01-01

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

  3. Energy and Technology Review: Unlocking the mysteries of DNA repair

    SciTech Connect

    Quirk, W.A.

    1993-04-01

    DNA, the genetic blueprint, has the remarkable property of encoding its own repair following diverse types of structural damage induced by external agents or normal metabolism. We are studying the interplay of DNA damaging agents, repair genes, and their protein products to decipher the complex biochemical pathways that mediate such repair. Our research focuses on repair processes that correct DNA damage produced by chemical mutagens and radiation, both ionizing and ultraviolet. The most important type of DNA repair in human cells is called excision repair. This multistep process removes damaged or inappropriate pieces of DNA -- often as a string of 29 nucleotides containing the damage -- and replaces them with intact ones. We have isolated, cloned, and mapped several human repair genes associated with the nucleotide excision repair pathway and involved in the repair of DNA damage after exposure to ultraviolet light or mutagens in cooked food. We have shown that a defect in one of these repair genes, ERCC2, is responsible for the repair deficiency in one of the groups of patients with the recessive genetic disorder xeroderma pigmentosum (XP group D). We are exploring ways to purify sufficient quantities (milligrams) of the protein products of these and other repair genes so that we can understand their functions. Our long-term goals are to link defective repair proteins to human DNA repair disorders that predispose to cancer, and to produce DNA-repair-deficient mice that can serve as models for the human disorders.

  4. A history of the DNA repair and mutagenesis field: The discovery of base excision repair.

    PubMed

    Friedberg, Errol C

    2016-01-01

    This article reviews the early history of the discovery of an DNA repair pathway designated as base excision repair (BER), since in contrast to the enzyme-catalyzed removal of damaged bases from DNA as nucleotides [called nucleotide excision repair (NER)], BER involves the removal of damaged or inappropriate bases, such as the presence of uracil instead of thymine, from DNA as free bases.

  5. The nucleosome: orchestrating DNA damage signaling and repair within chromatin.

    PubMed

    Agarwal, Poonam; Miller, Kyle M

    2016-10-01

    DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.

  6. Importance of DNA repair in tumor suppression

    NASA Astrophysics Data System (ADS)

    Brumer, Yisroel; Shakhnovich, Eugene I.

    2004-12-01

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

  7. Stability of nucleosome placement in newly repaired regions of DNA

    SciTech Connect

    Nissen, K.A.; Lan, S.Y.; Smerdon, M.J.

    1986-07-05

    Rearrangements of chromatin structure during excision repair of UV-damaged DNA appear to involve unfolding of nucleosomal DNA while repair is taking place, followed by refolding of this DNA into a native nucleosome structure. Recently, we found that repair patches are not distributed uniformly along the DNA in nucleosome core particles immediately following their refolding into nucleosomes. Therefore, the distribution of repair patches in nucleosome core DNA was used to monitor the stability of nucleosome placement in these regions. Our results indicate that in nondividing human cells undergoing excision repair there is a slow change in the positioning of nucleosomes in newly repaired regions of chromatin, resulting in the eventual randomization of repair patches in nucleosome core DNA. Furthermore, the nonrandom placement of nucleosomes observed just after the refolding event is not re-established during DNA replication. Possible mechanisms for this change in nucleosome placement along the DNA are discussed.

  8. Envisioning the molecular choreography of DNA base excision repair.

    PubMed

    Parikh, S S; Mol, C D; Hosfield, D J; Tainer, J A

    1999-02-01

    Recent breakthroughs integrate individual DNA repair enzyme structures, biochemistry and biology to outline the structural cell biology of the DNA base excision repair pathways that are essential to genome integrity. Thus, we are starting to envision how the actions, movements, steps, partners and timing of DNA repair enzymes, which together define their molecular choreography, are elegantly controlled by both the nature of the DNA damage and the structural chemistry of the participating enzymes and the DNA double helix.

  9. SUMO-mediated regulation of DNA damage repair and responses

    PubMed Central

    Sarangi, Prabha; Zhao, Xiaolan

    2015-01-01

    Sumoylation plays important roles during DNA damage repair and responses. Recent broad-scope and substrate-based studies have shed light on the regulation and significance of sumoylation during these processes. An emerging paradigm is that sumoylation of many DNA metabolism proteins is controlled by DNA engagement. Such “on-site modification” can explain low substrate modification levels and has important implications in sumoylation mechanisms and effects. New studies also suggest that sumoylation can regulate a process through an ensemble effect or via major substrates. Additionally, we describe new trends in the functional effects of sumoylation, such as bi-directional changes in biomolecule binding and multi-level coordination with other modifications. These emerging themes and models will stimulate our thinking and research in sumoylation and genome maintenance. PMID:25778614

  10. DNA repair genes of mammalian cells

    SciTech Connect

    Thompson, L.H.; Brookman, K.W.; Salazar, E.P.; Fuscoe, J.C.; Weber, C.A.

    1985-09-27

    In the CHO cell line various mutations affecting DNA repair have been obtained. Mutants that belong to five genetic complementation groups for UV sensitivity and resemble the cells from individuals having the cancer-prone genetic disorder xeroderma pigmentosum were previously identified. Each mutant is defective in the incision step of nucleotide excision repair and hypersensitive to bulky DNA lesions. A sixth genetic complementation group for UV sensitivity has now been identified with UV27-1. These UV mutants can be divided into two subgroups; only Groups 2 and 4 are extremely sensitive to mitomycin C and other DNA cross-linking agents. The clear-cut phenotypes of the CHO mutants have allowed us to construct hybrid cells by fusion with human lymphocytes and thereby identify which human chromosomes carry genes that correct the CHO mutations. The first two mutants analyzed, UV20 (excision-repair deficient; UV Group 2) and EM9, which has very high SCE, are both corrected by chromosome 19. 46 refs., 3 figs.

  11. DNA Repair Defects and Chromosomal Aberrations

    NASA Technical Reports Server (NTRS)

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

    2009-01-01

    Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

  12. Targeting DNA repair pathways for cancer treatment: what's new?

    PubMed

    Kelley, Mark R; Logsdon, Derek; Fishel, Melissa L

    2014-05-01

    Disruptions in DNA repair pathways predispose cells to accumulating DNA damage. A growing body of evidence indicates that tumors accumulate progressively more mutations in DNA repair proteins as cancers progress. DNA repair mechanisms greatly affect the response to cytotoxic treatments, so understanding those mechanisms and finding ways to turn dysregulated repair processes against themselves to induce tumor death is the goal of all DNA repair inhibition efforts. Inhibition may be direct or indirect. This burgeoning field of research is replete with promise and challenge, as more intricacies of each repair pathway are discovered. In an era of increasing concern about healthcare costs, use of DNA repair inhibitors can prove to be highly effective stewardship of R&D resources and patient expenses.

  13. The RecQ DNA helicases in DNA repair.

    PubMed

    Bernstein, Kara A; Gangloff, Serge; Rothstein, Rodney

    2010-01-01

    The RecQ helicases are conserved from bacteria to humans and play a critical role in genome stability. In humans, loss of RecQ gene function is associated with cancer predisposition and/or premature aging. Recent experiments have shown that the RecQ helicases function during distinct steps during DNA repair; DNA end resection, displacement-loop (D-loop) processing, branch migration, and resolution of double Holliday junctions (dHJs). RecQ function in these different processing steps has important implications for its role in repair of double-strand breaks (DSBs) that occur during DNA replication and meiosis, as well as at specific genomic loci such as telomeres.

  14. Ancient bacteria show evidence of DNA repair

    PubMed Central

    Johnson, Sarah Stewart; Hebsgaard, Martin B.; Christensen, Torben R.; Mastepanov, Mikhail; Nielsen, Rasmus; Munch, Kasper; Brand, Tina; Gilbert, M. Thomas P.; Zuber, Maria T.; Bunce, Michael; Rønn, Regin; Gilichinsky, David; Froese, Duane; Willerslev, Eske

    2007-01-01

    Recent claims of cultivable ancient bacteria within sealed environments highlight our limited understanding of the mechanisms behind long-term cell survival. It remains unclear how dormancy, a favored explanation for extended cellular persistence, can cope with spontaneous genomic decay over geological timescales. There has been no direct evidence in ancient microbes for the most likely mechanism, active DNA repair, or for the metabolic activity necessary to sustain it. In this paper, we couple PCR and enzymatic treatment of DNA with direct respiration measurements to investigate long-term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence of bacterial survival in samples up to half a million years in age, making this the oldest independently authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence that this long-term survival is closely tied to cellular metabolic activity and DNA repair that over time proves to be superior to dormancy as a mechanism in sustaining bacteria viability. PMID:17728401

  15. Nucleotide excision repair of DNA: The very early history.

    PubMed

    Friedberg, Errol C

    2011-07-15

    This article, taken largely from the book Correcting the Blueprint of Life: An Historical Account of the Discovery of DNA Repair Mechanisms, summarizes the very early history of the discovery of nucleotide excision repair.

  16. DNA Repair in Human Cells Exposed to Combinations of Carcinogenic Agents

    SciTech Connect

    Setlow, R. B.; Ahmed, F. E.

    1980-01-01

    Normal human and XP2 fibroblasts were treated with UV plus UV-mimetic chemicals. The UV dose used was sufficient to saturate the UV excision repair system. Excision repair after combined treatments was estimated by unscheduled DNA synthesis, BrdUrd photolysis, and the loss of sites sensitive to a UV specific endonuclease. Since the repair of damage from UV and its mimetics is coordinately controlled we expected that there would be similar rate-limiting steps in the repair of UV and chemical damage and that after a combined treatment the total amount of repair would be the same as from UV or the chemicals separately. The expectation was not fulfilled. In normal cells repair after a combined treatment was additive whereas in XP cells repair after a combined treatment was usually less than after either agent separately. The chemicals tested were AAAF, DMBA-epoxide, 4NQO, and ICR-170.

  17. DNA-mediated charge transport for DNA repair

    PubMed Central

    Boon, Elizabeth M.; Livingston, Alison L.; Chmiel, Nikolas H.; David, Sheila S.; Barton, Jacqueline K.

    2003-01-01

    MutY, like many DNA base excision repair enzymes, contains a [4Fe4S]2+ cluster of undetermined function. Electrochemical studies of MutY bound to a DNA-modified gold electrode demonstrate that the [4Fe4S] cluster of MutY can be accessed in a DNA-mediated redox reaction. Although not detectable without DNA, the redox potential of DNA-bound MutY is ≈275 mV versus NHE, which is characteristic of HiPiP iron proteins. Binding to DNA is thus associated with a change in [4Fe4S]3+/2+ potential, activating the cluster toward oxidation. Given that DNA charge transport chemistry is exquisitely sensitive to perturbations in base pair structure, such as mismatches, we propose that this redox process of MutY bound to DNA exploits DNA charge transport and provides a DNA signaling mechanism to scan for mismatches and lesions in vivo. PMID:14559969

  18. Endonucleases involved in repair and recombination of DNA

    SciTech Connect

    Linn, S.M.

    1988-01-01

    When our DOE support began as a contract in 1970, from the AEC, it was our intent to begin to understand how several enzymes which we had detected in E. coli might be involved in DNA recombination and repair. These studies led to our characterization of the recBC DNase (exonuclease 5) as well as endonucleases 3 and 5. As research supported by that contract progressed, we expanded our interests to include mammalian enzymes involved in base excision repair, most notably AP endonucleases, DNA glycosylases and DNA purine insertase. A logical next step involved the inclusion of DNA polymerases into our studies of repair. Current progress includes research on: isolation of xeroderma pigmentosum correction factors; isolation of ultraviolet (UV) endonucleases; mitochondrial repair enzymes; alkylation damage repair; comparisons of repair in normal diploid, transformed, and non-mitotic cells; and repair reactions by DNA polymerases.

  19. A brief history of the DNA repair field.

    PubMed

    Friedberg, Errol C

    2008-01-01

    The history of the repair of damaged DNA can be traced to the mid-1930s. Since then multiple DNA repair mechanisms, as well as other biological responses to DNA damage, have been discovered and their regulation has been studied. This article briefly recounts the early history of this field.

  20. HSP90 regulates DNA repair via the interaction between XRCC1 and DNA polymerase β

    PubMed Central

    Fang, Qingming; Inanc, Burcu; Schamus, Sandy; Wang, Xiao-hong; Wei, Leizhen; Brown, Ashley R.; Svilar, David; Sugrue, Kelsey F.; Goellner, Eva M.; Zeng, Xuemei; Yates, Nathan A.; Lan, Li; Vens, Conchita; Sobol, Robert W.

    2014-01-01

    Cellular DNA repair processes are crucial to maintain genome stability and integrity. In DNA base excision repair, a tight heterodimer complex formed by DNA polymerase β (Polβ) and XRCC1 is thought to facilitate repair by recruiting Polβ to DNA damage sites. Here we show that disruption of the complex does not impact DNA damage response or DNA repair. Instead, the heterodimer formation is required to prevent ubiquitylation and degradation of Polβ. In contrast, the stability of the XRCC1 monomer is protected from CHIP-mediated ubiquitylation by interaction with the binding partner HSP90. In response to cellular proliferation and DNA damage, proteasome and HSP90-mediated regulation of Polβ and XRCC1 alters the DNA repair complex architecture. We propose that protein stability, mediated by DNA repair protein complex formation, functions as a regulatory mechanism for DNA repair pathway choice in the context of cell cycle progression and genome surveillance. PMID:25423885

  1. Mechanism of DNA loading by the DNA repair helicase XPD

    PubMed Central

    Constantinescu-Aruxandei, Diana; Petrovic-Stojanovska, Biljana; Penedo, J. Carlos; White, Malcolm F.; Naismith, James H.

    2016-01-01

    The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5′ to 3′ helicase with an essential iron–sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase from Thermoplasma acidophilum has been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD from Sulfolobus acidocaldiarius that lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD. PMID:26896802

  2. DNA repair and radiation sensitivity in mammalian cells

    SciTech Connect

    Chen, D.J.C.; Stackhouse, M. ); Chen, D.S. . Dept. of Radiation Oncology)

    1993-01-01

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

  3. DNA repair and radiation sensitivity in mammalian cells

    SciTech Connect

    Chen, D.J.C.; Stackhouse, M.; Chen, D.S.

    1993-02-01

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

  4. Structure of the DNA repair helicase XPD

    PubMed Central

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

    2012-01-01

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

  5. Participation of DNA repair in the response to 5-fluorouracil

    PubMed Central

    Wyatt, Michael D.; Wilson, David M.

    2008-01-01

    The anti-metabolite 5-fluorouracil (5-FU) is employed clinically to manage solid tumors including colorectal and breast cancer. Intracellular metabolites of 5-FU can exert cytotoxic effects via inhibition of thymidylate synthetase, or through incorporation into RNA and DNA, events that ultimately activate apoptosis. In this review, we cover the current data implicating DNA repair processes in cellular responsiveness to 5-FU treatment. Evidence points to roles for base excision repair (BER) and mismatch repair (MMR). However, mechanistic details remain unexplained, and other pathways have not been exhaustively interrogated. Homologous recombination is of particular interest, because it resolves unrepaired DNA intermediates not properly dealt with by BER or MMR. Furthermore, crosstalk among DNA repair pathways and S-phase checkpoint signaling has not been examined. Ongoing efforts aim to design approaches and reagents that (i) approximate repair capacity and (ii) mediate strategic regulation of DNA repair in order to improve the efficacy of current anti-cancer treatments. PMID:18979208

  6. Nonuniform distribution of excision repair synthesis in nucleosome core DNA

    SciTech Connect

    Lan, S.Y.; Smerdon, M.J.

    1985-12-17

    We have studied the distribution in nucleosome core DNA of nucleotides incorporated by excision repair synthesis occurring immediately after UV irradiation in human cells. The differences previously observed for whole nuclei between the DNase I digestion profiles of repaired DNA (following its refolding into a nucleosome structure) and bulk DNA are obtained for isolated nucleosome core particles. Analysis of the differences obtained indicates that they could reflect a significant difference in the level of repair-incorporated nucleotides at different sites within the core DNA region. To test this possibility directly, we have used exonuclease III digestion of very homogeneous sized core particle DNA to map the distribution of repair synthesis in these regions. Results indicate that in a significant fraction of the nucleosomes the 5' and 3' ends of the core DNA are markedly enhanced in repair-incorporated nucleotides relative to the central region of the core particle. A best fit analysis indicates that a good approximation of the data is obtained for a distribution where the core DNA is uniformly labeled from the 5' end to position 62 and from position 114 to the 3' end, with the 52-base central region being devoid of repair-incorporated nucleotides. This distribution accounts for all of the quantitative differences observed previously between repaired DNA and bulk DNA following the rapid phase of nucleosome rearrangement when it is assumed that linker DNA and the core DNA ends are repaired with equal efficiency and the nucleosome structure of newly repaired DNA is identical with that of bulk chromatin. The 52-base central region that is devoid of repair synthesis contains the lowest frequency cutting sites for DNase I in vitro, as well as the only internal locations where two (rather than one) histones interact with a 10-base segment of each DNA strand.

  7. DNA Repair and Genome Maintenance in Bacillus subtilis

    PubMed Central

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

    2012-01-01

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

  8. Princess Takamatsu Symposium on DNA Repair and Human Cancers

    PubMed Central

    Loeb, Lawrence A.; Nishimura, Susumu

    2013-01-01

    The 40th International Symposium of the Princess Takamatsu Cancer Research Fund, entitled “DNA Repair and Human Cancers” was held on November 10–12, 2009 at Hotel Grand Palace, Tokyo, Japan. The meeting focused on the role of DNA repair in preventing mutations by endogenous and exogenous DNA damage and increasing the efficacy of chemotherapeutic agents by interfering with DNA repair. The fourteen presentations by the speakers from U.S.A., four from U.K., one each from Italy, The Netherlands and France, and thirteen from Japan, covered most aspects of DNA repair spanning DNA damage, molecular structures of repair enzymes, and clinical studies on inhibition of DNA repair processes. Extensive time was reserved for discussions with the active participation of the 150 invited Japanese scientists. The choice of a symposium on DNA repair in human cancers resulted in part from the excellent basic and clinical studies that have been carried out for many years in Japan, and the general lack of recognition vs. the importance of DNA repair in understanding carcinogenesis. PMID:20460534

  9. DNA mismatch repair and the DNA damage response

    PubMed Central

    Li, Zhongdao; Pearlman, Alexander H.; Hsieh, Peggy

    2015-01-01

    This review discusses the role of DNA mismatch repair (MMR) in the DNA damage response (DDR) that triggers cell cycle arrest and, in some cases, apoptosis. Although the focus is on findings from mammalian cells, much has been learned from studies in other organisms including bacteria and yeast [1,2]. MMR promotes a DDR mediated by a key signaling kinase, ATM and Rad3-related (ATR), in response to various types of DNA damage including some encountered in widely used chemotherapy regimes. An introduction to the DDR mediated by ATR reveals its immense complexity and highlights the many biological and mechanistic questions that remain. Recent findings and future directions are highlighted. PMID:26704428

  10. Alternative DNA base pairing through metal coordination.

    PubMed

    Clever, Guido H; Shionoya, Mitsuhiko

    2012-01-01

    Base-pairing in the naturally occurring DNA and RNA oligonucleotide duplexes is based on π-stacking, hydrogen bonding, and shape complementarity between the nucleobases adenine, thymine, guanine, and cytosine as well as on the hydrophobic-hydrophilic balance in aqueous media. This complex system of multiple supramolecular interactions is the product of a long-term evolutionary process and thus highly optimized to serve its biological functions such as information storage and processing. After the successful implementation of automated DNA synthesis, chemists have begun to introduce artificial modifications inside the core of the DNA double helix in order to study various aspects of base pairing, generate new base pairs orthogonal to the natural ones, and equip the biopolymer with entirely new functions. The idea to replace the hydrogen bonding interactions with metal coordination between ligand-like nucleosides and suitable transition metal ions culminated in the development of a plethora of artificial base-pairing systems termed "metal base-pairs" which were shown to strongly enhance the DNA duplex stability. Furthermore, they show great potential for the use of DNA as a molecular wire in nanoscale electronic architectures. Although single electrons have proven to be transmitted by natural DNA over a distance of several base pairs, the high ohmic resistance of unmodified oligonucleotides was identified as a serious obstacle. By exchanging some or all of the Watson-Crick base pairs in DNA with metal complexes, this problem may be solved. In the future, these research efforts are supposed to lead to DNA-like materials with superior conductivity for nano-electronic applications. Other fields of potential application such as DNA-based supramolecular architecture and catalysis may be strongly influenced by these developments as well. This text is meant to illustrate the basic concepts of metal-base pairing and give an outline over recent developments in this field.

  11. Transcript RNA supports precise repair of its own DNA gene.

    PubMed

    Keskin, Havva; Meers, Chance; Storici, Francesca

    2016-01-01

    The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells transcribe abundant amount of RNA with a wide range of functions, it has been difficult to uncover whether RNA can serve as a template for DNA repair and recombination. An increasing number of experimental evidences suggest a direct role of RNA in DNA modification. Recently, we demonstrated that endogenous transcript RNA can serve as a template to repair a DNA double-strand break (DSB), the most harmful DNA lesion, not only indirectly via formation of a DNA copy (cDNA) intermediate, but also directly in a homology driven mechanism in budding yeast. These results point out that the transfer of genetic information from RNA to DNA is more general than previously thought. We found that transcript RNA is more efficient in repairing a DSB in its own DNA (in cis) than in a homologous but ectopic locus (in trans). Here, we summarize current knowledge about the process of RNA-driven DNA repair and recombination, and provide further data in support of our model of DSB repair by transcript RNA in cis. We show that a DSB is precisely repaired predominately by transcript RNA and not by residual cDNA in conditions in which formation of cDNA by reverse transcription is inhibited. Additionally, we demonstrate that defects in ribonuclease (RNase) H stimulate precise DSB repair by homologous RNA or cDNA sequence, and not by homologous DNA sequence carried on a plasmid. These results highlight an antagonistic role of RNase H in RNA-DNA recombination. Ultimately, we discuss several questions that should be addressed to better understand mechanisms and implications of RNA-templated DNA repair and recombination.

  12. DNA repair systems as targets of cadmium toxicity

    SciTech Connect

    Giaginis, Constantinos; Gatzidou, Elisavet; Theocharis, Stamatios . E-mail: theocharis@ath.forthnet.gr

    2006-06-15

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

  13. DNA repair mechanisms in cancer development and therapy

    PubMed Central

    Torgovnick, Alessandro; Schumacher, Björn

    2015-01-01

    DNA damage has been long recognized as causal factor for cancer development. When erroneous DNA repair leads to mutations or chromosomal aberrations affecting oncogenes and tumor suppressor genes, cells undergo malignant transformation resulting in cancerous growth. Genetic defects can predispose to cancer: mutations in distinct DNA repair systems elevate the susceptibility to various cancer types. However, DNA damage not only comprises a root cause for cancer development but also continues to provide an important avenue for chemo- and radiotherapy. Since the beginning of cancer therapy, genotoxic agents that trigger DNA damage checkpoints have been applied to halt the growth and trigger the apoptotic demise of cancer cells. We provide an overview about the involvement of DNA repair systems in cancer prevention and the classes of genotoxins that are commonly used for the treatment of cancer. A better understanding of the roles and interactions of the highly complex DNA repair machineries will lead to important improvements in cancer therapy. PMID:25954303

  14. DNA Damage Repair in the Context of Plant Chromatin1

    PubMed Central

    2015-01-01

    The integrity of DNA molecules is constantly challenged. All organisms have developed mechanisms to detect and repair multiple types of DNA lesions. The basic principles of DNA damage repair (DDR) in prokaryotes and unicellular and multicellular eukaryotes are similar, but the association of DNA with nucleosomes in eukaryotic chromatin requires mechanisms that allow access of repair enzymes to the lesions. This is achieved by chromatin-remodeling factors, and their necessity for efficient DDR has recently been demonstrated for several organisms and repair pathways. Plants share many features of chromatin organization and DNA repair with fungi and animals, but they differ in other, important details, which are both interesting and relevant for our understanding of genome stability and genetic diversity. In this Update, we compare the knowledge of the role of chromatin and chromatin-modifying factors during DDR in plants with equivalent systems in yeast and humans. We emphasize plant-specific elements and discuss possible implications. PMID:26089404

  15. Repair of damaged DNA in vivo: Final technical report

    SciTech Connect

    Hanawalt, P.C.

    1987-09-01

    This contract was initiated in 1962 with the US Atomic Energy Commission to carry out basic research on the effects of radiation on the process of DNA replication in bacteria. Within the first contract year we discovered repair replication at the same time that Setlow and Carrier discovered pyrimidine dimer excision. These discoveries led to the elucidation of the process of excision-repair, one of the most important mechanisms by which living systems, including humans, respond to structural damage in their genetic material. We improved methodology for distinguishing repair replication from semiconservative replication and instructed others in these techniques. Painter then was the first to demonstrate repair replication in ultraviolet irradiated human cells. He, in turn, instructed James Cleaver who discovered that skin fibroblasts from patients with xeroderma pigmentosum were defective in excision-repair. People with this genetic defect are extremely sensitive to sunlight and they develop carcinomas and melanomas of the skin with high frequency. The existence of this hereditary disease attests to the importance of DNA repair in man. We certainly could not survive in the normal ultraviolet flux from the sun if our DNA were not continuously monitored for damage and repaired. Other hereditary diseases such as ataxia telangiectasia, Cockayne's syndrome, Blooms syndrome and Fanconi's anemia also involve deficiencies in DNA damage processing. The field of DNA repair has developed rapidly as we have learned that most environmental chemical carcinogens as well as radiation produce repairable damage in DNA. 251 refs.

  16. DNA repair investigations using siRNA.

    PubMed

    Miller, Holly; Grollman, Arthur P

    2003-06-11

    Small interfering RNA (siRNA) is a revolutionary tool for the experimental modulation of gene expression, in many cases making redundant the need for specific gene mutations and allowing examination of the effect of modulating essential genes. It has now been shown that siRNA phenotypes resulting from stable transfection with short hairpin RNA (shRNA) can be transmitted through the mouse germ line and Rosenquist and his colleagues have used shRNA, which is processed in vivo to siRNA, to create germline transgenic mice in which a target DNA repair gene has been silenced. Here, Holly Miller and Arthur P. Grollman give the background of these discoveries, provide an overview of current uses, and look at future applications of this research.

  17. Mystery of DNA repair: the role of the MRN complex and ATM kinase in DNA damage repair.

    PubMed

    Czornak, Kamila; Chughtai, Sanaullah; Chrzanowska, Krystyna H

    2008-01-01

    Genomes are subject to a number of exogenous or endogenous DNA-damaging agents that cause DNA double-strand breaks (DSBs). These critical DNA lesions can result in cell death or a wide variety of genetic alterations, including deletions, translocations, loss of heterozygosity, chromosome loss, or chromosome fusions, which enhance genome instability and can trigger carcinogenesis. The cells have developed an efficient mechanism to cope with DNA damages by evolving the DNA repair machinery. There are 2 major DSB repair mechanisms: nonhomologous end joining (NHEJ) and homologous recombination (HR). One element of the repair machinery is the MRN complex, consisting of MRE11, RAD50 and NBN (previously described as NBS1), which is involved in DNA replication, DNA repair, and signaling to the cell cycle checkpoints. A number of kinases, like ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad-3-related), and DNA PKcs (DNA protein kinase catalytic subunit), phosphorylate various protein targets in order to repair the damage. If the damage cannot be repaired, they direct the cell to apoptosis. The MRN complex as well as repair kinases are also involved in telomere maintenance and genome stability. The dysfunction of particular elements involved in the repair mechanisms leads to genome instability disorders, like ataxia telangiectasia (A-T), A-T-like disorder (ATLD) and Nijmegen breakage syndrome (NBS). The mutated genes responsible for these disorders code for proteins that play key roles in the process of DNA repair. Here we present a detailed review of current knowledge on the MRN complex, kinases engaged in DNA repair, and genome instability disorders.

  18. Cloning of Salmonella typhimurium DNA encoding mutagenic DNA repair

    SciTech Connect

    Thomas, S.M.; Sedgwick, S.G. )

    1989-11-01

    Mutagenic DNA repair in Escherichia coli is encoded by the umuDC operon. Salmonella typhimurium DNA which has homology with E. coli umuC and is able to complement E. coli umuC122::Tn5 and umuC36 mutations has been cloned. Complementation of umuD44 mutants and hybridization with E. coli umuD also occurred, but these activities were much weaker than with umuC. Restriction enzyme mapping indicated that the composition of the cloned fragment is different from the E. coli umuDC operon. Therefore, a umu-like function of S. typhimurium has been found; the phenotype of this function is weaker than that of its E. coli counterpart, which is consistent with the weak mutagenic response of S. typhimurium to UV compared with the response in E. coli.

  19. DNA repair in cancer: emerging targets for personalized therapy

    PubMed Central

    Abbotts, Rachel; Thompson, Nicola; Madhusudan, Srinivasan

    2014-01-01

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

  20. DNA base excision repair nanosystem engineering: model development.

    PubMed

    Sokhansanj, B A

    2005-01-01

    DNA base damage results from a combination of endogenous sources, (normal metabolism, increased metabolism due to obesity, stress from diseases such as arthritis and diabetes, and ischemia) and the environment (ingested toxins, ionizing radiation, etc.). If unrepaired DNA base damage can lead to diminished cell function, and potentially diseases and eventually mutations that lead to cancer. Sophisticated DNA repair mechanisms have evolved in all living cells to preserve the integrity of inherited genetic information and transcriptional control. Understanding a system like DNA repair is greatly enhanced by using engineering methods, in particular modeling interactions and using predictive simulation to analyze the impact of perturbations. We describe the use of such a "nanosystem engineering" approach to analyze the DNA base excision repair pathway in human cells, and use simulation to predict the impact of varying enzyme concentration on DNA repair capacity.

  1. DNA repair: Dynamic defenders against cancer and aging

    SciTech Connect

    Fuss, Jill O.; Cooper, Priscilla K.

    2006-04-01

    You probably weren't thinking about your body's cellular DNA repair systems the last time you sat on the beach in the bright sunshine. Fortunately, however, while you were subjecting your DNA to the harmful effects of ultraviolet light, your cells were busy repairing the damage. The idea that our genetic material could be damaged by the sun was not appreciated in the early days of molecular biology. When Watson and Crick discovered the structure of DNA in 1953 [1], it was assumed that DNA is fundamentally stable since it carries the blueprint of life. However, over 50 years of research have revealed that our DNA is under constant assault by sunlight, oxygen, radiation, various chemicals, and even our own cellular processes. Cleverly, evolution has provided our cells with a diverse set of tools to repair the damage that Mother Nature causes. DNA repair processes restore the normal nucleotide sequence and DNA structure of the genome after damage [2]. These responses are highly varied and exquisitely regulated. DNA repair mechanisms are traditionally characterized by the type of damage repaired. A large variety of chemical modifications can alter normal DNA bases and either lead to mutations or block transcription if not repaired, and three distinct pathways exist to remove base damage. Base excision repair (BER) corrects DNA base alterations that do not distort the overall structure of the DNA helix such as bases damaged by oxidation resulting from normal cellular metabolism. While BER removes single damaged bases, nucleotide excision repair (NER) removes short segments of nucleotides (called oligonucleotides) containing damaged bases. NER responds to any alteration that distorts the DNA helix and is the mechanism responsible for repairing bulky base damage caused by carcinogenic chemicals such as benzo [a]pyrene (found in cigarette smoke and automobile exhaust) as well as covalent linkages between adjacent pyrimidine bases resulting from the ultraviolet (UV

  2. Situation-dependent repair of DNA damage in yeast

    SciTech Connect

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

    1985-01-01

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

  3. RNF4 is required for DNA double-strand break repair in vivo.

    PubMed

    Vyas, R; Kumar, R; Clermont, F; Helfricht, A; Kalev, P; Sotiropoulou, P; Hendriks, I A; Radaelli, E; Hochepied, T; Blanpain, C; Sablina, A; van Attikum, H; Olsen, J V; Jochemsen, A G; Vertegaal, A C O; Marine, J-C

    2013-03-01

    Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial roles in regulating the dynamic assembly of protein complexes at these sites. However, how SUMOylation influences protein ubiquitylation at DSBs is poorly understood. We show herein that Rnf4, an E3 ubiquitin ligase that targets SUMO-modified proteins, accumulates in DSB repair foci and is required for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation-induced DNA damage and signaling, and that Rnf4-deficient cells and mice exhibit increased sensitivity to genotoxic stress. Mechanistically, we show that Rnf4 targets SUMOylated MDC1 and SUMOylated BRCA1, and is required for the loading of Rad51, an enzyme required for HR repair, onto sites of DNA damage. Similarly to inactivating mutations in other key regulators of HR repair, Rnf4 deficiency leads to age-dependent impairment in spermatogenesis. These findings identify Rnf4 as a critical component of the DDR in vivo and support the possibility that Rnf4 controls protein localization at DNA damage sites by integrating SUMOylation and ubiquitylation events.

  4. Bacterial DNA repair genes and their eukaryotic homologues: 5. The role of recombination in DNA repair and genome stability.

    PubMed

    Nowosielska, Anetta

    2007-01-01

    Recombinational repair is a well conserved DNA repair mechanism present in all living organisms. Repair by homologous recombination is generally accurate as it uses undamaged homologous DNA molecule as a repair template. In Escherichia coli homologous recombination repairs both the double-strand breaks and single-strand gaps in DNA. DNA double-strand breaks (DSB) can be induced upon exposure to exogenous sources such as ionizing radiation or endogenous DNA-damaging agents including reactive oxygen species (ROS) as well as during natural biological processes like conjugation. However, the bulk of double strand breaks are formed during replication fork collapse encountering an unrepaired single strand gap in DNA. Under such circumstances DNA replication on the damaged template can be resumed only if supported by homologous recombination. This functional cooperation of homologous recombination with replication machinery enables successful completion of genome duplication and faithful transmission of genetic material to a daughter cell. In eukaryotes, homologous recombination is also involved in essential biological processes such as preservation of genome integrity, DNA damage checkpoint activation, DNA damage repair, DNA replication, mating type switching, transposition, immune system development and meiosis. When unregulated, recombination can lead to genome instability and carcinogenesis.

  5. Connecting the Dots: From DNA Damage and Repair to Aging

    PubMed Central

    Pan, Mei-Ren; Li, Kaiyi; Lin, Shiaw-Yih; Hung, Wen-Chun

    2016-01-01

    Mammalian cells evolve a delicate system, the DNA damage response (DDR) pathway, to monitor genomic integrity and to prevent the damage from both endogenous end exogenous insults. Emerging evidence suggests that aberrant DDR and deficient DNA repair are strongly associated with cancer and aging. Our understanding of the core program of DDR has made tremendous progress in the past two decades. However, the long list of the molecules involved in the DDR and DNA repair continues to grow and the roles of the new “dots” are under intensive investigation. Here, we review the connection between DDR and DNA repair and aging and discuss the potential mechanisms by which deficient DNA repair triggers systemic effects to promote physiological or pathological aging. PMID:27164092

  6. Effect of aging and dietary restriction on DNA repair

    SciTech Connect

    Weraarchakul, N.; Strong, R.; Wood, W.G.; Richardson, A.

    1989-03-01

    DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.

  7. The Repeat Expansion Diseases: the dark side of DNA repair?

    PubMed Central

    Zhao, Xiao-Nan; Usdin, Karen

    2015-01-01

    DNA repair normally protects the genome against mutations that threaten genome integrity and thus cell viability. However, growing evidence suggests that in the case of the Repeat Expansion Diseases, disorders that result from an increase in the size of a disease-specific microsatellite, the disease-causing mutation is actually the result of aberrant DNA repair. A variety of proteins from different DNA repair pathways have thus far been implicated in this process. This review will summarize recent findings from patients and from mouse models of these diseases that shed light on how these pathways may interact to cause repeat expansion. PMID:26002199

  8. Noncanonical views of homology-directed DNA repair

    PubMed Central

    Verma, Priyanka; Greenberg, Roger A.

    2016-01-01

    DNA repair is essential to maintain genomic integrity and initiate genetic diversity. While gene conversion and classical nonhomologous end-joining are the most physiologically predominant forms of DNA repair mechanisms, emerging lines of evidence suggest the usage of several noncanonical homology-directed repair (HDR) pathways in both prokaryotes and eukaryotes in different contexts. Here we review how these alternative HDR pathways are executed, specifically focusing on the determinants that dictate competition between them and their relevance to cancers that display complex genomic rearrangements or maintain their telomeres by homology-directed DNA synthesis. PMID:27222516

  9. DNA double-strand break repair pathway choice and cancer.

    PubMed

    Aparicio, Tomas; Baer, Richard; Gautier, Jean

    2014-07-01

    Since DNA double-strand breaks (DSBs) contribute to the genomic instability that drives cancer development, DSB repair pathways serve as important mechanisms for tumor suppression. Thus, genetic lesions, such as BRCA1 and BRCA2 mutations, that disrupt DSB repair are often associated with cancer susceptibility. In addition, recent evidence suggests that DSB "mis-repair", in which DSBs are resolved by an inappropriate repair pathway, can also promote genomic instability and presumably tumorigenesis. This notion has gained currency from recent cancer genome sequencing studies which have uncovered numerous chromosomal rearrangements harboring pathological DNA repair signatures. In this perspective, we discuss the factors that regulate DSB repair pathway choice and their consequences for genome stability and cancer.

  10. Methods to alter levels of a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-10-17

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  11. Induced DNA repair pathway in mammalian cells

    SciTech Connect

    Overberg, R.

    1985-01-01

    The survival of cultured rat kangaroo cells (PtK-2) and human xeroderma pigmentosum cells incubated with 5 ..mu..M cycloheximide subsequent to ultraviolet irradiation is lower than that of cells incubated without cycloheximide. The drop in survival is considerably larger than that produced by incubation of unirradiated cells with cycloheximide. The phenomenon was also observed when PtK-2 cells were incubated with emetine, another protein synthesis inhibitor, or with 5,6-dichloro-1-..beta..-D-ribofuranosylbenzimidazole, a RNA synthesis inhibitor. PtK cells which received a preliminary UV treatment followed by an incubation period without cycloheximide and then a second irradiation and 24 hour incubation with cycloheximide, survived the effects of the second irradiation better than cells which were incubated in the presence of cycloheximide after the first and second UV irradiation. The application of cycloheximide for 24 hours after UV irradiation of PtK cells resulted in one-half as many 6-thioguanine resistant cells as compared to the number of 6-thioguanine resistant cells found when cycloheximide was not used. These experiments indicate that a UV-inducible cycloheximide-sensitive DNA repair pathway is present in PtK and xeroderma pigmentosum cells, which is error-prone in PtK cells.

  12. WHERE MULTIFUNCTIONAL DNA REPAIR PROTEINS MEET: MAPPING THE INTERACTION DOMAINS BETWEEN XPG AND WRN

    SciTech Connect

    Rangaraj, K.; Cooper, P.K.; Trego, K.S.

    2009-01-01

    The rapid recognition and repair of DNA damage is essential for the maintenance of genomic integrity and cellular survival. Multiple complex and interconnected DNA damage responses exist within cells to preserve the human genome, and these repair pathways are carried out by a specifi c interplay of protein-protein interactions. Thus a failure in the coordination of these processes, perhaps brought about by a breakdown in any one multifunctional repair protein, can lead to genomic instability, developmental and immunological abnormalities, cancer and premature aging. This study demonstrates a novel interaction between two such repair proteins, Xeroderma pigmentosum group G protein (XPG) and Werner syndrome helicase (WRN), that are both highly pleiotropic and associated with inherited genetic disorders when mutated. XPG is a structure-specifi c endonuclease required for the repair of UV-damaged DNA by nucleotide excision repair (NER), and mutations in XPG result in the diseases Xeroderma pigmentosum (XP) and Cockayne syndrome (CS). A loss of XPG incision activity results in XP, whereas a loss of non-enzymatic function(s) of XPG causes CS. WRN is a multifunctional protein involved in double-strand break repair (DSBR), and consists of 3’–5’ DNA-dependent helicase, 3’–5’ exonuclease, and single-strand DNA annealing activities. Nonfunctional WRN protein leads to Werner syndrome, a premature aging disorder with increased cancer incidence. Far Western analysis was used to map the interacting domains between XPG and WRN by denaturing gel electrophoresis, which separated purifi ed full length and recombinant XPG and WRN deletion constructs, based primarily upon the length of each polypeptide. Specifi c interacting domains were visualized when probed with the secondary protein of interest which was then detected by traditional Western analysis using the antibody of the secondary protein. The interaction between XPG and WRN was mapped to the C-terminal region of

  13. Robustness of DNA Repair through Collective Rate Control

    PubMed Central

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

    2014-01-01

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

  14. New insights into the mechanism of DNA mismatch repair

    PubMed Central

    Reyes, Gloria X.; Schmidt, Tobias T.; Kolodner, Richard D.; Hombauer, Hans

    2015-01-01

    The genome of all organisms is constantly being challenged by endogenous and exogenous sources of DNA damage. Errors like base:base mismatches or small insertions and deletions, primarily introduced by DNA polymerases during DNA replication are repaired by an evolutionary conserved DNA mismatch repair (MMR) system. The MMR system, together with the DNA replication machinery, promote repair by an excision and resynthesis mechanism during or after DNA replication, increasing replication fidelity by upto-three orders of magnitude. Consequently, inactivation of MMR genes results in elevated mutation rates that can lead to increased cancer susceptibility in humans. In this review, we summarize our current understanding of MMR with a focus on the different MMR protein complexes, their function and structure. We also discuss how recent findings have provided new insights in the spatio-temporal regulation and mechanism of MMR. PMID:25862369

  15. Repair of DNA Double-Strand Breaks in Heterochromatin

    PubMed Central

    Watts, Felicity Z.

    2016-01-01

    DNA double-strand breaks (DSBs) are among the most damaging lesions in DNA, since, if not identified and repaired, they can lead to insertions, deletions or chromosomal rearrangements. DSBs can be in the form of simple or complex breaks, and may be repaired by one of a number of processes, the nature of which depends on the complexity of the break or the position of the break within the chromatin. In eukaryotic cells, nuclear DNA is maintained as either euchromatin (EC) which is loosely packed, or in a denser form, much of which is heterochromatin (HC). Due to the less accessible nature of the DNA in HC as compared to that in EC, repair of damage in HC is not as straightforward as repair in EC. Here we review the literature on how cells deal with DSBs in HC. PMID:27999260

  16. p53 in the DNA damage repair process

    PubMed Central

    Williams, Ashley B.; Schumacher, Björn

    2016-01-01

    The cells in the human body are continuously challenged by a variety of genotoxic attacks. Erroneous repair of the DNA can lead to mutations and chromosomal aberrations that can alter the functions of tumor suppressor genes or oncogenes, thus causing cancer development. As a central tumor suppressor, p53 guards the genome by orchestrating a variety of DNA damage response (DDR) mechanisms. Already early in metazoan evolution, p53 started controlling the apoptotic demise of genomically compromised cells. p53 plays a prominent role as a facilitator of DNA repair by halting the cell cycle to allow time for the repair machineries to restore genome stability. In addition, p53 took on diverse roles to also directly impact the activity of various DNA repair systems. It thus appears as if p53 is multitasking in protecting from cancer development by maintaining genome stability. PMID:27048304

  17. Molecular mechanisms of DNA repair inhibition by caffeine

    SciTech Connect

    Selby, C.P.; Sancar, A. )

    1990-05-01

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

  18. The multifaceted influence of histone deacetylases on DNA damage signalling and DNA repair

    PubMed Central

    Roos, Wynand Paul; Krumm, Andrea

    2016-01-01

    Histone/protein deacetylases play multiple roles in regulating gene expression and protein activation and stability. Their deregulation during cancer initiation and progression cause resistance to therapy. Here, we review the role of histone deacetylases (HDACs) and the NAD+ dependent sirtuins (SIRTs) in the DNA damage response (DDR). These lysine deacetylases contribute to DNA repair by base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), homologous recombination (HR) and interstrand crosslink (ICL) repair. Furthermore, we discuss possible mechanisms whereby these histone/protein deacetylases facilitate the switch between DNA double-strand break (DSB) repair pathways, how SIRTs play a central role in the crosstalk between DNA repair and cell death pathways due to their dependence on NAD+, and the influence of small molecule HDAC inhibitors (HDACi) on cancer cell resistance to genotoxin based therapies. Throughout the review, we endeavor to identify the specific HDAC targeted by HDACi leading to therapy sensitization. PMID:27738139

  19. Impact of Alternative DNA Structures on DNA Damage, DNA Repair, and Genetic Instability

    PubMed Central

    Wang, Guliang; Vasquez, Karen M.

    2014-01-01

    Repetitive genomic sequences can adopt a number of alternative DNA structures that differ from the canonical B-form duplex (i.e. non-B DNA). These non-B DNA-forming sequences have been shown to have many important biological functions related to DNA metabolic processes; for example, they may have regulatory roles in DNA transcription and replication. In addition to these regulatory functions, non-B DNA can stimulate genetic instability in the presence or absence of DNA damage, via replication-dependent and/or replication-independent pathways. This review focuses on the interactions of non-B DNA conformations with DNA repair proteins and how these interactions impact genetic instability. PMID:24767258

  20. Transcript-RNA-templated DNA recombination and repair.

    PubMed

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

    2014-11-20

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

  1. A monofunctional platinum complex coordinated to a rhodium metalloinsertor selectively binds mismatched DNA in the minor groove.

    PubMed

    Weidmann, Alyson G; Barton, Jacqueline K

    2015-10-05

    We report the synthesis and characterization of a bimetallic complex derived from a new family of potent and selective metalloinsertors containing an unusual Rh-O axial coordination. This complex incorporates a monofunctional platinum center containing only one labile site for coordination to DNA, rather than two, and coordinates DNA nonclassically through adduct formation in the minor groove. This conjugate displays bifunctional, interdependent binding of mismatched DNA via metalloinsertion at a mismatch as well as covalent platinum binding. DNA sequencing experiments revealed that the preferred site of platinum coordination is not the traditional N7-guanine site in the major groove, but rather N3-adenine in the minor groove. The complex also displays enhanced cytotoxicity in mismatch repair-deficient and mismatch repair-proficient human colorectal carcinoma cell lines compared to the chemotherapeutic cisplatin, and it triggers cell death via an apoptotic pathway, rather than the necrotic pathway induced by rhodium metalloinsertors.

  2. A Monofunctional Platinum Complex Coordinated to a Rhodium Metalloinsertor Selectively Binds Mismatched DNA in the Minor Groove

    PubMed Central

    Weidmann, Alyson G.; Barton, Jacqueline K.

    2015-01-01

    We report the synthesis and characterization of a bimetallic complex derived from a new family of potent and selective metalloinsertors containing an unusual Rh—O axial coordination. This complex incorporates a monofunctional platinum center containing only one labile site for coordination to DNA, rather than two, and coordinates DNA non-classically through adduct formation in the minor groove. This conjugate displays bifunctional, interdependent binding of mismatched DNA via metalloinsertion at a mismatch as well as covalent platinum binding. DNA sequencing experiments revealed that the preferred site of platinum coordination is not the traditional N7-guanine site in the major groove, but rather N3-adenine in the minor groove. The complex also displays enhanced cytotoxicity in mismatch repair-deficient and mismatch repair-proficient human colorectal carcinoma cell lines compared to the chemotherapeutic cisplatin, and triggers cell death via an apoptotic pathway, rather than the necrotic pathway induced by rhodium metalloinsertors. PMID:26397309

  3. Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes

    PubMed Central

    Morales, Maria E.; Derbes, Rebecca S.; Ade, Catherine M.; Ortego, Jonathan C.; Stark, Jeremy; Deininger, Prescott L.; Roy-Engel, Astrid M.

    2016-01-01

    Heavy metals such as cadmium, arsenic and nickel are classified as carcinogens. Although the precise mechanism of carcinogenesis is undefined, heavy metal exposure can contribute to genetic damage by inducing double strand breaks (DSBs) as well as inhibiting critical proteins from different DNA repair pathways. Here we take advantage of two previously published culture assay systems developed to address mechanistic aspects of DNA repair to evaluate the effects of heavy metal exposures on competing DNA repair outcomes. Our results demonstrate that exposure to heavy metals significantly alters how cells repair double strand breaks. The effects observed are both specific to the particular metal and dose dependent. Low doses of NiCl2 favored resolution of DSBs through homologous recombination (HR) and single strand annealing (SSA), which were inhibited by higher NiCl2 doses. In contrast, cells exposed to arsenic trioxide preferentially repaired using the “error prone” non-homologous end joining (alt-NHEJ) while inhibiting repair by HR. In addition, we determined that low doses of nickel and cadmium contributed to an increase in mutagenic recombination-mediated by Alu elements, the most numerous family of repetitive elements in humans. Sequence verification confirmed that the majority of the genetic deletions were the result of Alu-mediated non-allelic recombination events that predominantly arose from repair by SSA. All heavy metals showed a shift in the outcomes of alt-NHEJ repair with a significant increase of non-templated sequence insertions at the DSB repair site. Our data suggest that exposure to heavy metals will alter the choice of DNA repair pathway changing the genetic outcome of DSBs repair. PMID:26966913

  4. Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes.

    PubMed

    Morales, Maria E; Derbes, Rebecca S; Ade, Catherine M; Ortego, Jonathan C; Stark, Jeremy; Deininger, Prescott L; Roy-Engel, Astrid M

    2016-01-01

    Heavy metals such as cadmium, arsenic and nickel are classified as carcinogens. Although the precise mechanism of carcinogenesis is undefined, heavy metal exposure can contribute to genetic damage by inducing double strand breaks (DSBs) as well as inhibiting critical proteins from different DNA repair pathways. Here we take advantage of two previously published culture assay systems developed to address mechanistic aspects of DNA repair to evaluate the effects of heavy metal exposures on competing DNA repair outcomes. Our results demonstrate that exposure to heavy metals significantly alters how cells repair double strand breaks. The effects observed are both specific to the particular metal and dose dependent. Low doses of NiCl2 favored resolution of DSBs through homologous recombination (HR) and single strand annealing (SSA), which were inhibited by higher NiCl2 doses. In contrast, cells exposed to arsenic trioxide preferentially repaired using the "error prone" non-homologous end joining (alt-NHEJ) while inhibiting repair by HR. In addition, we determined that low doses of nickel and cadmium contributed to an increase in mutagenic recombination-mediated by Alu elements, the most numerous family of repetitive elements in humans. Sequence verification confirmed that the majority of the genetic deletions were the result of Alu-mediated non-allelic recombination events that predominantly arose from repair by SSA. All heavy metals showed a shift in the outcomes of alt-NHEJ repair with a significant increase of non-templated sequence insertions at the DSB repair site. Our data suggest that exposure to heavy metals will alter the choice of DNA repair pathway changing the genetic outcome of DSBs repair.

  5. Mitochondrial DNA repair: a novel therapeutic target for heart failure.

    PubMed

    Marín-García, José

    2016-09-01

    Mitochondria play a crucial role in a variety of cellular processes ranging from energy metabolism, generation of reactive oxygen species (ROS) and Ca(2+) handling to stress responses, cell survival and death. Malfunction of the organelle may contribute to the pathogenesis of neuromuscular, cancer, premature aging and cardiovascular diseases (CVD), including myocardial ischemia, cardiomyopathy and heart failure (HF). Mitochondria contain their own genome organized into DNA-protein complexes, called "mitochondrial nucleoids," along with multiprotein machineries, which promote mitochondrial DNA (mtDNA) replication, transcription and repair. Although the mammalian organelle possesses almost all known nuclear DNA repair pathways, including base excision repair, mismatch repair and recombinational repair, the proximity of mtDNA to the main sites of ROS production and the lack of protective histones may result in increased susceptibility to various types of mtDNA damage. These include accumulation of mtDNA point mutations and/or deletions and decreased mtDNA copy number, which will impair mitochondrial function and finally, may lead to CVD including HF.

  6. Xeroderma Pigmentosum: defective DNA repair causes skin cancer and neurodegeneration

    SciTech Connect

    Robbins, J.H.

    1988-07-15

    Xeroderma pigmentosum is a rare autosomal recessive disease with numerous malignancies on sun-exposed areas of the skin and eye because of an inability to repair DNA damage inflicted by harmful ultraviolet (UV) radiation of the sun. Because it is the only disease in which cancer is known to result from defective DNA repair, XP has received intense clinical and biochemical study during the last two decades. Furthermore, some patients with XP develop a primary neuronal degeneration, probably due to the inability of nerve cells to repair damage to their DNA caused by intraneuronal metabolites and physicochemical events that mimic the effects of UV radiation. Studies of XP neurodegeneration and DNA-repair defects have led to the conclusion that efficient DNA repair is required to prevent premature death of human nerve cells. Since XP neurodegeneration has similarities to premature death of nerve cells that occurs in such neurodegenerative disorders, XP may be the prototype for these more common neurodegenerations. Recent studies indicate that these degenerations also may have DNA-repair defects.

  7. Purification of mammalian DNA repair protein XRCC1

    SciTech Connect

    Chen, I.

    1995-11-01

    Malfunctioning DNA repair systems lead to cancer mutations, and cell death. XRCC1 (X-ray Repair Cross Complementing) is a human DNA repair gene that has been found to fully correct the x-ray repair defect in Chinese hamster ovary (CHO) cell mutant EM9. The corresponding protein (XRCC1) encoded by this gene has been linked to a DNA repair pathway known as base excision repair, and affects the activity of DNA ligase III. Previously, an XRCC1 cDNA minigene (consisting of the uninterrupted coding sequence for XRCC1 protein followed by a decahistidine tag) was constructed and cloned into vector pET-16b for the purpose of: (1) overproduction of XRCC1 in both prokaryotic and eukaryotic cells; and (2) to facilitate rapid purification of XRCC1 from these systems. A vector is basically a DNA carrier that allows recombinant protein to be cloned and overexpressed in host cells. In this study, XRCC1 protein was overexpressed in E. coli and purified by immobilized metal affinity chromatography. Currently, the XRCC1 minigene is being inserted into a new vector [pET-26b(+)] in hopes to increase overexpression and improve purification. Once purified XRCC1 can be crystallized for structural studies, or studied in vitro for its biological function.

  8. DNA repair proficiency: A potential susceptibility factor for breast cancer

    SciTech Connect

    Helzlsouer, K.J.; Perry, H.; Harris, E.L. |

    1994-09-01

    A family study and a case-control study were conducted to examine the association between sub-optimal repair of ionizing radiation induced DNA damage and the development of breast cancer. A familial cluster of breast cancer was investigated in which breast cancer occurred in 4 of 6 sisters, some of whom were exposed to ionizing radiation from repeated chest fluoroscopic examinations during adolescence and early adulthood. DNA repair proficiency was measured among available family members and correlated with their history of radiation exposure. DNA repair proficiency was also measured among 16 breast cancer cases, 5 women with a family history of breast cancer and 12 controls. The results of the family study suggest an association between poor DNA repair proficiency and increased sensitivity to the carcinogenic effects of early radiation exposure on breast tissue. The case-control study showed that a significantly higher percentage of women with breast cancer (63%) and women with a family history of breast cancer (80%) had poor repair of ionizing radiation induced DNA damage than control women (17%) (P-value=0.02). Sub-optimal repair of DNA damage may be a host susceptibility factor predisposing individuals to breast cancer through increased sensitivity to carcinogenic damage from environmental exposures such as ionizing radiation.

  9. New approaches to biochemical radioprotection: antioxidants and DNA repair enhancement

    NASA Astrophysics Data System (ADS)

    Riklis, E.; Emerit, I.; Setlow, R. B.

    Chemical repair may be provided by radioprotective compounds present during exposure to ionizing radiation. Considering DNA as the most sensitive target it is feasible to biochemically improve protection by enhancing DNA repair mechanisms. Protection of DNA by reducing the amount of damage (by radical scavenging and chemical repair) followed by enhanced repair of DNA will provide much improved protection and recovery. Furthermore, in cases of prolonged exposure, such as is possible in prolonged space missions, or of unexpected variations in the intensity of radiation, as is possible when encountering solar flares, it is important to provide long-acting protection, and this may be provided by antioxidants and well functioning DNA repair systems. It has also become important to provide protection from the potentially damaging action of long-lived clastogenic factors which have been found in plasma of exposed persons from Hiroshima & Nagasaki, radiation accidents, radiotherapy patients and recently in ``liquidators'' - persons involved in salvage operations at the Chernobyl reactor. The clastogenic factor, which causes chromatid breaks in non-exposed plasma, might account for late effects and is posing a potential carcinogenic hazard /1/. The enzyme superoxide dismutase (SOD) has been shown to eliminate the breakage factor from cultured plasma of exposed persons /2/. Several compounds have been shown to enhance DNA repair: WR-2721 /3/, nicotinamide /4/, glutathione monoester (Riklis et al., unpublished) and others. The right combination of such compounds may prove effective in providing protection from a wide range of radiation exposures over a long period of time.

  10. DNA in motion during double-strand break repair.

    PubMed

    Miné-Hattab, Judith; Rothstein, Rodney

    2013-11-01

    DNA organization and dynamics profoundly affect many biological processes such as gene regulation and DNA repair. In this review, we present the latest studies on DNA mobility in the context of DNA damage. Recent studies demonstrate that DNA mobility is dramatically increased in the presence of double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae. As a consequence, chromosomes explore a larger nuclear volume, facilitating homologous pairing but also increasing the rate of ectopic recombination. Increased DNA dynamics is dependent on several homologous recombination (HR) proteins and we are just beginning to understand how chromosome dynamics is regulated after DNA damage.

  11. Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

    PubMed Central

    Singh, K K; Samson, L

    1995-01-01

    Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7761422

  12. The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once

    PubMed Central

    Lafrance-Vanasse, Julien

    2014-01-01

    To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by Watson-Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure twice these nucleases

  13. DNA repair mechanisms in dividing and non-dividing cells.

    PubMed

    Iyama, Teruaki; Wilson, David M

    2013-08-01

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

  14. DNA repair mechanisms in dividing and non-dividing cells

    PubMed Central

    Iyama, Teruaki; Wilson, David M.

    2013-01-01

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

  15. DNA damage and repair in human skin in situ

    SciTech Connect

    Sutherland, B.M.; Gange, R.W.; Freeman, S.E.; Sutherland, J.C.

    1987-01-01

    Understanding the molecular and cellular origins of sunlight-induced skin cancers in man requires knowledge of the damages inflicted on human skin during sunlight exposure, as well as the ability of cells in skin to repair or circumvent such damage. Although repair has been studied extensively in procaryotic and eucaryotic cells - including human cells in culture - there are important differences between repair by human skin cells in culture and human skin in situ: quantitative differences in rates of repair, as well as qualitative differences, including the presence or absence of repair mechanisms. Quantitation of DNA damage and repair in human skin required the development of new approaches for measuring damage at low levels in nanogram quantities of non-radioactive DNA. The method allows for analysis of multiple samples and the resulting data should be related to behavior of the DNA molecules by analytic expressions. Furthermore, it should be possible to assay a variety of lesions using the same methodology. The development of new analysis methods, new technology, and new biochemical probes for the study of DNA damage and repair are described. 28 refs., 4 figs.

  16. Differential DNA lesion formation and repair in heterochromatin and euchromatin

    PubMed Central

    Han, Chunhua; Srivastava, Amit Kumar; Cui, Tiantian; Wang, Qi-En; Wani, Altaf A.

    2016-01-01

    Discretely orchestrated chromatin condensation is important for chromosome protection from DNA damage. However, it is still unclear how different chromatin states affect the formation and repair of nucleotide excision repair (NER) substrates, e.g. ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPD) and the pyrimidine (6-4) pyrimidone photoproducts (6-4PP), as well as cisplatin-induced intrastrand crosslinks (Pt-GG). Here, by using immunofluorescence and chromatin immunoprecipitation assays, we have demonstrated that CPD, which cause minor distortion of DNA double helix, can be detected in both euchromatic and heterochromatic regions, while 6-4PP and Pt-GG, which cause major distortion of DNA helix, can exclusively be detected in euchromatin, indicating that the condensed chromatin environment specifically interferes with the formation of these DNA lesions. Mechanistic investigation revealed that the class III histone deacetylase SIRT1 is responsible for restricting the formation of 6-4PP and Pt-GG in cells, probably by facilitating the maintenance of highly condensed heterochromatin. In addition, we also showed that the repair of CPD in heterochromatin is slower than that in euchromatin, and DNA damage binding protein 2 (DDB2) can promote the removal of CPD from heterochromatic region. In summary, our data provide evidence for differential formation and repair of DNA lesions that are substrates of NER. Both the sensitivity of DNA to damage and the kinetics of repair can be affected by the underlying level of chromatin compaction. PMID:26717995

  17. DNA Repair and Cytokines: TGF-β, IL-6, and Thrombopoietin as Different Biomarkers of Radioresistance

    PubMed Central

    Centurione, Lucia; Aiello, Francesca B.

    2016-01-01

    Double strand breaks (DSBs) induced by radiotherapy are highly cytotoxic lesions, leading to chromosomal aberrations and cell death. Ataxia-telangiectasia-mutated (ATM)-dependent DNA-damage response, non-homologous end joining, and homologous recombination pathways coordinately contribute to repairing DSBs in higher eukaryotes. It is known that the expression of DSB repair genes is increased in tumors, which is one of the main reasons for radioresistance. The inhibition of DSB repair pathways may be useful to increase tumor cell radiosensitivity and may target stem cell-like cancer cells, known to be the most radioresistant tumor components. Commonly overexpressed in neoplastic cells, cytokines confer radioresistance by promoting proliferation, survival, invasion, and angiogenesis. Unfortunately, tumor irradiation increases the expression of various cytokines displaying these effects, including transforming growth factor-beta and interleukin-6. Recently, the capabilities of these cytokines to support DNA repair pathways and the ATM-dependent DNA response have been demonstrated. Thrombopoietin, essential for megakaryopoiesis and very important for hematopoietic stem cell (HSC) homeostasis, has also been found to promote DNA repair in a highly selective manner. These findings reveal a novel mechanism underlying cytokine-related radioresistance, which may be clinically relevant. Therapies targeting specific cytokines may be used to improve radiosensitivity. Specific inhibitors may be chosen in consideration of different tumor microenvironments. Thrombopoietin may be useful in fending off irradiation-induced loss of HSCs. PMID:27500125

  18. Global genome nucleotide excision repair is organized into domains that promote efficient DNA repair in chromatin

    PubMed Central

    Yu, Shirong; Evans, Katie; Bennett, Mark; Webster, Richard M.; Leadbitter, Matthew; Teng, Yumin; Waters, Raymond

    2016-01-01

    The rates at which lesions are removed by DNA repair can vary widely throughout the genome, with important implications for genomic stability. To study this, we measured the distribution of nucleotide excision repair (NER) rates for UV-induced lesions throughout the budding yeast genome. By plotting these repair rates in relation to genes and their associated flanking sequences, we reveal that, in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organized within the genome. Furthermore, by comparing genome-wide DNA repair rates in wild-type cells and cells defective in the global genome–NER (GG-NER) subpathway, we establish how this alters the distribution of NER rates throughout the genome. We also examined the genomic locations of GG-NER factor binding to chromatin before and after UV irradiation, revealing that GG-NER is organized and initiated from specific genomic locations. At these sites, chromatin occupancy of the histone acetyl-transferase Gcn5 is controlled by the GG-NER complex, which regulates histone H3 acetylation and chromatin structure, thereby promoting efficient DNA repair of UV-induced lesions. Chromatin remodeling during the GG-NER process is therefore organized into these genomic domains. Importantly, loss of Gcn5 significantly alters the genomic distribution of NER rates; this has implications for the effects of chromatin modifiers on the distribution of mutations that arise throughout the genome. PMID:27470111

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

    PubMed

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

    2013-07-01

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

  20. Interindividual variation with respect to DNA repair in human cells

    SciTech Connect

    Leonard, R.C.; Leonard, J.C.; Bender, M.A.; Wieland, J.; Setlow, R.B.

    1989-01-01

    Ecogenetics is the study of genetically determined differences among individuals in their susceptibility to the actions of physical, chemical, and biological agents in the environment. An individual's most basic level of response to these environmental agents may be the ability to repair physical and chemical damage to DNA. We have been engaged in a survey of DNA-repair measurements in a healthy working population in order to determine the extent of the population variability in these endpoints and to assess the value of these screening protocols in identifying individuals who are at the extremes of the distribution. In addition, we are measuring intraindividual variation over time, as well as the correlations between measurements of different repair systems. The endpoints that we have chosen to use are cytogenetic responses (SCE's and micronucleus formation) and DNA excision repair (unscheduled DNA synthesis and removal of O{sup 6} guanine methylation) in human peripheral lymphocytes exposed to 254 nm ultraviolet light, x-rays, the bifunctional alkylating agent mitomycin C, or the monofunctional alkylating agent N-methyl-N-nitro-nitrosoguanidine (MNNG). These four test mutagens produce spectra of DNA lesions eliciting different types of DNA repair. 3 refs., 1 tab.

  1. Structural aspects of DNA repair: the role of restricted diffusion.

    PubMed

    Minsky, Abraham

    2003-10-01

    DNA repair and protection processes impose arduous demands upon cellular systems. The high-fidelity recombinational repair pathway entails a rapid genome-wide search for sequence homology. The efficiency of this transaction is intriguing in light of the uniquely adverse diffusion traits of the involved species. DNA protection in cells exposed to continuous stress or prolonged starvation is equally enigmatic, because the ability of such cells to deploy energy-dependent enzymatic repair processes is hampered as a result of progressive perturbation of the intracellular energy balance. DNA repair in radio-resistant bacteria, which involves accurate chromosome reconstruction from multiple fragments, is similarly associated with apparently insurmountable logistical obstacles. The studies reviewed here imply that the mechanisms deployed to overcome these intrinsic hurdles have a basic common denominator. In all these cases, condensed and ordered chromatin assemblies are formed, within which molecular diffusion is restricted and confined. Restricted diffusion thus appears as a general strategy that is exploited by nature to facilitate homologous search, to promote energy-independent DNA protection through physical DNA sequestration and attenuated accessibility to damaging agents, and to enable error-free repair of multiple double-strand DNA breaks.

  2. Recombinant methods for screening human DNA excision repair proficiency

    SciTech Connect

    Athas, W.F.

    1988-01-01

    A method for measuring DNA excision repair in response to ultraviolet radiation (UV)-induced DNA damage has been developed, validated, and field-tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiologic studies seeking to investigate associations between excision repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the belief that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum (XP) primarily is a result of the reduced capacity of patients cells to repair UV-induced DNA damage. For assay, UV-irradiated non-replicating recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (CAT) indicator gene is introduced into lymphocytes using DEAE-dextran short-term transfection conditions. Exposure to UV induces transcriptionally-inactivating DNA photoproducts in the plasmid DNA which inactivate CAT gene expression. Excision repair of the damaged CAT gene is monitored indirectly as a function of reactivated CAT enzyme activity following a 40 hour repair/expression incubation period.

  3. Exploiting DNA repair defects for novel cancer therapies

    PubMed Central

    van Gent, Dik C.; Kanaar, Roland

    2016-01-01

    Most human tumors accumulate a multitude of genetic changes due to defects in the DNA damage response. Recently, small-molecule inhibitors have been developed that target cells with specific DNA repair defects, providing hope for precision treatment of such tumors. Here we discuss the rationale behind these therapies and how an important bottleneck—patient selection—can be approached. PMID:27418635

  4. RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage–induced cell senescence

    PubMed Central

    Cekan, Pavol; Hasegawa, Keisuke; Pan, Yu; Tubman, Emily; Odde, David; Chen, Jin-Qiu; Herrmann, Michelle A.; Kumar, Sheetal; Kalab, Petr

    2016-01-01

    The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase–regulated nuclear–cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage–induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β–dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP–regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage. PMID:26864624

  5. Inducible repair of oxidative DNA damage in Escherichia coli.

    PubMed

    Demple, B; Halbrook, J

    Hydrogen peroxide is lethal to many cell types, including the bacterium Escherichia coli. Peroxides yield transient radical species that can damage DNA and cause mutations. Such partially reduced oxygen species are occasionally released during cellular respiration and are generated by lethal and mutagenic ionizing radiation. Because cells live in an environment where the threat of oxidative DNA damage is continual, cellular mechanisms may have evolved to avoid and repair this damage. Enzymes are known which evidently perform these functions. We report here that resistance to hydrogen peroxide toxicity can be induced in E. coli, that this novel induction is specific and occurs, in part, at the level of DNA repair.

  6. DNA repair in murine embryonic stem cells and differentiated cells

    SciTech Connect

    Tichy, Elisia D. Stambrook, Peter J.

    2008-06-10

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

  7. Electron Transfer Mechanisms of DNA Repair by Photolyase

    NASA Astrophysics Data System (ADS)

    Zhong, Dongping

    2015-04-01

    Photolyase is a flavin photoenzyme that repairs two DNA base damage products induced by ultraviolet (UV) light: cyclobutane pyrimidine dimers and 6-4 photoproducts. With femtosecond spectroscopy and site-directed mutagenesis, investigators have recently made significant advances in our understanding of UV-damaged DNA repair, and the entire enzymatic dynamics can now be mapped out in real time. For dimer repair, six elementary steps have been characterized, including three electron transfer reactions and two bond-breaking processes, and their reaction times have been determined. A unique electron-tunneling pathway was identified, and the critical residues in modulating the repair function at the active site were determined. The dynamic synergy between the elementary reactions for maintaining high repair efficiency was elucidated, and the biological nature of the flavin active state was uncovered. For 6-4 photoproduct repair, a proton-coupled electron transfer repair mechanism has been revealed. The elucidation of electron transfer mechanisms and two repair photocycles is significant and provides a molecular basis for future practical applications, such as in rational drug design for curing skin cancer.

  8. COORDINATING DNA POLYMERASE TRAFFIC DURING HIGH AND LOW FIDELITY SYNTHESIS

    PubMed Central

    Sutton, Mark D.

    2009-01-01

    With the discovery that organisms possess multiple DNA polymerases (Pols) displaying different fidelities, processivities, and activities came the realization that mechanisms must exist to manage the actions of these diverse enzymes to prevent gratuitous mutations. Although many of the Pols encoded by most organisms are largely accurate, and participate in DNA replication and DNA repair, a sizeable fraction display a reduced fidelity, and act to catalyze potentially error-prone translesion DNA synthesis (TLS) past lesions that persist in the DNA. Striking the proper balance between use of these different enzymes during DNA replication, DNA repair, and TLS is essential for ensuring accurate duplication of the cell’s genome. This review highlights mechanisms that organisms utilize to manage the actions of their different Pols. A particular emphasis is placed on discussion of current models for how different Pols switch places with each other at the replication fork during high fidelity replication and potentially error-pone TLS. PMID:19540941

  9. DNA double-strand break repair pathway choice in Dictyostelium.

    PubMed

    Hsu, Duen-Wei; Kiely, Rhian; Couto, C Anne-Marie; Wang, Hong-Yu; Hudson, Jessica J R; Borer, Christine; Pears, Catherine J; Lakin, Nicholas D

    2011-05-15

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). The mechanisms that govern whether a DSB is repaired by NHEJ or HR remain unclear. Here, we characterise DSB repair in the amoeba Dictyostelium. HR is the principal pathway responsible for resistance to DSBs during vegetative cell growth, a stage of the life cycle when cells are predominantly in G2. However, we illustrate that restriction-enzyme-mediated integration of DNA into the Dictyostelium genome is possible during this stage of the life cycle and that this is mediated by an active NHEJ pathway. We illustrate that Dclre1, a protein with similarity to the vertebrate NHEJ factor Artemis, is required for NHEJ independently of DNA termini complexity. Although vegetative dclre1(-) cells are not radiosensitive, they exhibit delayed DSB repair, further supporting a role for NHEJ during this stage of the life cycle. By contrast, cells lacking the Ku80 component of the Ku heterodimer that binds DNA ends to facilitate NHEJ exhibit no such defect and deletion of ku80 suppresses the DSB repair defect of dclre1(-) cells through increasing HR efficiency. These data illustrate a functional NHEJ pathway in vegetative Dictyostelium and the importance of Ku in regulating DSB repair choice during this phase of the life cycle.

  10. Hsp90: A New Player in DNA Repair?

    PubMed Central

    Pennisi, Rosa; Ascenzi, Paolo; di Masi, Alessandra

    2015-01-01

    Heat shock protein 90 (Hsp90) is an evolutionary conserved molecular chaperone that, together with Hsp70 and co-chaperones makes up the Hsp90 chaperone machinery, stabilizing and activating more than 200 proteins, involved in protein homeostasis (i.e., proteostasis), transcriptional regulation, chromatin remodeling, and DNA repair. Cells respond to DNA damage by activating complex DNA damage response (DDR) pathways that include: (i) cell cycle arrest; (ii) transcriptional and post-translational activation of a subset of genes, including those associated with DNA repair; and (iii) triggering of programmed cell death. The efficacy of the DDR pathways is influenced by the nuclear levels of DNA repair proteins, which are regulated by balancing between protein synthesis and degradation as well as by nuclear import and export. The inability to respond properly to either DNA damage or to DNA repair leads to genetic instability, which in turn may enhance the rate of cancer development. Multiple components of the DNA double strand breaks repair machinery, including BRCA1, BRCA2, CHK1, DNA-PKcs, FANCA, and the MRE11/RAD50/NBN complex, have been described to be client proteins of Hsp90, which acts as a regulator of the diverse DDR pathways. Inhibition of Hsp90 actions leads to the altered localization and stabilization of DDR proteins after DNA damage and may represent a cell-specific and tumor-selective radiosensibilizer. Here, the role of Hsp90-dependent molecular mechanisms involved in cancer onset and in the maintenance of the genome integrity is discussed and highlighted. PMID:26501335

  11. Isolating human DNA repair genes using rodent-cell mutants

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  12. Repair of mismatched basepairs in mammalian DNA

    SciTech Connect

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

    1991-08-01

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

  13. DNA Repair Is Associated with Information Content in Bacteria, Archaea, and DNA Viruses.

    PubMed

    Acosta, Sharlene; Carela, Miguelina; Garcia-Gonzalez, Aurian; Gines, Mariela; Vicens, Luis; Cruet, Ricardo; Massey, Steven E

    2015-01-01

    The concept of a "proteomic constraint" proposes that DNA repair capacity is positively correlated with the information content of a genome, which can be approximated to the size of the proteome (P). This in turn implies that DNA repair genes are more likely to be present in genomes with larger values of P. This stands in contrast to the common assumption that informational genes have a core function and so are evenly distributed across organisms. We examined the presence/absence of 18 DNA repair genes in bacterial genomes. A positive relationship between gene presence and P was observed for 17 genes in the total dataset, and 16 genes when only nonintracellular bacteria were examined. A marked reduction of DNA repair genes was observed in intracellular bacteria, consistent with their reduced value of P. We also examined archaeal and DNA virus genomes, and show that the presence of DNA repair genes is likewise related to a larger value of P. In addition, the products of the bacterial genes mutY, vsr, and ndk, involved in the correction of GC/AT mutations, are strongly associated with reduced genome GC content. We therefore propose that a reduction in information content leads to a loss of DNA repair genes and indirectly to a reduction in genome GC content in bacteria by exposure to the underlying AT mutation bias. The reduction in P may also indirectly lead to the increase in substitution rates observed in intracellular bacteria via loss of DNA repair genes.

  14. Control of gene editing by manipulation of DNA repair mechanisms.

    PubMed

    Danner, Eric; Bashir, Sanum; Yumlu, Saniye; Wurst, Wolfgang; Wefers, Benedikt; Kühn, Ralf

    2017-04-03

    DNA double-strand breaks (DSBs) are produced intentionally by RNA-guided nucleases to achieve genome editing through DSB repair. These breaks are repaired by one of two main repair pathways, classic non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter being restricted to the S/G2 phases of the cell cycle and notably less frequent. Precise genome editing applications rely on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence modifications. Here, we give an overview of HDR- and c-NHEJ-mediated DSB repair in gene editing and summarize the current efforts to promote HDR over c-NHEJ.

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

    PubMed Central

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

    2011-01-01

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

  16. DNA repair and aging: the impact of the p53 family

    PubMed Central

    Nicolai, Sara; Rossi, Antonello; Di Daniele, Nicola; Melino, Gerry; Annicchiarico-Petruzzelli, Margherita; Raschellà, Giuseppe

    2015-01-01

    Cells are constantly exposed to endogenous and exogenous factors that threaten the integrity of their DNA. The maintenance of genome stability is of paramount importance in the prevention of both cancer and aging processes. To deal with DNA damage, cells put into operation a sophisticated and coordinated mechanism, collectively known as DNA damage response (DDR). The DDR orchestrates different cellular processes, such as DNA repair, senescence and apoptosis. Among the key factors of the DDR, the related proteins p53, p63 and p73, all belonging to the same family of transcription factors, play multiple relevant roles. Indeed, the members of this family are directly involved in the induction of cell cycle arrest that is necessary to allow the cells to repair. Alternatively, they can promote cell death in case of prolonged or irreparable DNA damage. They also take part in a more direct task by modulating the expression of core factors involved in the process of DNA repair or by directly interacting with them. In this review we will analyze the fundamental roles of the p53 family in the aging process through their multifaceted function in DDR. PMID:26668111

  17. DNA repair and aging: the impact of the p53 family.

    PubMed

    Nicolai, Sara; Rossi, Antonello; Di Daniele, Nicola; Melino, Gerry; Annicchiarico-Petruzzelli, Margherita; Raschellà, Giuseppe

    2015-12-01

    Cells are constantly exposed to endogenous and exogenous factors that threaten the integrity of their DNA. The maintenance of genome stability is of paramount importance in the prevention of both cancer and aging processes. To deal with DNA damage, cells put into operation a sophisticated and coordinated mechanism, collectively known as DNA damage response (DDR). The DDR orchestrates different cellular processes, such as DNA repair, senescence and apoptosis. Among the key factors of the DDR, the related proteins p53, p63 and p73, all belonging to the same family of transcription factors, play multiple relevant roles. Indeed, the members of this family are directly involved in the induction of cell cycle arrest that is necessary to allow the cells to repair. Alternatively, they can promote cell death in case of prolonged or irreparable DNA damage. They also take part in a more direct task by modulating the expression of core factors involved in the process of DNA repair or by directly interacting with them. In this review we will analyze the fundamental roles of the p53 family in the aging process through their multifaceted function in DDR.

  18. Emerging critical roles of Fe-S clusters in DNA replication and repair

    PubMed Central

    Fuss, Jill O.; Tsai, Chi-Lin; Ishida, Justin P.; Tainer, John A.

    2015-01-01

    Fe-S clusters are partners in the origin of life that predate cells, acetyl-CoA metabolism, DNA, and the RNA world. The double helix solved the mystery of DNA replication by base pairing for accurate copying. Yet, for genome stability necessary to life, the double helix has equally important implications for damage repair. Here we examine striking advances that uncover Fe-S cluster roles both in copying the genetic sequence by DNA polymerases and in crucial repair processes for genome maintenance, as mutational defects cause cancer and degenerative disease. Moreover, we examine an exciting, controversial role for Fe-S clusters in a third element required for life – the long-range coordination and regulation of replication and repair events. By their ability to delocalize electrons over both Fe and S centers, Fe-S clusters have unbeatable features for protein conformational control and charge transfer via double-stranded DNA that may fundamentally transform our understanding of life, replication, and repair. PMID:25655665

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

    SciTech Connect

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

    2005-01-01

    Archaea are found in some of the most extreme environments on earth and represent a third domain of life distinct from Eukarya and Eubacteria. The hyperthermophilic archaeon Sulfolobus solfataricus, isolated from acidic hot springs (80oC, pH 3) in Yellowstone National Park, has emerged as a potential model system for studying human DNA repair processes. Archaea are more closely related to Eukarya than to Eubacteria, suggesting that archaeal DNA repair machinery may model the complex human system much more closely than that of other prokaryotes. DNA repair requires coordinated protein-protein interactions that are frequently transient. Protein complexes that are transient at extreme temperatures where archaea thrive may be more stable at room temperature, allowing for the characterization of otherwise short-lived complexes. However, characterization of these systems in archaea has been limited by the absence of a stable in vivo transformation and expression system. The work presented here is a pilot study in gene cloning and recombinant protein expression in S. solfataricus. Three genes associated with DNA repair were selected for expression: MRE11, PCNA1, and a putative CSB homologue. Though preparation of these recombinant genes followed standard methods, preparation of a suitable vector proved more challenging. The shuttle vector pSSV64, derived from the SSV1 virus and the E. coli vector pBSSK+, was most successfully isolated from the DH5α E. coli strain. Currently, alternative vectors are being designed for more efficient genetic manipulations in S. solfataricus.

  20. Emerging critical roles of Fe-S clusters in DNA replication and repair.

    PubMed

    Fuss, Jill O; Tsai, Chi-Lin; Ishida, Justin P; Tainer, John A

    2015-06-01

    Fe-S clusters are partners in the origin of life that predate cells, acetyl-CoA metabolism, DNA, and the RNA world. The double helix solved the mystery of DNA replication by base pairing for accurate copying. Yet, for genome stability necessary to life, the double helix has equally important implications for damage repair. Here we examine striking advances that uncover Fe-S cluster roles both in copying the genetic sequence by DNA polymerases and in crucial repair processes for genome maintenance, as mutational defects cause cancer and degenerative disease. Moreover, we examine an exciting, controversial role for Fe-S clusters in a third element required for life - the long-range coordination and regulation of replication and repair events. By their ability to delocalize electrons over both Fe and S centers, Fe-S clusters have unbeatable features for protein conformational control and charge transfer via double-stranded DNA that may fundamentally transform our understanding of life, replication, and repair. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

  1. Defective DNA repair mechanisms in prostate cancer: impact of olaparib

    PubMed Central

    De Felice, Francesca; Tombolini, Vincenzo; Marampon, Francesco; Musella, Angela; Marchetti, Claudia

    2017-01-01

    The field of prostate oncology has continued to change dramatically. It has truly become a field that is intensely linked to molecular genetic alterations, especially DNA-repair defects. Germline breast cancer 1 gene (BRCA1) and breast cancer 2 gene (BRCA2) mutations are implicated in the highest risk of prostate cancer (PC) predisposition and aggressiveness. Poly adenosine diphosphate ribose polymerase (PARP) proteins play a key role in DNA repair mechanisms and represent a valid target for new therapies. Olaparib is an oral PARP inhibitor that blocks DNA repair pathway and coupled with BRCA mutated-disease results in tumor cell death. In phase II clinical trials, including patients with advanced castration-resistant PC, olaparib seems to be efficacious and well tolerated. Waiting for randomized phase III trials, olaparib should be considered as a promising treatment option for PC. PMID:28280302

  2. Metabolism, Genomics, and DNA Repair in the Mouse Aging Liver

    PubMed Central

    Lebel, Michel; de Souza-Pinto, Nadja C.; Bohr, Vilhelm A.

    2011-01-01

    The liver plays a pivotal role in the metabolism of nutrients, drugs, hormones, and metabolic waste products, thereby maintaining body homeostasis. The liver undergoes substantial changes in structure and function within old age. Such changes are associated with significant impairment of many hepatic metabolic and detoxification activities, with implications for systemic aging and age-related disease. It has become clear, using rodent models as biological tools, that genetic instability in the form of gross DNA rearrangements or point mutations accumulate in the liver with age. DNA lesions, such as oxidized bases or persistent breaks, increase with age and correlate well with the presence of senescent hepatocytes. The level of DNA damage and/or mutation can be affected by changes in carcinogen activation, decreased ability to repair DNA, or a combination of these factors. This paper covers some of the DNA repair pathways affecting liver homeostasis with age using rodents as model systems. PMID:21559242

  3. An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation

    PubMed Central

    Grin, Inga; Ishchenko, Alexander A.

    2016-01-01

    Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM. PMID:26843430

  4. DNA damage and repair in telomeres: relation to aging.

    PubMed Central

    Kruk, P A; Rampino, N J; Bohr, V A

    1995-01-01

    We have established a method for the detection of DNA damage and its repair in human telomeres, the natural ends of chromosomes which are necessary for replication and critical for chromosomal stability. We find that ultraviolet light-induced pyrimidine dimers in telomeric DNA are repaired less efficiently than endogenous genes but more efficiently than inactive, noncoding regions. We have also measured telomeric length, telomeric DNA damage, and its repair in relation to the progression of aging. Telomeres are shorter in fibroblasts from an old donor compared to fibroblasts from a young donor, shortest in cells from a patient with the progeroid disorder Werner syndrome, and relatively long in fibroblasts from a patient with Alzheimer disease. Telomeric DNA repair efficiency is lower in cells from an old donor than in cells from a young donor, normal in Alzheimer cells, and slightly lower in Werner cells. It is possible that this decline in telomeric repair with aging is of functional significance to an age-related decline in genomic stability. Images Fig. 1 Fig. 2 PMID:7816828

  5. FANCD2 binds CtIP and regulates DNA-end resection during DNA interstrand crosslink repair.

    PubMed

    Unno, Junya; Itaya, Akiko; Taoka, Masato; Sato, Koichi; Tomida, Junya; Sakai, Wataru; Sugasawa, Kaoru; Ishiai, Masamichi; Ikura, Tsuyoshi; Isobe, Toshiaki; Kurumizaka, Hitoshi; Takata, Minoru

    2014-05-22

    The Fanconi anemia (FA) pathway is critically involved in the maintenance of hematopoietic stem cells and the suppression of carcinogenesis. A key FA protein, FANCD2, is monoubiquitinated and accumulates in chromatin in response to DNA interstrand crosslinks (ICLs), where it coordinates DNA repair through mechanisms that are still poorly understood. Here, we report that CtIP protein directly interacts with FANCD2. A region spanning amino acids 166 to 273 of CtIP and monoubiquitination of FANCD2 are both essential for the FANCD2-CtIP interaction and mitomycin C (MMC)-induced CtIP foci. Remarkably, both FANCD2 and CtIP are critical for MMC-induced RPA2 hyperphosphorylation, an event that accompanies end resection of double-strand breaks. Collectively, our results reveal a role of monoubiquitinated FANCD2 in end resection that depends on its binding to CtIP during ICL repair.

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

    SciTech Connect

    Protic, M.

    1994-12-31

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

  7. DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair

    PubMed Central

    Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

    2016-01-01

    DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell’s genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy. PMID:27589807

  8. DNA double strand break repair, aging and the chromatin connection.

    PubMed

    Gorbunova, Vera; Seluanov, Andrei

    2016-06-01

    Are DNA damage and mutations possible causes or consequences of aging? This question has been hotly debated by biogerontologists for decades. The importance of DNA damage as a possible driver of the aging process went from being widely recognized to then forgotten, and is now slowly making a comeback. DNA double strand breaks (DSBs) are particularly relevant to aging because of their toxicity, increased frequency with age and the association of defects in their repair with premature aging. Recent studies expand the potential impact of DNA damage and mutations on aging by linking DNA DSB repair and age-related chromatin changes. There is overwhelming evidence that increased DNA damage and mutations accelerate aging. However, an ultimate proof of causality would be to show that enhanced genome and epigenome stability delays aging. This is not an easy task, as improving such complex biological processes is infinitely more difficult than disabling it. We will discuss the possibility that animal models with enhanced DNA repair and epigenome maintenance will be generated in the near future.

  9. Ultraviolet irradiation of monkey cells enhances the repair of DNA adducts in alpha DNA

    SciTech Connect

    Leadon, S.A.; Hanawalt, P.C.

    1984-11-01

    Excision repair of bulky adducts in alpha DNA of African green monkey cells has previously been shown to be deficient relative to that in the overall genome. We have found that u.v. irradiation of these cells results in the enhanced removal of both aflatoxin B1 (AFB1) and acetylaminofluorene (AAF) adducts from the alpha DNA sequences without affecting repair in the bulk of the DNA. The degree of enhanced removal of AFB1 is dependent upon the u.v. dose and the time interval between irradiation and AFB1 treatment. The u.v. enhancement is not inhibited by cycloheximide. Exposure of the cells to dimethylsulfate or gamma-rays does not affect AFB1 adduct repair. The formation and removal of N-acetoxy-2-acetylaminofluorene (NA-AAF) adducts from alpha and bulk DNA was studied in detail. A higher initial level of the acetylated C8 adduct of guanine was found in alpha DNA than in bulk DNA. Although both the acetylated and deacetylated C8 adducts were removed from the two DNA species, the level of repair was significantly greater in the bulk DNA. Irradiation of cells with u.v. prior to treatment with NA-AAF enhanced the removal of both adducts from alpha DNA with little or no effect on repair in bulk DNA. We conclude that the presence of u.v. photoproducts or some intermediate in their processing alters the chromatin structure of alpha DNA thereby rendering bulky adducts accessible to repair enzymes. In addition, the differential formation and repair of AAF adducts in alpha DNA compared with that in the bulk of the genome supports the hypothesis of an altered chromatin structure for alpha domains.

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

    PubMed

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

    1996-06-27

    The impact of microgravity on cellular repair processes was tested in the space experiments REPAIR and KINETICS, which were performed during the IML-2 mission in the Biorack of ESA: (a) survival of spores of Bacillus subtilis HA101 after UV-irradiation (up to 340 J m-2) in the experiment REPAIR; (b) in the experiment KINETICS the kinetics of DNA repair in three different test systems: rejoining of X-ray-induced DNA strand breaks (B1) in cells of Escherichia coli B/r (120 Gy) and (B2) in human fibroblasts (5 and 10 Gy) as well as (B3) induction of the SOS response after gamma-irradiation (300 Gy) of cells of Escherichia coli PQ37. Cells were irradiated prior to the space mission and were kept in a non-metabolic state (metabolically inactive spores of B. subtilis on membrane filters, frozen cells of E. coli and human fibroblasts) until incubation in orbit. Germination and growth of B. subtilis were initiated by humidification, E. coli and fibroblasts were thawed up and incubated at 37 degrees C for defined repair periods (up to 4.5 h), thereafter they were frozen again for laboratory analysis. Relevant controls were performed in-flight (1 x g reference centrifuge) and on ground (1 x g and 1.4 x g) The results show no significant differences between the microgravity samples and the corresponding controls neither in the survival curves nor in the kinetics of DNA strand break rejoining and induction of the SOS response (proven by Student's t-test, 2 P = 0.05). These observations provide evidence that in the microgravity environment cells are able to repair radiation-induced DNA damage close to normality. The results suggest that a disturbance of cellular repair processes in the microgravity environment might not be the explanation for the reported synergism of radiation and microgravity.

  11. DNA-damage repair; the good, the bad, and the ugly.

    PubMed

    Hakem, Razqallah

    2008-02-20

    Organisms have developed several DNA-repair pathways as well as DNA-damage checkpoints to cope with the frequent challenge of endogenous and exogenous DNA insults. In the absence or impairment of such repair or checkpoint mechanisms, the genomic integrity of the organism is often compromised. This review will focus on the functional consequences of impaired DNA-repair pathways. Although each pathway is addressed individually, it is essential to note that cross talk exists between repair pathways, and that there are instances in which a DNA-repair protein is involved in more than one pathway. It is also important to integrate DNA-repair process with DNA-damage checkpoints and cell survival, to gain a better understanding of the consequences of compromised DNA repair at both cellular and organismic levels. Functional consequences associated with impaired DNA repair include embryonic lethality, shortened life span, rapid ageing, impaired growth, and a variety of syndromes, including a pronounced manifestation of cancer.

  12. Polymorphisms in DNA repair genes and associations with cancer risk.

    PubMed

    Goode, Ellen L; Ulrich, Cornelia M; Potter, John D

    2002-12-01

    Common polymorphisms in DNA repair genes may alter protein function and an individual's capacity to repair damaged DNA; deficits in repair capacity may lead to genetic instability and carcinogenesis. To establish our overall understanding of possible in vivo relationships between DNA repair polymorphisms and the development of cancer, we performed a literature review of epidemiological studies that assessed associations between such polymorphisms and risk of cancer. Thirty studies of polymorphisms in OGG1, XRCC1, ERCC1, XPC, XPD, XPF, BRCA2, and XRCC3 were identified in the April 30, 2002 MEDLINE database (National Center for Biotechnology Information. PubMed Database: http://www.ncbi.nlm.nih.gov/entrez). These studies focused on adult glioma, bladder cancer, breast cancer, esophageal cancer, lung cancer, prostate cancer, skin cancer (melanoma and nonmelanoma), squamous cell carcinoma of the head and neck, and stomach cancer. We found that a small proportion of the published studies were large and population-based. Nonetheless, published data were consistent with associations between: (a) the OGG1 S326C variant and increased risk of various types of cancer; (b) the XRCC1 R194W variant and reduced risk of various types of cancer; and (c) the BRCA2 N372H variant and increased risk of breast cancer. Suggestive results were seen for polymorphisms in other genes; however, small sample sizes may have contributed to false-positive or false-negative findings. We conclude that large, well-designed studies of common polymorphisms in DNA repair genes are needed. Such studies may benefit from analysis of multiple genes or polymorphisms and from the consideration of relevant exposures that may influence the likelihood of cancer in the presence of reduced DNA repair capacity.

  13. Repair of Topoisomerase I-Mediated DNA Damage

    PubMed Central

    Pommier, Yves; Barcelo, Juana; Rao, V. Ashutosh; Sordet, Olivier; Jobson, Andrew G.; Thibaut, Laurent; Miao, Zheyong; Seiler, Jennifer; Zhang, Hongliang; Marchand, Christophe; Agama, Keli; Redon, Christophe

    2008-01-01

    Topoisomerase I (Top1) is an abundant and essential enzyme. Top1 is the selective target of camptothecins, which are effective anticancer agents. Top1-DNA cleavage complexes can also be trapped by various endogenous and exogenous DNA lesions including mismatches, abasic sites and carcinogenic adducts. Tyrosyl-DNA phosphodiesterase (Tdp1) is one of the repair enzymes for Top1-DNA covalent complexes. Tdp1 forms a multiprotein complex that includes poly(ADP) ribose polymerase (PARP). PARP-deficient cells are hypersensitive to camptothecins and functionally deficient for Tdp1. We will review recent developments in several pathways involved in the repair of Top1 cleavage complexes and the role of Chk1 and Chk2 checkpoint kinases in the cellular responses to Top1 inhibitors. The genes conferring camptothecin hypersensitivity are compiled for humans, budding yeast and fission yeast. PMID:16891172

  14. DEK is required for homologous recombination repair of DNA breaks

    PubMed Central

    Smith, Eric A.; Gole, Boris; Willis, Nicholas A.; Soria, Rebeca; Starnes, Linda M.; Krumpelbeck, Eric F.; Jegga, Anil G.; Ali, Abdullah M.; Guo, Haihong; Meetei, Amom R.; Andreassen, Paul R.; Kappes, Ferdinand; Vinnedge, Lisa M. Privette; Daniel, Jeremy A.; Scully, Ralph; Wiesmüller, Lisa; Wells, Susanne I.

    2017-01-01

    DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition. PMID:28317934

  15. DEK is required for homologous recombination repair of DNA breaks.

    PubMed

    Smith, Eric A; Gole, Boris; Willis, Nicholas A; Soria, Rebeca; Starnes, Linda M; Krumpelbeck, Eric F; Jegga, Anil G; Ali, Abdullah M; Guo, Haihong; Meetei, Amom R; Andreassen, Paul R; Kappes, Ferdinand; Vinnedge, Lisa M Privette; Daniel, Jeremy A; Scully, Ralph; Wiesmüller, Lisa; Wells, Susanne I

    2017-03-20

    DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.

  16. Maintenance of DNA and repair of Apurinic sites.

    PubMed

    Verly, W G

    1975-01-01

    Escherichia coli cells contain an enzyme which hydrolyzes a phosphodiester bond near each apurinic site in double-stranded DNA. This endonuclease is specific for apurinic sites; it has no effect on normal DNA, and its action on alkylated DNA is restricted to apurinic sites. In vitro incubation with the endonuclease for apurinic sites, DNA polymerase I, and ligase permits repair of DNA containing apurinic sites. The endonuclease for apurinic sites might thus play a role in cell survival after a treatment with alkylating agents; as DNA spontaneously loses purines, the enzyme might also play a role in the maintance of a normal DNA in every cell. Indeed, an endonuclease for apurinic sites has been found not only in bacteria but also in animal and plant cells; it is very active in thermophilic bacteria.

  17. Chromatin remodeling in DNA double-strand break repair.

    PubMed

    Bao, Yunhe; Shen, Xuetong

    2007-04-01

    ATP-dependent chromatin remodeling complexes use ATP hydrolysis to remodel nucleosomes and have well-established functions in transcription. However, emerging lines of evidence suggest that chromatin remodeling complexes are important players in DNA double-strand break (DSB) repair as well. The INO80 and SWI2 subfamilies of chromatin remodeling complexes have been found to be recruited to the double-strand lesions and to function directly in both homologous recombination and non-homologous end-joining, the two major conserved DSB repair pathways. Improperly repaired DSBs are implicated in cancer development in higher organisms. Understanding how chromatin remodeling complexes contribute to DSB repair should provide new insights into the mechanisms of carcinogenesis and might suggest new targets for cancer treatment.

  18. Roles of chromatin remodellers in DNA double strand break repair.

    PubMed

    Jeggo, Penny A; Downs, Jessica A

    2014-11-15

    Now that we have a good understanding of the DNA double strand break (DSB) repair mechanisms and DSB-induced damage signalling, attention is focusing on the changes to the chromatin environment needed for efficient DSB repair. Mutations in chromatin remodelling complexes have been identified in cancers, making it important to evaluate how they impact upon genomic stability. Our current understanding of the DSB repair pathways suggests that each one has distinct requirements for chromatin remodelling. Moreover, restricting the extent of chromatin modifications could be a significant factor regulating the decision of pathway usage. In this review, we evaluate the distinct DSB repair pathways for their potential need for chromatin remodelling and review the roles of ATP-driven chromatin remodellers in the pathways.

  19. Tumor suppressor and deubiquitinase BAP1 promotes DNA double-strand break repair

    PubMed Central

    Yu, Helen; Pak, Helen; Hammond-Martel, Ian; Ghram, Mehdi; Rodrigue, Amélie; Daou, Salima; Barbour, Haithem; Corbeil, Luc; Hébert, Josée; Drobetsky, Elliot; Masson, Jean Yves; Di Noia, Javier M.; Affar, El Bachir

    2014-01-01

    The cellular response to highly genotoxic DNA double-strand breaks (DSBs) involves the exquisite coordination of multiple signaling and repair factors. Here, we conducted a functional RNAi screen and identified BAP1 as a deubiquitinase required for efficient assembly of the homologous recombination (HR) factors BRCA1 and RAD51 at ionizing radiation (IR) -induced foci. BAP1 is a chromatin-associated protein frequently inactivated in cancers of various tissues. To further investigate the role of BAP1 in DSB repair, we used a gene targeting approach to knockout (KO) this deubiquitinase in chicken DT40 cells. We show that BAP1-deficient cells are (i) sensitive to IR and other agents that induce DSBs, (ii) defective in HR-mediated immunoglobulin gene conversion, and (iii) exhibit an increased frequency of chromosomal breaks after IR treatment. We also show that BAP1 is recruited to chromatin in the proximity of a single site-specific I-SceI–induced DSB. Finally, we identified six IR-induced phosphorylation sites in BAP1 and showed that mutation of these residues inhibits BAP1 recruitment to DSB sites. We also found that both BAP1 catalytic activity and its phosphorylation are critical for promoting DNA repair and cellular recovery from DNA damage. Our data reveal an important role for BAP1 in DSB repair by HR, thereby providing a possible molecular basis for its tumor suppressor function. PMID:24347639

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

    PubMed

    Mukherjee, Anirban; Vasquez, Karen M

    2011-08-01

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

  1. Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription.

    PubMed

    Nadkarni, Aditi; Burns, John A; Gandolfi, Alberto; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E; Scicchitano, David A

    2016-01-08

    DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER.

  2. Roles of PTEN with DNA Repair in Parkinson's Disease.

    PubMed

    Ogino, Mako; Ichimura, Mayuko; Nakano, Noriko; Minami, Akari; Kitagishi, Yasuko; Matsuda, Satoru

    2016-06-15

    Oxidative stress is considered to play key roles in aging and pathogenesis of many neurodegenerative diseases such as Parkinson's disease, which could bring DNA damage by cells. The DNA damage may lead to the cell apoptosis, which could contribute to the degeneration of neuronal tissues. Recent evidence suggests that PTEN (phosphatase and tensin homolog on chromosome 10) may be involved in the pathophysiology of the neurodegenerative disorders. Since PTEN expression appears to be one dominant determinant of the neuronal cell death, PTEN should be a potential molecular target of novel therapeutic strategies against Parkinson's disease. In addition, defects in DNA damage response and DNA repair are often associated with modulation of hormone signaling pathways. Especially, many observations imply a role for estrogen in a regulation of the DNA repair action. In the present review, we have attempted to summarize the function of DNA repair molecules at a viewpoint of the PTEN signaling pathway and the hormone related functional modulation of cells, providing a broad interpretation on the molecular mechanisms for treatment of Parkinson's disease. Particular attention will be paid to the mechanisms proposed to explain the health effects of food ingredients against Parkinson's disease related to reduce oxidative stress for an efficient therapeutic intervention.

  3. Oxidative DNA damage and repair in teratogenesis and neurodevelopmental deficits.

    PubMed

    Wells, Peter G; McCallum, Gordon P; Lam, Kyla C H; Henderson, Jeffrey T; Ondovcik, Stephanie L

    2010-06-01

    Several teratogenic agents, including ionizing radiation and xenobiotics such as phenytoin, benzo[a]pyrene, thalidomide, and methamphetamine, can initiate the formation of reactive oxygen species (ROS) that oxidatively damage cellular macromolecules including DNA. Oxidative DNA damage, and particularly the most prevalent 8-oxoguanine lesion, may adversely affect development, likely via alterations in gene transcription rather than via a mutational mechanism. Contributions from oxidative DNA damage do not exclude roles for alternative mechanisms of initiation like receptor-mediated processes or the formation of covalent xenobiotic-macromolecular adducts, damage to other macromolecular targets like proteins and lipids, and other effects of ROS like altered signal transduction. Even in the absence of teratogen exposure, endogenous developmental oxidative stress can have embryopathic consequences in the absence of key pathways for detoxifying ROS or repairing DNA damage. Critical proteins in pathways for DNA damage detection/repair signaling, like p53 and ataxia telangiectasia mutated, and DNA repair itself, like oxoguanine glycosylase 1 and Cockayne syndrome B, can often, but not always, protect the embryo from ROS-initiating teratogens. Protection may be variably dependent upon such factors as the nature of the teratogen and its concentration within the embryo, the stage of development, the species, strain, gender, target tissue and cell type, among other factors.

  4. Autophagy positively regulates DNA damage recognition by nucleotide excision repair.

    PubMed

    Qiang, Lei; Zhao, Baozhong; Shah, Palak; Sample, Ashley; Yang, Seungwon; He, Yu-Ying

    2016-01-01

    Macroautophagy (hereafter autophagy) is a cellular catabolic process that is essential for maintaining tissue homeostasis and regulating various normal and pathologic processes in human diseases including cancer. One cancer-driving process is accumulation of genetic mutations due to impaired DNA damage repair, including nucleotide excision repair. Here we show that autophagy positively regulates nucleotide excision repair through enhancing DNA damage recognition by the DNA damage sensor proteins XPC and DDB2 via 2 pathways. First, autophagy deficiency downregulates the transcription of XPC through TWIST1-dependent activation of the transcription repressor complex E2F4-RBL2. Second, autophagy deficiency impairs the recruitment of DDB2 to ultraviolet radiation (UV)-induced DNA damage sites through TWIST1-mediated inhibition of EP300. In mice, the pharmacological autophagy inhibitor Spautin-1 promotes UVB-induced tumorigenesis, whereas the autophagy inducer rapamycin reduces UVB-induced tumorigenesis. These findings demonstrate the crucial role of autophagy in maintaining proper nucleotide excision repair in mammalian cells and suggest a previously unrecognized tumor-suppressive mechanism of autophagy in cancer.

  5. p53 downregulates the Fanconi anaemia DNA repair pathway

    PubMed Central

    Jaber, Sara; Toufektchan, Eléonore; Lejour, Vincent; Bardot, Boris; Toledo, Franck

    2016-01-01

    Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53Δ31, a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53Δ31/Δ31 fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53Δ31/Δ31 fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop. PMID:27033104

  6. Oxidative Stress, DNA Repair and Prostate Cancer Risk

    DTIC Science & Technology

    2010-08-01

    progressed smoothly for all three specific aims. 15. SUBJECT TERMS microRNA ovarian cancer 16. SECURITY CLASSIFICATION OF: 17. LIMITATION... factors for prostate cancer are associated with elevated levels of ROS (advancing age, inflammation, androgen, high-fat diet), or decreased...TITLE: Oxidative Stress, DNA Repair and Prostate Cancer Risk PRINCIPAL INVESTIGATOR: Hua Zhao, Ph.D

  7. UV Radiation Damage and Bacterial DNA Repair Systems

    ERIC Educational Resources Information Center

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

    2006-01-01

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

  8. DNA Repair-Protein Relocalization After Heavy Ion Exposure

    NASA Technical Reports Server (NTRS)

    Metting, N. F.

    1999-01-01

    Ionizing radiation is good at making DNA double strand breaks, and high linear energy transfer (LET) radiations such as heavy ion particles are particularly efficient. For this reason, the proteins belonging to repair systems that deal with double strand breaks are of particular interest. One such protein is Ku, a component in the non-homologous recombination repair system. The Ku protein is an abundant, heterodimeric DNA end-binding complex, composed of one 70 and one 86 kDa subunit. Ku protein binds to DNA ends, nicks, gaps, and regions of transition between single and double-stranded structure. These binding properties suggest an important role in DNA repair. The Ku antigen is important in this study because it is present in relatively large copy numbers and it is part of a double-strand-break repair system. More importantly, we consistently measure an apparent upregulation in situ that is not verified by whole-cell-lysate immunoblot measurements. This apparent upregulation is triggered by very low doses of radiation, thus showing a potentially useful high sensitivity. However, elucidation of the mechanism underlying this phenomenon is still to be done.

  9. Base excision repair in Archaea: back to the future in DNA repair.

    PubMed

    Grasso, Stefano; Tell, Gianluca

    2014-09-01

    Together with Bacteria and Eukarya, Archaea represents one of the three domain of life. In contrast with the morphological difference existing between Archaea and Eukarya, these two domains are closely related. Phylogenetic analyses confirm this evolutionary relationship showing that most of the proteins involved in DNA transcription and replication are highly conserved. On the contrary, information is scanty about DNA repair pathways and their mechanisms. In the present review the most important proteins involved in base excision repair, namely glycosylases, AP lyases, AP endonucleases, polymerases, sliding clamps, flap endonucleases, and ligases, will be discussed and compared with bacterial and eukaryotic ones. Finally, possible applications and future perspectives derived from studies on Archaea and their repair pathways, will be taken into account.

  10. Measurement of DNA repair deficiency in workers exposed to benzene

    SciTech Connect

    Hallberg, L.M.; Au, W.W.; El Zein, R.; Grossman, L.

    1996-05-01

    We hypothesize that chronic exposure to environmental toxicants can induce genetic damage causing DNA repair deficiencies and leading to the postulated mutator phenotype of carcinogenesis. To test our hypothesis, a host cell reactivation (HCR) assay was used in which pCMVcat plasmids were damaged with UV light (175, 350 J/m{sup 2} UV light), inactivating the chloramphenicol acetyltransferase reporter gene, and then transfected into lymphocytes. Transfected lymphocytes were therefore challenged to repair the damaged plasmids, reactivating the reporter gene. Xeroderma pigmentosum (XP) and Gaucher cell lines were used as positive and negative controls for the HCR assay. The Gaucher cell line repaired normally but XP cell lines demonstrated lower repair activity. Additionally, the repair activity of the XP heterozygous cell line showed intermediate repair compared to the homozygous XP and Gaucher cells. We used HCR to measure the effects of benzene exposure on 12 exposed and 8 nonexposed workers from a local benzene plant. Plasmids 175 J/m{sup 2} and 350 J/m{sup 2} were repaired with a mean frequency of 66% and 58%, respectively, in control workers compared to 71% and 62% in exposed workers. Conversely, more of the exposed workers were grouped into the reduced repair category than controls. These differences in repair capacity between exposed and control workers were, however, not statistically significant. The lack of significant differences between the exposed and control groups may be due to extremely low exposure to benzene (<0.3 ppm), small population size, or a lack of benzene genotoxicity at these concentrations. These results are consistent with a parallel hprt gene mutation assay. 26 refs., 4 figs., 2 tabs.

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

    PubMed Central

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

    2012-01-01

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

  12. GADD45α inhibition of DNMT1 dependent DNA methylation during homology directed DNA repair

    PubMed Central

    Lee, Bongyong; Morano, Annalisa; Porcellini, Antonio; Muller, Mark T.

    2012-01-01

    In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45α. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45α, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45α during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45α increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45α specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45α. Since GADD45α binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair. PMID:22135303

  13. Assays for DNA double-strand break repair by microhomology-based end-joining repair mechanisms.

    PubMed

    Kostyrko, Kaja; Mermod, Nicolas

    2016-04-07

    DNA double stranded breaks (DSBs) are one of the most deleterious types of DNA lesions. The main pathways responsible for repairing these breaks in eukaryotic cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). However, a third group of still poorly characterized DSB repair pathways, collectively termed microhomology-mediated end-joining (MMEJ), relies on short homologies for the end-joining process. Here, we constructed GFP reporter assays to characterize and distinguish MMEJ variant pathways, namely the simple MMEJ and the DNA synthesis-dependent (SD)-MMEJ mechanisms. Transfection of these assay vectors in Chinese hamster ovary (CHO) cells and characterization of the repaired DNA sequences indicated that while simple MMEJ is able to mediate relatively efficient DSB repair if longer microhomologies are present, the majority of DSBs were repaired using the highly error-prone SD-MMEJ pathway. To validate the involvement of DNA synthesis in the repair process, siRNA knock-down of different genes proposed to play a role in MMEJ were performed, revealing that the knock-down of DNA polymerase θ inhibited DNA end resection and repair through simple MMEJ, thus favoring the other repair pathway. Overall, we conclude that this approach provides a convenient assay to study MMEJ-related DNA repair pathways.

  14. DNA repair in spermatocytes and spermatids of the mouse

    SciTech Connect

    Sega, G.A.

    1982-01-01

    When male mice are exposed to chemical agents that reach the germ cells several outcomes are possible in terms of the germ cell unscheduled DNA synthesis (UDS) response and removal of DNA adducts. It is possible that: the chemical binds to the DNA and induces a UDS response with concomittant removal of DNA adducts; the chemical binds to the DNA but no UDS response is induced; or the chemical does not bind to DNA and no UDS is induced. Many mutagens have been shown to induce a UDS response in postgonial germ cell stages of the male mouse up through midspermatids, but the relationship between this UDS and the repair of genetic damage within the germ cells is still unknown. While some mutagens appear to have an effect only in germ-cell stages where no UDS occurs, others are able to induce genetic damage in stages where UDS has been induced.

  15. E2F-7 couples DNA damage-dependent transcription with the DNA repair process.

    PubMed

    Zalmas, Lykourgos-Panagiotis; Coutts, Amanda S; Helleday, Thomas; La Thangue, Nicholas B

    2013-09-15

    The cellular response to DNA damage, mediated by the DNA repair process, is essential in maintaining the integrity and stability of the genome. E2F-7 is an atypical member of the E2F family with a role in negatively regulating transcription and cell cycle progression under DNA damage. Surprisingly, we found that E2F-7 makes a transcription-independent contribution to the DNA repair process, which involves E2F-7 locating to and binding damaged DNA. Further, E2F-7 recruits CtBP and HDAC to the damaged DNA, altering the local chromatin environment of the DNA lesion. Importantly, the E2F-7 gene is a target for somatic mutation in human cancer and tumor-derived mutant alleles encode proteins with compromised transcription and DNA repair properties. Our results establish that E2F-7 participates in 2 closely linked processes, allowing it to directly couple the expression of genes involved in the DNA damage response with the DNA repair machinery, which has relevance in human malignancy.

  16. Hindered DNA coordinated motion in nanochannels

    NASA Astrophysics Data System (ADS)

    Chen, Yeng-Long; Lin, Yu-Hui; Luzhbin, Dmytro

    2014-03-01

    We investigate the relaxation dynamics of long DNA molecules confined in micro- and nano-channels with Brownian dynamics simulations. Prior experiment by Reisner et al. found that the stretch fluctuation correlation time (trelax) of DNA molecules in nanochannels increases as the channel height (H) decreases for H greater than the DNA Kuhn length (σk), and trelax decreases as H decreases for H <σk . Our simulations capture this behavior, and quantitatively agree with the experimental results within the error bars. The scaling-law dependence of trelax on H in different regimes is verified. Rouse mode analysis of the chain relaxation mechanism further shows that segmental relaxation on length scale longer than σk are hindered and the dynamics of segments shorter than H dominate the chain relaxation processes. We also find that the inclusion of intra-chain hydrodynamic interactions affect segmental relaxation. The implications for DNA translocation through nanopores and nanochannels are discussed.

  17. Hypomorphic PCNA mutation underlies a human DNA repair disorder

    PubMed Central

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

    2014-01-01

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

  18. Molecular Understanding of Efficient DNA Repair Machinery of Photolyase

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

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

  19. Cycling with BRCA2 from DNA repair to mitosis

    SciTech Connect

    Lee, Hyunsook

    2014-11-15

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

  20. Eukaryotic Mismatch Repair in Relation to DNA Replication.

    PubMed

    Kunkel, Thomas A; Erie, Dorothy A

    2015-01-01

    Three processes act in series to accurately replicate the eukaryotic nuclear genome. The major replicative DNA polymerases strongly prevent mismatch formation, occasional mismatches that do form are proofread during replication, and rare mismatches that escape proofreading are corrected by mismatch repair (MMR). This review focuses on MMR in light of increasing knowledge about nuclear DNA replication enzymology and the rate and specificity with which mismatches are generated during leading- and lagging-strand replication. We consider differences in MMR efficiency in relation to mismatch recognition, signaling to direct MMR to the nascent strand, mismatch removal, and the timing of MMR. These studies are refining our understanding of relationships between generating and repairing replication errors to achieve accurate replication of both DNA strands of the nuclear genome.

  1. Breaking bad: The mutagenic effect of DNA repair

    PubMed Central

    2015-01-01

    Species survival depends on the faithful replication of genetic information, which is continually monitored and maintained by DNA repair pathways thatcorrect replication errors and the thousands of lesions that arise daily from the inherent chemical lability of DNA and the effects of genotoxic agents. Nonetheless,neutrally evolving DNA (not under purifying selection) accumulates base substitutions with time (the neutral mutation rate). Thus, repair processes are not 100% efficient. The neutral mutation rate varies both between and within chromosomes. For example it is 10 – 50 fold higher at CpGsthan at non-CpG positions. Interestingly, the neutral mutation rate at non-CpG sites is positively correlated with CpG content. Althoughthe basis of this correlation was not immediately apparent,some bioinformatic results were consistent with the induction of non-CpGmutations byDNA repairat flanking CpG sites. Recent studies with a model system showed that in vivo repair of preformed lesions (mismatches, abasic sites, single stranded nicks) can in factinduce mutations in flanking DNA. Mismatch repair (MMR) is an essential component for repair-induced mutations, which can occur as distant as 5 kb from the introduced lesions. Most, but not all, mutations involved the C of TpCpN (G of NpGpA) which is the target sequence of the C-preferringsingle-stranded DNA specific APOBEC deaminases. APOBEC-mediated mutations are not limited to our model system: Recent studies by others showed that some tumors harbor mutations with the same signature, as can intermediates in RNA-guided endonuclease-mediated genome editing. APOBEC deaminases participate in normal physiological functions such as generating mutations that inactivate viruses or endogenous retrotransposons, or that enhance immunoglobulin diversity in B cells. The recruitment of normally physiological errorprone processes during DNA repairwould have important implications for disease, aging and evolution. This perspective briefly

  2. The Ageing Brain: Effects on DNA Repair and DNA Methylation in Mice

    PubMed Central

    Langie, Sabine A. S.; Cameron, Kerry M.; Ficz, Gabriella; Oxley, David; Tomaszewski, Bartłomiej; Gorniak, Joanna P.; Maas, Lou M.; Godschalk, Roger W. L.; van Schooten, Frederik J.; Reik, Wolf; von Zglinicki, Thomas; Mathers, John C.

    2017-01-01

    Base excision repair (BER) may become less effective with ageing resulting in accumulation of DNA lesions, genome instability and altered gene expression that contribute to age-related degenerative diseases. The brain is particularly vulnerable to the accumulation of DNA lesions; hence, proper functioning of DNA repair mechanisms is important for neuronal survival. Although the mechanism of age-related decline in DNA repair capacity is unknown, growing evidence suggests that epigenetic events (e.g., DNA methylation) contribute to the ageing process and may be functionally important through the regulation of the expression of DNA repair genes. We hypothesize that epigenetic mechanisms are involved in mediating the age-related decline in BER in the brain. Brains from male mice were isolated at 3–32 months of age. Pyrosequencing analyses revealed significantly increased Ogg1 methylation with ageing, which correlated inversely with Ogg1 expression. The reduced Ogg1 expression correlated with enhanced expression of methyl-CpG binding protein 2 and ten-eleven translocation enzyme 2. A significant inverse correlation between Neil1 methylation at CpG-site2 and expression was also observed. BER activity was significantly reduced and associated with increased 8-oxo-7,8-dihydro-2′-deoxyguanosine levels. These data indicate that Ogg1 and Neil1 expression can be epigenetically regulated, which may mediate the effects of ageing on DNA repair in the brain. PMID:28218666

  3. Cell specificity in DNA binding and repair of chemical carcinogens.

    PubMed Central

    Swenberg, J A; Rickert, D E; Baranyi, B L; Goodman, J I

    1983-01-01

    Many animal models for organ specific neoplasia have been developed and used to study the pathogenesis of cancer. Morphologic studies have usually concentrated on the response of target cells, whereas biochemical investigations have usually employed whole organ homogenates. Since hepatocytes comprise nearly 90% of the liver's mass and 70-80% of its DNA, alterations in DNA replication, covalent binding and DNA repair of nonparenchymal cells are usually obscured when whole organ homogenates are used. By utilizing cell separation methods, we have been able to demonstrate differences between hepatocyte and nonparenchymal cell replication. DNA damage and repair following exposure to a variety of hepatocarcinogen. Differences in removal of simple O6-alkylguanine and DNA replication correlate with cell specific carcinogenesis of simply alkylating agents. For several other procarcinogens, including 2-acetylaminofluorene and dinitroluene, cell specificity appears to reside primarily in the differential metabolic competence of hepatocytes and nonparenchymal cells. This results in greater covalent binding of the carcinogen to hepatocyte DNA, although the DNA adducts are removed at a similar rate in both cell types. Images FIGURE 1. PMID:6832089

  4. DNA damage and repair after high LET radiation

    NASA Astrophysics Data System (ADS)

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

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

  5. The Ku heterodimer: function in DNA repair and beyond.

    PubMed

    Fell, Victoria L; Schild-Poulter, Caroline

    2015-01-01

    Ku is an abundant, highly conserved DNA binding protein found in both prokaryotes and eukaryotes that plays essential roles in the maintenance of genome integrity. In eukaryotes, Ku is a heterodimer comprised of two subunits, Ku70 and Ku80, that is best characterized for its central role as the initial DNA end binding factor in the "classical" non-homologous end joining (C-NHEJ) pathway, the main DNA double-strand break (DSB) repair pathway in mammals. Ku binds double-stranded DNA ends with high affinity in a sequence-independent manner through a central ring formed by the intertwined strands of the Ku70 and Ku80 subunits. At the break, Ku directly and indirectly interacts with several C-NHEJ factors and processing enzymes, serving as the scaffold for the entire DNA repair complex. There is also evidence that Ku is involved in signaling to the DNA damage response (DDR) machinery to modulate the activation of cell cycle checkpoints and the activation of apoptosis. Interestingly, Ku is also associated with telomeres, where, paradoxically to its DNA end-joining functions, it protects the telomere ends from being recognized as DSBs, thereby preventing their recombination and degradation. Ku, together with the silent information regulator (Sir) complex is also required for transcriptional silencing through telomere position effect (TPE). How Ku associates with telomeres, whether it is through direct DNA binding, or through protein-protein interactions with other telomere bound factors remains to be determined. Ku is central to the protection of organisms through its participation in C-NHEJ to repair DSBs generated during V(D)J recombination, a process that is indispensable for the establishment of the immune response. Ku also functions to prevent tumorigenesis and senescence since Ku-deficient mice show increased cancer incidence and early onset of aging. Overall, Ku function is critical to the maintenance of genomic integrity and to proper cellular and organismal

  6. Patching Broken DNA: Nucleosome Dynamics and the Repair of DNA Breaks.

    PubMed

    Gursoy-Yuzugullu, Ozge; House, Nealia; Price, Brendan D

    2016-05-08

    The ability of cells to detect and repair DNA double-strand breaks (DSBs) is dependent on reorganization of the surrounding chromatin structure by chromatin remodeling complexes. These complexes promote access to the site of DNA damage, facilitate processing of the damaged DNA and, importantly, are essential to repackage the repaired DNA. Here, we will review the chromatin remodeling steps that occur immediately after DSB production and that prepare the damaged chromatin template for processing by the DSB repair machinery. DSBs promote rapid accumulation of repressive complexes, including HP1, the NuRD complex, H2A.Z and histone methyltransferases at the DSB. This shift to a repressive chromatin organization may be important to inhibit local transcription and limit mobility of the break and to maintain the DNA ends in close contact. Subsequently, the repressive chromatin is rapidly dismantled through a mechanism involving dynamic exchange of the histone variant H2A.Z. H2A.Z removal at DSBs alters the acidic patch on the nucleosome surface, promoting acetylation of the H4 tail (by the NuA4-Tip60 complex) and shifting the chromatin to a more open structure. Further, H2A.Z removal promotes chromatin ubiquitination and recruitment of additional DSB repair proteins to the break. Modulation of the nucleosome surface and nucleosome function during DSB repair therefore plays a vital role in processing of DNA breaks. Further, the nucleosome surface may function as a central hub during DSB repair, directing specific patterns of histone modification, recruiting DNA repair proteins and modulating chromatin packing during processing of the damaged DNA template.

  7. Canonical DNA Repair Pathways Influence R-Loop-Driven Genome Instability.

    PubMed

    Stirling, Peter C; Hieter, Philip

    2016-07-22

    DNA repair defects create cancer predisposition in humans by fostering a higher rate of mutations. While DNA repair is quite well characterized, recent studies have identified previously unrecognized relationships between DNA repair and R-loop-mediated genome instability. R-loops are three-stranded nucleic acid structures in which RNA binds to genomic DNA to displace a loop of single-stranded DNA. Mutations in homologous recombination, nucleotide excision repair, crosslink repair, and DNA damage checkpoints have all now been linked to formation and function of transcription-coupled R-loops. This perspective will summarize recent literature linking DNA repair to R-loop-mediated genomic instability and discuss how R-loops may contribute to mutagenesis in DNA-repair-deficient cancers.

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

    PubMed Central

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

    2012-01-01

    Many current chemotherapies function by damaging genomic DNA in rapidly dividing cells ultimately leading to cell death. This therapeutic approach differentially targets cancer cells that generally display rapid cell division compared to normal tissue cells. However, although these treatments are initially effective in arresting tumor growth and reducing tumor burden, resistance and disease progression eventually occur. A major mechanism underlying this resistance is increased levels of cellular DNA repair. Most cells have complex mechanisms in place to repair DNA damage that occurs due to environmental exposures or normal metabolic processes. These systems, initially overwhelmed when faced with chemotherapy induced DNA damage, become more efficient under constant selective pressure and as a result chemotherapies become less effective. Thus, inhibiting DNA repair pathways using target specific small molecule inhibitors may overcome cellular resistance to DNA damaging chemotherapies. Non-homologous end joining a major mechanism for the repair of double-strand breaks (DSB) in DNA is regulated in part by the serine/threonine kinase, DNA dependent protein kinase (DNA-PK). The DNA-PK holoenzyme acts as a scaffold protein tethering broken DNA ends and recruiting other repair molecules. It also has enzymatic activity that may be involved in DNA damage signaling. Because of its’ central role in repair of DSBs, DNA-PK has been the focus of a number of small molecule studies. In these studies specific DNA-PK inhibitors have shown efficacy in synergizing chemotherapies in vitro. However, compounds currently known to specifically inhibit DNA-PK are limited by poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives. Future improvement in DNA-PK inhibition will likely be achieved by designing new molecules based on the recently reported crystallographic structure of DNA-PK. Computer based drug

  9. BRCA Mutations, DNA Repair Deficiency, and Ovarian Aging.

    PubMed

    Oktay, Kutluk; Turan, Volkan; Titus, Shiny; Stobezki, Robert; Liu, Lin

    2015-09-01

    Oocyte aging has a significant impact on reproductive outcomes both quantitatively and qualitatively. However, the molecular mechanisms underlying the age-related decline in reproductive success have not been fully addressed. BRCA is known to be involved in homologous DNA recombination and plays an essential role in double-strand DNA break repair. Given the growing body of laboratory and clinical evidence, we performed a systematic review on the current understanding of the role of DNA repair in human reproduction. We find that BRCA mutations negatively affect ovarian reserve based on convincing evidence from in vitro and in vivo results and prospective studies. Because decline in the function of the intact gene occurs at an earlier age, women with BRCA1 mutations exhibit accelerated ovarian aging, unlike those with BRCA2 mutations. However, because of the still robust function of the intact allele in younger women and because of the masking of most severe cases by prophylactic oophorectomy or cancer, it is less likely one would see an effect of BRCA mutations on fertility until later in reproductive age. The impact of BRCA2 mutations on reproductive function may be less visible because of the delayed decline in the function of normal BRCA2 allele. BRCA1 function and ataxia-telangiectasia-mutated (ATM)-mediated DNA repair may also be important in the pathogenesis of age-induced increase in aneuploidy. BRCA1 is required for meiotic spindle assembly, and cohesion function between sister chromatids is also regulated by ATM family member proteins. Taken together, these findings strongly suggest the implication of BRCA and DNA repair malfunction in ovarian aging.

  10. Pathophysiology of Bronchoconstriction: Role of Oxidatively Damaged DNA Repair

    PubMed Central

    Bacsi, Attila; Pan, Lang; Ba, Xueqing; Boldogh, Istvan

    2016-01-01

    Purpose of review To provide an overview on the present understanding of roles of oxidative DNA damage repair in cell signaling underlying bronchoconstriction common to, but not restricted to various forms of asthma and chronic obstructive pulmonary disease Recent findings Bronchoconstriction is a tightening of smooth muscle surrounding the bronchi and bronchioles with consequent wheezing and shortness of breath. Key stimuli include air pollutants, viral infections, allergens, thermal and osmotic changes, and shear stress of mucosal epithelium, triggering a wide range of cellular, vascular and neural events. Although activation of nerve fibers, the role of G-proteins, protein kinases and Ca++, and molecular interaction within contracting filaments of muscle are well defined, the overarching mechanisms by which a wide range of stimuli initiate these events are not fully understood. Many, if not all, stimuli increase levels of reactive oxygen species (ROS), which are signaling and oxidatively modifying macromolecules, including DNA. The primary ROS target in DNA is guanine, and 8-oxoguanine is one of the most abundant base lesions. It is repaired by 8-oxoguanine DNA glycosylase1 (OGG1) during base excision repair processes. The product, free 8-oxoG base, is bound by OGG1 with high affinity, and the complex then functions as an activator of small GTPases, triggering pathways for inducing gene expression and contraction of intracellular filaments in mast and smooth muscle cells. Summary Oxidative DNA damage repair-mediated cell activation signaling result in gene expression that “primes” the mucosal epithelium and submucosal tissues to generate mediators of airway smooth muscle contractions. PMID:26694039

  11. N-Butyrate alters chromatin accessibility to DNA repair enzymes

    SciTech Connect

    Smith, P.J.

    1986-03-01

    Current evidence suggests that the complex nature of mammalian chromatin can result in the concealment of DNA damage from repair enzymes and their co-factors. Recently it has been proposed that the acetylation of histone proteins in chromatin may provide a surveillance system whereby damaged regions of DNA become exposed due to changes in chromatin accessibility. This hypothesis has been tested by: (i) using n-butyrate to induce hyperacetylation in human adenocarcinoma (HT29) cells; (ii) monitoring the enzymatic accessibility of chromatin in permeabilised cells; (iii) measuring u.v. repair-associated nicking of DNA in intact cells and (iv) determining the effects of n-butyrate on cellular sensitivity to DNA damaging agents. The results indicate that the accessibility of chromatin to Micrococcus luteus u.v. endonuclease is enhanced by greater than 2-fold in n-butyrate-treated cells and that there is a corresponding increase in u.v. repair incision rates in intact cells exposed to the drug. Non-toxic levels of n-butyrate induce a block to G1 phase transit and there is a significant growth delay on removal of the drug. Resistance of HT29 cells to u.v.-radiation and adriamycin is enhanced in n-butyrate-treated cells whereas X-ray sensitivity is increased. Although changes in the responses of cells to DNA damaging agents must be considered in relation to the effects of n-butyrate on growth rate and cell-cycle distribution, the results are not inconsistent with the proposal that increased enzymatic-accessibility/repair is biologically favourable for the resistance of cells to u.v.-radiation damage. Overall the results support the suggested operation of a histone acetylation-based chromatin surveillance system in human cells.

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

    PubMed Central

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

    2014-01-01

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

  13. Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

    PubMed

    Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

    2013-10-01

    Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

  14. DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism

    PubMed Central

    Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Morano, Annalisa; Zuchegna, Candida; Romano, Antonella; Muller, Mark T.; Gottesman, Max E.; Porcellini, Antonio; Avvedimento, Enrico V.

    2016-01-01

    We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5′ and 3′ ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells. PMID:27629060

  15. Rational design of human DNA ligase inhibitors that target cellular DNA replication and repair.

    PubMed

    Chen, Xi; Zhong, Shijun; Zhu, Xiao; Dziegielewska, Barbara; Ellenberger, Tom; Wilson, Gerald M; MacKerell, Alexander D; Tomkinson, Alan E

    2008-05-01

    Based on the crystal structure of human DNA ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA ligase I. Notably, a subset of these compounds was also active against the other human DNA ligases. Three compounds that differed in their specificity for the three human DNA ligases were analyzed further. L82 inhibited DNA ligase I, L67 inhibited DNA ligases I and III, and L189 inhibited DNA ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents.

  16. Deficient repair of chemical adducts in alpha DNA of monkey cells

    SciTech Connect

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

    1982-03-01

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

  17. β2-spectrin depletion impairs DNA damage repair

    PubMed Central

    Horikoshi, Nobuo; Pandita, Raj K.; Mujoo, Kalpana; Hambarde, Shashank; Sharma, Dharmendra; Mattoo, Abid R.; Chakraborty, Sharmistha; Charaka, Vijaya; Hunt, Clayton R.; Pandita, Tej K.

    2016-01-01

    β2-Spectrin (β2SP/SPTBN1, gene SPTBN1) is a key TGF-β/SMAD3/4 adaptor and transcriptional cofactor that regulates TGF-β signaling and can contribute to liver cancer development. Here we report that cells deficient in β2-Spectrin (β2SP) are moderately sensitive to ionizing radiation (IR) and extremely sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with IR or ICL agents (formaldehyde, cisplatin, camptothecin, mitomycin), β2SP deficient cells displayed a higher frequency of cells with delayed γ-H2AX removal and a higher frequency of residual chromosome aberrations. Following hydroxyurea (HU)-induced replication stress, β2SP-deficient cells displayed delayed disappearance of γ-H2AX foci along with defective repair factor recruitment (MRE11, CtIP, RAD51, RPA, and FANCD2) as well as defective restart of stalled replication forks. Repair factor recruitment is a prerequisite for initiation of DNA damage repair by the homologous recombination (HR) pathway, which was also defective in β2SP deficient cells. We propose that β2SP is required for maintaining genomic stability following replication fork stalling, whether induced by either ICL damage or replicative stress, by facilitating fork regression as well as DNA damage repair by homologous recombination. PMID:27248179

  18. Generation of DNA single-strand displacement by compromised nucleotide excision repair

    PubMed Central

    Godon, Camille; Mourgues, Sophie; Nonnekens, Julie; Mourcet, Amandine; Coin, Fréderic; Vermeulen, Wim; Mari, Pierre-Olivier; Giglia-Mari, Giuseppina

    2012-01-01

    Nucleotide excision repair (NER) is a precisely coordinated process essential to avoid DNA damage-induced cellular malfunction and mutagenesis. Here, we investigate the mechanistic details and effects of the NER machinery when it is compromised by a pathologically significant mutation in a subunit of the repair/transcription factor TFIIH, namely XPD. In contrast to previous studies, we find that no single- or double-strand DNA breaks are produced at early time points after UV irradiation of cells bearing a specific XPD mutation, despite the presence of a clear histone H2AX phosphorylation (γH2AX) signal in the UV-exposed areas. We show that the observed γH2AX signal can be explained by the presence of longer single-strand gaps possibly generated by strand displacement. Our in vivo measurements also indicate a strongly reduced TFIIH-XPG binding that could promote single-strand displacement at the site of UV lesions. This finding not only highlights the crucial role of XPG's interactions with TFIIH for proper NER, but also sheds new light on how a faulty DNA repair process can induce extreme genomic instability in human patients. PMID:22863773

  19. CDK1 Enhances Mitochondrial Bioenergetics for Radiation-Induced DNA Repair

    PubMed Central

    Qin, Lili; Fan, Ming; Candas, Demet; Jiang, Guochun; Papadopoulos, Stelios; Tian, Lin; Woloschak, Gayle; Grdina, David J.; Li, Jian Jian

    2015-01-01

    SUMMARY Nuclear DNA repair capacity is a critical determinant of cell fate under genotoxic stress conditions. DNA repair is a well-defined energy consuming process; however, it is unclear how DNA repair is fueled and whether mitochondrial energy production contributes to nuclear DNA repair. Here, we report a dynamic enhancement of oxygen consumption and mitochondrial ATP generation in irradiated normal cells, paralleled with increased mitochondrial relocation of cell cycle kinase CDK1 and nuclear DNA repair. The basal and radiation-induced mitochondrial ATP generation is significantly reduced in cells harboring CDK1 phosphorylation deficient mutant complex I subunits. Similarly, mitochondrial ATP generation and nuclear DNA repair are also severely compromised in cells harboring mitochondrial-targeted kinase deficient CDK1. These results demonstrate a mechanism governing the communication between mitochondria and nucleus, by which CDK1 boosts mitochondrial bioenergetics to meet the increased cellular fuel demand for DNA repair and cell survival under genotoxic stress. PMID:26670043

  20. Replication factor C recruits DNA polymerase delta to sites of nucleotide excision repair but is not required for PCNA recruitment.

    PubMed

    Overmeer, René M; Gourdin, Audrey M; Giglia-Mari, Ambra; Kool, Hanneke; Houtsmuller, Adriaan B; Siegal, Gregg; Fousteri, Maria I; Mullenders, Leon H F; Vermeulen, Wim

    2010-10-01

    Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3' termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5' phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA.

  1. The Cartography of UV-induced DNA Damage Formation and DNA Repair.

    PubMed

    Hu, Jinchuan; Adar, Sheera

    2017-01-01

    DNA damage presents a barrier to DNA-templated biochemical processes, including gene expression and faithful DNA replication. Compromised DNA repair leads to mutations, enhancing the risk for genetic diseases and cancer development. Conventional experimental approaches to study DNA damage required a researcher to choose between measuring bulk damage over the entire genome, with little or no resolution regarding a specific location, and obtaining data specific to a locus of interest, without a global perspective. Recent advances in high-throughput genomic tools overcame these limitations and provide high-resolution measurements simultaneously across the genome. In this review, we discuss the available methods for measuring DNA damage and their repair, focusing on genomewide assays for pyrimidine photodimers, the major types of damage induced by ultraviolet irradiation. These new genomic assays will be a powerful tool in identifying key components of genome stability and carcinogenesis.

  2. How are base excision DNA repair pathways deployed in vivo?

    PubMed Central

    Thapar, Upasna; Demple, Bruce

    2017-01-01

    Since the discovery of the base excision repair (BER) system for DNA more than 40 years ago, new branches of the pathway have been revealed at the biochemical level by in vitro studies. Largely for technical reasons, however, the confirmation of these subpathways in vivo has been elusive. We review methods that have been used to explore BER in mammalian cells, indicate where there are important knowledge gaps to fill, and suggest a way to address them. PMID:28357058

  3. Cisplatin pharmacogenetics, DNA repair polymorphisms, and esophageal cancer outcomes

    PubMed Central

    Bradbury, Penelope A.; Kulke, Matthew H.; Heist, Rebecca S.; Zhou, Wei; Ma, Clement; Xu, Wei; Marshall, Ariela L.; Zhai, Rihong; Hooshmand, Susanne M.; Asomaning, Kofi; Su, Li; Shepherd, Frances A.; Lynch, Thomas J.; Wain, John C.; Christiani, David C.; Liu, Geoffrey

    2011-01-01

    Objectives Genetic variations or polymorphisms within genes of the nucleotide excision repair (NER) pathway alter DNA repair capacity. Reduced DNA repair (NER) capacity may result in tumors that are more susceptible to cisplatin chemotherapy, which functions by causing DNA damage. We investigated the potential predictive significance of functional NER single nucleotide polymorphisms in esophageal cancer patients treated with (n = 262) or without (n = 108) cisplatin. Methods Four NER polymorphisms XPD Asp312Asn; XPD Lys751Gln, ERCC1 8092C/A, and ERCC1 codon 118C/T were each assessed in polymorphism–cisplatin treatment interactions for overall survival (OS), with progression-free survival (PFS) as a secondary endpoint. Results No associations with ERCC1 118 were found. Polymorphism–cisplatin interactions were highly significant in both OS (P = 0.002, P = 0.0001, and P < 0.0001) and PFS (P = 0.006, P = 0.008, and P = 0.0007) for XPD 312, XPD 751, and ERCC1 8092, respectively. In cisplatin-treated patients, variant alleles of XPD 312, XPD 751, and ERCC1 8092 were each associated with significantly improved OS (and PFS): adjusted hazard ratios of homozygous variants versus wild-type ranged from 0.22 [95% confidence interval (CI): 0.1–0.5] to 0.31 (95% CI: 0.1–0.7). In contrast, in patients who did not receive cisplatin, variant alleles of XPD 751 and ERCC1 8092 had significantly worse survival, with adjusted hazard ratios of homozygous variants ranging from 2.47 (95% CI: 1.1–5.5) to 3.73 (95% CI: 1.6–8.7). Haplotype analyses affirmed these results. Conclusion DNA repair polymorphisms are associated with OS and PFS, and if validated may predict for benefit from cisplatin therapy in patients with esophageal cancer. PMID:19620936

  4. Repair Machinery for Radiation-Induced DNA Damage

    DTIC Science & Technology

    2000-07-01

    significant defect in the repair of certain DNA damages, but of which damages needs to be determined. We have selected Chinese Hamster Ovary ( CHO ) as...chromosome (BAC) genomic fragment, which we isolated from a CHO BAC library, revealed that APE1 exists as a single copy gene in AA8 (see Appendix, Figure... cells , we first determined the APE1 gene copy number in the CHO AA8 cell line. Fluorescence in situ hybridization with an APE1 bacterial artificial

  5. How are base excision DNA repair pathways deployed in vivo?

    PubMed

    Thapar, Upasna; Demple, Bruce

    2017-01-01

    Since the discovery of the base excision repair (BER) system for DNA more than 40 years ago, new branches of the pathway have been revealed at the biochemical level by in vitro studies. Largely for technical reasons, however, the confirmation of these subpathways in vivo has been elusive. We review methods that have been used to explore BER in mammalian cells, indicate where there are important knowledge gaps to fill, and suggest a way to address them.

  6. Oxidative Stress, DNA Repair, and Prostate Cancer Risk

    DTIC Science & Technology

    2011-08-01

    have concluded that DRC is not a risk factor for prostate cancer microRNA prostate cancer Hua.Zhao@RoswellPark.org Table of Contents...known and suspected risk factors for prostate cancer are associated with elevated levels of reactive oxygen species (ROS) (advancing age, inflammation...association between DNA repair capacity and prostate cancer risk might be due to the fact of using surrogate tissues , not the target tissues . In this study

  7. Long noncoding RNA LINP1 regulates double strand DNA break repair in triple negative breast cancer

    PubMed Central

    Zhang, Youyou; He, Qun; Hu, Zhongyi; Feng, Yi; Fan, Lingling; Tang, Zhaoqing; Yuan, Jiao; Shan, Weiwei; Li, Chunsheng; Hu, Xiaowen; Tanyi, Janos L; Fan, Yi; Huang, Qihong; Montone, Kathleen; Dang, Chi V; Zhang, Lin

    2016-01-01

    Long noncoding RNAs (lncRNAs), which are transcripts that are larger than 200 nucleotides but do not appear to have protein-coding potential, play critical roles during tumorigenesis by functioning as scaffolds to regulate protein-protein, protein-DNA or protein-RNA interactions. Using a clinically guided genetic screening approach, we identified (lncRNA in Non-homologous end joining [NHEJ] pathway 1) as a lncRNA that is overexpressed in human triple-negative breast cancer. We found that LINP1 enhances double-strand DNA break repair by serving as a scaffold that links Ku80 and DNA-PKcs, thereby coordinating the NHEJ pathway. Importantly, blocking LINP1, which is regulated by the p53 and epidermal growth factor receptor (EGFR) signaling, increases sensitivity of tumor cell response to radiotherapy in breast cancer. PMID:27111890

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

    SciTech Connect

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

    1987-10-01

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

  9. DNA Repair Decline During Mouse Spermiogenesis Results in the Accumulation of Heritable DNA Damage

    SciTech Connect

    Marchetti, Francesco; Marchetti, Francesco; Wyrobek, Andrew J.

    2007-12-01

    The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7-1 dbf). Analysis of chromosomal aberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

  10. DNA repair decline during mouse spermiogenesis results in the accumulation of heritable DNA damage

    SciTech Connect

    Marchetti, Francesco; Marchetti, Francesco; Wryobek, Andrew J

    2008-02-21

    The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7- 1 dbf). Analysis of chromosomalaberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

  11. Formamidopyrimidines in DNA: mechanisms of formation, repair, and biological effects.

    PubMed

    Dizdaroglu, Miral; Kirkali, Güldal; Jaruga, Pawel

    2008-12-15

    Oxidatively induced damage to DNA results in a plethora of lesions comprising modified bases and sugars, DNA-protein cross-links, tandem lesions, strand breaks, and clustered lesions. Formamidopyrimidines, 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), are among the major lesions generated in DNA by hydroxyl radical attack, UV radiation, or photosensitization under numerous in vitro and in vivo conditions. They are formed by one-electron reduction of C8-OH-adduct radicals of purines and thus have a common precursor with 8-hydroxypurines generated upon one-electron oxidation. Methodologies using mass spectrometry exist to accurately measure FapyAde and FapyGua in vitro and in vivo. Formamidopyrimidines are repaired by base excision repair. Numerous prokaryotic and eukaryotic DNA glycosylases are highly specific for removal of these lesions from DNA in the first step of this repair pathway, indicating their biological importance. FapyAde and FapyGua are bypassed by DNA polymerases with the insertion of the wrong intact base opposite them, leading to mutagenesis. In mammalian cells, the mutagenicity of FapyGua exceeds that of 8-hydroxyguanine, which is thought to be the most mutagenic of the oxidatively induced lesions in DNA. The background and formation levels of the former in vitro and in vivo equal or exceed those of the latter under various conditions. FapyAde and FapyGua exist in living cells at significant background levels and are abundantly generated upon exposure to oxidative stress. Mice lacking the genes that encode specific DNA glycosylases accumulate these lesions in different organs and, in some cases, exhibit a series of pathological conditions including metabolic syndrome and cancer. Animals exposed to environmental toxins accumulate formamidopyrimidines in their organs. Here, we extensively review the mechanisms of formation, measurement, repair, and biological effects of formamidopyrimidines

  12. DNA repair inhibition by UVA photoactivated fluoroquinolones and vemurafenib

    PubMed Central

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

    2014-01-01

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

  13. Clinical Radiation Sensitivity With DNA Repair Disorders: An Overview

    SciTech Connect

    Pollard, Julianne M.; Gatti, Richard A.

    2009-08-01

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

  14. Protein–DNA charge transport: Redox activation of a DNA repair protein by guanine radical

    PubMed Central

    Yavin, Eylon; Boal, Amie K.; Stemp, Eric D. A.; Boon, Elizabeth M.; Livingston, Alison L.; O'Shea, Valerie L.; David, Sheila S.; Barton, Jacqueline K.

    2005-01-01

    DNA charge transport (CT) chemistry provides a route to carry out oxidative DNA damage from a distance in a reaction that is sensitive to DNA mismatches and lesions. Here, DNA-mediated CT also leads to oxidation of a DNA-bound base excision repair enzyme, MutY. DNA-bound Ru(III), generated through a flash/quench technique, is found to promote oxidation of the [4Fe-4S]2+ cluster of MutY to [4Fe-4S]3+ and its decomposition product [3Fe-4S]1+. Flash/quench experiments monitored by EPR spectroscopy reveal spectra with g = 2.08, 2.06, and 2.02, characteristic of the oxidized clusters. Transient absorption spectra of poly(dGC) and [Ru(phen)2dppz]3+ (dppz = dipyridophenazine), generated in situ, show an absorption characteristic of the guanine radical that is depleted in the presence of MutY with formation instead of a long-lived species with an absorption at 405 nm; we attribute this absorption also to formation of the oxidized [4Fe-4S]3+ and [3Fe-4S]1+ clusters. In ruthenium-tethered DNA assemblies, oxidative damage to the 5′-G of a 5′-GG-3′ doublet is generated from a distance but this irreversible damage is inhibited by MutY and instead EPR experiments reveal cluster oxidation. With ruthenium-tethered assemblies containing duplex versus single-stranded regions, MutY oxidation is found to be mediated by the DNA duplex, with guanine radical as an intermediate oxidant; guanine radical formation facilitates MutY oxidation. A model is proposed for the redox activation of DNA repair proteins through DNA CT, with guanine radicals, the first product under oxidative stress, in oxidizing the DNA-bound repair proteins, providing the signal to stimulate DNA repair. PMID:15738421

  15. A second DNA methyltransferase repair enzyme in Escherichia coli.

    PubMed Central

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

    1988-01-01

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

  16. Human premature aging, DNA repair and RecQ helicases.

    PubMed

    Brosh, Robert M; Bohr, Vilhelm A

    2007-01-01

    Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important roles in the DNA damage response. The RecQ family of DNA helicases is of particular interest since several human RecQ helicases are defective in diseases associated with premature aging and cancer. In this review, we will provide an update on our understanding of the specific roles of human RecQ helicases in the maintenance of genomic stability through their catalytic activities and protein interactions in various pathways of cellular nucleic acid metabolism with an emphasis on DNA replication and repair. We will also discuss the clinical features of the premature aging disorders associated with RecQ helicase deficiencies and how they relate to the molecular defects.

  17. Phosphorylation-Regulated Transitions in an Oligomeric State Control the Activity of the Sae2 DNA Repair Enzyme

    PubMed Central

    Fu, Qiong; Chow, Julia; Bernstein, Kara A.; Makharashvili, Nodar; Arora, Sucheta; Lee, Chia-Fang; Person, Maria D.; Rothstein, Rodney

    2014-01-01

    In the DNA damage response, many repair and signaling molecules mobilize rapidly at the sites of DNA double-strand breaks. This network of immediate responses is regulated at the level of posttranslational modifications that control the activation of DNA processing enzymes, protein kinases, and scaffold proteins to coordinate DNA repair and checkpoint signaling. Here we investigated the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DNA double-strand break repair. Sae2 is a target of multiple phosphorylation events, which we identified and characterized in vivo in the budding yeast Saccharomyces cerevisiae. Both cell cycle-dependent and DNA damage-dependent phosphorylation sites in Sae2 are important for the survival of DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We found that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by this series of phosphorylations. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. Overall, this analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by posttranslational modifications that regulate its solubility and oligomeric state. PMID:24344201

  18. Quantitative characterization of protein–protein complexes involved in base excision DNA repair

    PubMed Central

    Moor, Nina A.; Vasil'eva, Inna A.; Anarbaev, Rashid O.; Antson, Alfred A.; Lavrik, Olga I.

    2015-01-01

    Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein–protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process. PMID:26013813

  19. DNA repair within nucleosome cores of UV-irradiated human cells

    SciTech Connect

    Jensen, K.A.; Smerdon, M.J. )

    1990-05-22

    We have compared the distributions of repair synthesis and pyrimidine dimers (PD) in nucleosome core DNA during the early (fast) repair phase and the late (slow) repair phase of UV-irradiated human fibroblasts. As shown previously, repair synthesis is nonuniform in nucleosome core particles during the fast repair phase, and the distribution curve can be approximated by a model where repair synthesis occurs preferentially in the 5' and 3' end regions. In this report, we show that, during the slow repair phase, (3H)dThd-labeled repair patches are much more uniformly distributed in core DNA, although they appear to be preferentially located in sequences degraded slowly by exonuclease III. This change in distribution cannot be explained by an increase in patch size during slow repair, since the size of these patches actually decreases to about half the size measured during the fast repair phase. Furthermore, PD mapping within core DNA at the single-nucleotide level demonstrated that, at least within the 30-130-base region from the 5' end, there is little (or no) selective removal of PD during the fast repair phase. However, the nonuniform distribution of repair synthesis obtained during fast repair throughout most of the core DNA region (approximately 40-146 bases) is accounted for by the nonuniform distribution of PD in core DNA. The near-uniform distribution of repair synthesis observed during slow repair may result from more extensive nucleosome rearrangement and/or nucleosome modification during this phase.

  20. HDAC inhibitors: roles of DNA damage and repair.

    PubMed

    Robert, Carine; Rassool, Feyruz V

    2012-01-01

    Histone deacetylase inhibitors (HDACis) increase gene expression through induction of histone acetylation. However, it remains unclear whether specific gene expression changes determine the apoptotic response following HDACis administration. Herein, we discuss evidence that HDACis trigger in cancer and leukemia cells not only widespread histone acetylation but also actual increases in reactive oxygen species (ROS) and DNA damage that are further increased following treatment with DNA-damaging chemotherapies. While the origins of ROS production are not completely understood, mechanisms, including inflammation and altered antioxidant signaling, have been reported. While the generation of ROS is an explanation, at least in part, for the source of DNA damage observed with HDACi treatment, DNA damage can also be independently induced by changes in the DNA repair activity and chromatin remodeling factors. Recent development of sirtuin inhibitors (SIRTis) has shown that, similar to HDACis, these drugs induce increases in ROS and DNA damage used singly, or in combination with HDACis and other drugs. Thus, induction of apoptosis by HDACis/SIRTis may result through oxidative stress and DNA damage mechanisms in addition to direct activation of apoptosis-inducing genes. Nevertheless, while DNA damage and stress responses could be of interest as markers for clinical responses, they have yet to be validated as markers for responses to HDACi treatment in clinical trials, alone, and in combination.

  1. Bi-allelic alterations in DNA repair genes underpin homologous recombination DNA repair defects in breast cancer.

    PubMed

    Mutter, Robert W; Riaz, Nadeem; Ng, Charlotte K Y; Delsite, Rob; Piscuoglio, Salvatore; Edelweiss, Marcia; Martelotto, Luciano G; Sakr, Rita A; King, Tari A; Giri, Dilip D; Drobnjak, Maria; Brogi, Edi; Bindra, Ranjit; Bernheim, Giana; Lim, Raymond S; Blecua, Pedro; Desrichard, Alexis; Higginson, Dan; Towers, Russell; Jiang, Ruomu; Lee, William; Weigelt, Britta; Reis-Filho, Jorge S; Powell, Simon N

    2017-03-15

    Homologous recombination (HR) DNA repair deficient (HRD) breast cancers have been shown to be sensitive to DNA repair targeted therapies. Burgeoning evidence suggests that sporadic breast cancers, lacking germline BRCA1/BRCA2 mutations, may also be HRD. We developed a functional ex vivo RAD51-based test to identify HRD primary breast cancers. An integrated approach examining methylation, gene expression and whole-exome sequencing was employed to ascertain the etiology of HRD. Functional HRD breast cancers displayed genomic features of lack of competent HR, including large-scale state transitions and specific mutational signatures. Somatic and/or germline genetic alterations resulting in bi-allelic loss-of-function of HR genes underpinned functional HRD in 89% of cases, and were observed in only one of the 15 HR-proficient samples tested. These findings indicate the importance of a comprehensive genetic assessment of bi-allelic alterations in the HR pathway to deliver a precision medicine-based approach to select patients for therapies targeting tumor-specific DNA repair defects.

  2. Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions

    PubMed Central

    Brown, Maxwell W.; Kim, Yoori; Williams, Gregory M.; Huck, John D.; Surtees, Jennifer A.; Finkelstein, Ilya J.

    2016-01-01

    DNA-binding proteins search for specific targets via facilitated diffusion along a crowded genome. However, little is known about how crowded DNA modulates facilitated diffusion and target recognition. Here we use DNA curtains and single-molecule fluorescence imaging to investigate how Msh2–Msh3, a eukaryotic mismatch repair complex, navigates on crowded DNA. Msh2–Msh3 hops over nucleosomes and other protein roadblocks, but maintains sufficient contact with DNA to recognize a single lesion. In contrast, Msh2–Msh6 slides without hopping and is largely blocked by protein roadblocks. Remarkably, the Msh3-specific mispair-binding domain (MBD) licences a chimeric Msh2–Msh6(3MBD) to bypass nucleosomes. Our studies contrast how Msh2–Msh3 and Msh2–Msh6 navigate on a crowded genome and suggest how Msh2–Msh3 locates DNA lesions outside of replication-coupled repair. These results also provide insights into how DNA repair factors search for DNA lesions in the context of chromatin. PMID:26837705

  3. Repair of Heteroduplex DNA in Xenopus Laevis Oocytes

    PubMed Central

    Lehman, C. W.; Jeong-Yu, S.; Trautman, J. K.; Carroll, D.

    1994-01-01

    We have hypothesized that the inheritance of heteroallelic markers during recombination of homologous DNAs in Xenopus oocytes is determined by resolution of a heteroduplex intermediate containing multiple single-base mismatches. To test this idea, we prepared synthetic heteroduplexes carrying 8 separate mispairs in vitro and injected them into oocyte nuclei. DNA was recovered and analyzed directly, by Southern blot-hybridization, and indirectly, by cloning individual repair products in bacteria. Mismatch correction was quite efficient in the oocytes; markers on the same strand were commonly co-corrected, indicating a long-patch mechanism; and the distribution of markers was very similar to that obtained by recombination. This supports our interpretation of the recombination outcome in terms of a resection-annealing mechanism. The injected heteroduplexes carried strand breaks (nicks) as a result of their method of preparation. We tested the idea that mismatch correction might be nick-directed by ligating the strands of the heteroduplex substrate to form covalently closed circles. Repair in oocytes was still efficient, and long patches predominated; but the pattern of recovered markers was quite different than with the nicked substrate. This suggests that nicks, when present, do indeed direct repair, but that, in their absence, recognition of specific mismatches governs repair of the ligated heteroduplexes. PMID:7828827

  4. Herpes Simplex Virus Latency: The DNA Repair-Centered Pathway

    PubMed Central

    2017-01-01

    Like all herpesviruses, herpes simplex virus 1 (HSV1) is able to produce lytic or latent infections depending on the host cell type. Lytic infections occur in a broad range of cells while latency is highly specific for neurons. Although latency suggests itself as an attractive target for novel anti-HSV1 therapies, progress in their development has been slowed due in part to a lack of agreement about the basic biochemical mechanisms involved. Among the possibilities being considered is a pathway in which DNA repair mechanisms play a central role. Repair is suggested to be involved in both HSV1 entry into latency and reactivation from it. Here I describe the basic features of the DNA repair-centered pathway and discuss some of the experimental evidence supporting it. The pathway is particularly attractive because it is able to account for important features of the latent response, including the specificity for neurons, the specificity for neurons of the peripheral compared to the central nervous system, the high rate of genetic recombination in HSV1-infected cells, and the genetic identity of infecting and reactivated virus. PMID:28255301

  5. Targeting the DNA repair pathway in Ewing sarcoma.

    PubMed

    Stewart, Elizabeth; Goshorn, Ross; Bradley, Cori; Griffiths, Lyra M; Benavente, Claudia; Twarog, Nathaniel R; Miller, Gregory M; Caufield, William; Freeman, Burgess B; Bahrami, Armita; Pappo, Alberto; Wu, Jianrong; Loh, Amos; Karlström, Åsa; Calabrese, Chris; Gordon, Brittney; Tsurkan, Lyudmila; Hatfield, M Jason; Potter, Philip M; Snyder, Scott E; Thiagarajan, Suresh; Shirinifard, Abbas; Sablauer, Andras; Shelat, Anang A; Dyer, Michael A

    2014-11-06

    Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.

  6. Opportunities for translation: targeting DNA repair pathways in pancreatic cancer.

    PubMed

    Maginn, Elaina N; de Sousa, Camila H; Wasan, Harpreet S; Stronach, Euan A

    2014-08-01

    Pancreatic ductal adenocarcinoma (PDAC) remains one of the poorest prognosis neoplasms. It is typified by high levels of genomic aberrations and copy-number variation, intra-tumoural heterogeneity and resistance to conventional chemotherapy. Improved therapeutic options, ideally targeted against cancer-specific biological mechanisms, are urgently needed. Although induction of DNA damage and/or modulation of DNA damage response pathways are associated with the activity of a number of conventional PDAC chemotherapies, the effectiveness of this approach in the treatment of PDAC has not been comprehensively reviewed. Here, we review chemotherapeutic agents that have shown anti-cancer activity in PDAC and whose mechanisms of action involve modulation of DNA repair pathways. In addition, we highlight novel potential targets within these pathways based on the emerging understanding of PDAC biology and their exploitation as targets in other cancers.

  7. Disruption of Maternal DNA Repair Increases Sperm-DerivedChromosomal Aberrations

    SciTech Connect

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

    2007-02-07

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

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

    PubMed Central

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

    2015-01-01

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

  9. DNA DSB repair pathway choice: an orchestrated handover mechanism.

    PubMed

    Kakarougkas, A; Jeggo, P A

    2014-03-01

    DNA double strand breaks (DSBs) are potential lethal lesions but can also lead to chromosome rearrangements, a step promoting carcinogenesis. DNA non-homologous end-joining (NHEJ) is the major DSB rejoining process and occurs in all cell cycle stages. Homologous recombination (HR) can additionally function to repair irradiation-induced two-ended DSBs in G2 phase. In mammalian cells, HR predominantly uses a sister chromatid as a template for DSB repair; thus HR functions only in late S/G2 phase. Here, we review current insight into the interplay between HR and NHEJ in G2 phase. We argue that NHEJ represents the first choice pathway, repairing approximately 80% of X-ray-induced DSBs with rapid kinetics. However, a subset of DSBs undergoes end resection and repair by HR. 53BP1 restricts resection, thereby promoting NHEJ. During the switch from NHEJ to HR, 53BP1 is repositioned to the periphery of enlarged irradiation-induced foci (IRIF) via a BRCA1-dependent process. K63-linked ubiquitin chains, which also form at IRIF, are also repositioned as well as receptor-associated protein 80 (RAP80), a ubiquitin binding protein. RAP80 repositioning requires POH1, a proteasome component. Thus, the interfacing barriers to HR, 53BP1 and RAP80 are relieved by POH1 and BRCA1, respectively. Removal of RAP80 from the IRIF core is required for loss of the ubiquitin chains and 53BP1, and for efficient replication protein A foci formation. We propose that NHEJ is used preferentially to HR because it is a compact process that does not necessitate extensive chromatin changes in the DSB vicinity.

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

    PubMed

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

    2014-01-01

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

  11. RNase H enables efficient repair of R-loop induced DNA damage

    PubMed Central

    Amon, Jeremy D; Koshland, Douglas

    2016-01-01

    R-loops, three-stranded structures that form when transcripts hybridize to chromosomal DNA, are potent agents of genome instability. This instability has been explained by the ability of R-loops to induce DNA damage. Here, we show that persistent R-loops also compromise DNA repair. Depleting endogenous RNase H activity impairs R-loop removal in Saccharomyces cerevisiae, causing DNA damage that occurs preferentially in the repetitive ribosomal DNA locus (rDNA). We analyzed the repair kinetics of this damage and identified mutants that modulate repair. We present a model that the persistence of R-loops at sites of DNA damage induces repair by break-induced replication (BIR). This R-loop induced BIR is particularly susceptible to the formation of lethal repair intermediates at the rDNA because of a barrier imposed by RNA polymerase I. DOI: http://dx.doi.org/10.7554/eLife.20533.001 PMID:27938663

  12. Assay to detect chemically induced DNA repair in rat spermatocytes

    SciTech Connect

    Working, P.K.; Butterworth, B.E.

    1984-01-01

    An in vivo/in vitro DNA repair assay has been developed to quantitate chemically induced unscheduled DNA synthesis (UDS) in rat spermatocytes utilizing autoradiography. Male Fischer-344 rats were treated by i.p. injection or gavage with a variety of genotoxic agents dissolved in dimethyl sulfoxide, corn oil, or water. At selected times after treatment, spermatocytes were isolated by trypsin digestion of testes and cultured for 24 hr in the presence of /sup 3/H-thymidine. The direct-acting genotoxicants methyl methanesulfonate (MMS) and ethyl methanesulfonate and the chemotherapeutic agent cyclophosphamide (CPA) produced positive UDS responses in spermatocyes isolated l hr after i.p. injection. Other known genotoxicants--including dimethylnitrosamine, aflatoxin B/sub 1/, 2-acetylaminofluorene, 2, 6-dinitrotoluene, and l,6-dinitropyrene--failed to induce UDS, even with routes of administration and at times of exposure known to produce a positive response in hepatocytes. These results demonstrate that the in vivo/in vitro spermatocyte DNA repair assay may be useful as a predictive screen for germ cell mutagens.

  13. Differential Expression of DNA Double-Strand Break Repair Proteins in Breast Cells

    DTIC Science & Technology

    2002-07-01

    DNA-PK in human breast tissues by immuno-histochemistry and extended these studies to two other components of the NHEJ repair pathway, XRCC4 and DNA ... ligase IV, as well as other DNA repair components including NBS 1 and MRE11. In contrast to the original report, 90% of the epithelial cells in normal

  14. Differential Expression of DNA Double-Strand Break Repair Proteins in Breast Cells

    DTIC Science & Technology

    2003-07-01

    DNA-PK in human breast tissues by immuno-histochemistry and extended these studies to two other components of the NHEJ repair pathway, XRCC4 and DNA ... ligase IV, as well as other DNA repair components including NBSl and MRE11. In contrast to the original report, 90% of the epithelial cells in normal

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  16. Integrating Multi-omics Data to Dissect Mechanisms of DNA repair Dysregulation in Breast Cancer

    PubMed Central

    Liu, Chao; Rohart, Florian; Simpson, Peter T.; Khanna, Kum Kum; Ragan, Mark A.; Lê Cao, Kim-Anh

    2016-01-01

    DNA repair genes and pathways that are transcriptionally dysregulated in cancer provide the first line of evidence for the altered DNA repair status in tumours, and hence have been explored intensively as a source for biomarker discovery. The molecular mechanisms underlying DNA repair dysregulation, however, have not been systematically investigated in any cancer type. In this study, we performed a statistical analysis to dissect the roles of DNA copy number alteration (CNA), DNA methylation (DM) at gene promoter regions and the expression changes of transcription factors (TFs) in the differential expression of individual DNA repair genes in normal versus tumour breast samples. These gene-level results were summarised at pathway level to assess whether different DNA repair pathways are affected in distinct manners. Our results suggest that CNA and expression changes of TFs are major causes of DNA repair dysregulation in breast cancer, and that a subset of the identified TFs may exert global impacts on the dysregulation of multiple repair pathways. Our work hence provides novel insights into DNA repair dysregulation in breast cancer. These insights improve our understanding of the molecular basis of the DNA repair biomarkers identified thus far, and have potential to inform future biomarker discovery. PMID:27666291

  17. Current advances in DNA repair: regulation of enzymes and pathways involved in maintaining genomic stability.

    PubMed

    Neher, Tracy M; Turchi, John J

    2011-06-15

    Novel discoveries in the DNA repair field have lead to continuous and rapid advancement of our understanding of not only DNA repair but also DNA replication and recombination. Research in the field transcends numerous areas of biology, biochemistry, physiology, and medicine, making significant connections across these broad areas of study. From early studies conducted in bacterial systems to current analyses in eukaryotic systems and human disease, the innovative research into the mechanisms of repair machines and the consequences of ineffective DNA repair has impacted a wide scientific community. This Forum contains a select mix of primary research articles in addition to a number of timely reviews covering a subset of DNA repair pathways where recent advances and novel discoveries are improving our understanding of DNA repair, its regulation, and implications to human disease.

  18. Nicotinamide enhances repair of ultraviolet radiation-induced DNA damage in primary melanocytes.

    PubMed

    Thompson, Benjamin C; Surjana, Devita; Halliday, Gary M; Damian, Diona L

    2014-07-01

    Cutaneous melanoma is a significant cause of morbidity and mortality. Nicotinamide is a safe, widely available vitamin that reduces the immune suppressive effects of UV, enhances DNA repair in keratinocytes and has shown promise in the chemoprevention of non-melanoma skin cancer. Here, we report the effect of nicotinamide on DNA damage and repair in primary human melanocytes. Nicotinamide significantly enhanced the repair of oxidative DNA damage (8-oxo-7,8-dihydro-2'-deoxyguanosine) and cyclobutane pyrimidine dimers induced by UV exposure. It also enhanced the repair of 8-oxo-7,8-dihydro-2'-deoxyguanosine induced by the culture conditions in unirradiated melanocytes. A significant increase in the percentage of melanocytes undergoing unscheduled but not scheduled DNA synthesis was observed, confirming that nicotinamide enhances DNA repair in human melanocytes. In summary, nicotinamide, by enhancing DNA repair in melanocytes, is a potential agent for the chemoprevention of cutaneous melanoma.

  19. Using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro.

    PubMed

    Córdoba-Cañero, Dolores; Roldán-Arjona, Teresa; Ariza, Rafael R

    2012-01-01

    Base excision repair (BER) is a major pathway for the removal of endogenous and exogenous DNA damage. This repair mechanism is initiated by DNA glycosylases that excise the altered base, and continues through alternative routes that culminate in DNA resynthesis and ligation. In contrast to the information available for microbes and animals, our knowledge about this important DNA repair pathway in plants is very limited, partially due to a lack of biochemical approaches. Here we describe an in vitro assay to monitor BER in cell-free extracts from the model plant Arabidopsis thaliana. The assay uses labeled DNA substrates containing a single damaged base within a restriction site, and allows detection of fully repaired molecules as well as DNA repair intermediates. The method is easily applied to measure the repair activity of purified proteins and can be successfully used in combination with the extensive array of biological resources available for Arabidopsis.

  20. Early age decline in DNA repair capacity in the liver: in depth profile of differential gene expression

    PubMed Central

    Guedj, Avital; Geiger-Maor, Anat; Galun, Eithan; Amsalem, Hagai; Rachmilewitz, Jacob

    2016-01-01

    Aging is associated with progressive decline in cell function and with increased damage to macromolecular components. DNA damage, in the form of double-strand breaks (DSBs), increases with age and in turn, contributes to the aging process and age-related diseases. DNA strand breaks triggers a set of highly orchestrated signaling events known as the DNA damage response (DDR), which coordinates DNA repair. However, whether the accumulation of DNA damage with age is a result of decreased repair capacity, remains to be determined. In our study we showed that with age there is a decline in the resolution of foci containing γH2AX and pKAP-1 in diethylnitrosamine (DEN)-treated mouse livers, already evident at a remarkably early age of 6-months. Considerable age-dependent differences in global gene expression profiles in mice livers after exposure to DEN, further affirmed these age related differences in the response to DNA damage. Functional analysis identified p53 as the most overrepresented pathway that is specifically enhanced and prolonged in 6-month-old mice. Collectively, our results demonstrated an early decline in DNA damage repair that precedes ‘old age’, suggesting this may be a driving force contributing to the aging process rather than a phenotypic consequence of old age. PMID:27922819

  1. Solar UVB-induced DNA damage and photoenzymatic DNA repair in antarctic zooplankton

    SciTech Connect

    Malloy, K.D.; Holman, M.A.; Mitchell, D.

    1997-02-18

    The detrimental effects of elevated intensities of mid-UV radiation (UVB), a result of stratospheric ozone depletion during the austral spring, on the primary producers of the Antarctic marine ecosystem have been well documented. Here we report that natural populations of Antarctic zooplankton also sustain significant DNA damage [measured as cyclobutane pyrimidine dimers (CPDs)] during periods of increased UVB flux. This is the first direct evidence that increased solar UVB may result in damage to marine organisms other than primary producers in Antarctica. The extent of DNA damage in pelagic icefish eggs correlated with daily incident UVB irradiance, reflecting the difference between acquisition and repair of CPDs. Patterns of DNA damage in fish larvae did not correlated with daily UVB flux, possibly due to different depth distributions and/or different capacities for DNA repair. Clearance of CPDs by Antarctic fish and krill was mediated primarily by the photoenzymatic repair system. Although repair rates were large for all species evaluated, they were apparently inadequate to prevent the transient accumulation of substantial CPD burdens. The capacity for DNA repair in Antarctic organisms was highest in those species whose early life history stages occupy the water column during periods of ozone depletion (austral spring) and lowest in fish species whose eggs and larvae are abundant during winter. Although the potential reduction in fitness of Antarctic zooplankton resulting from DNA damage is unknown, we suggest that increased solar UV may reduce recruitment and adversely affect trophic transfer of productivity by affecting heterotrophic species as well as primary producers. 54 refs., 4 figs., 2 tabs.

  2. Differential impact of ionic and coordinate covalent chromium (Cr)-DNA binding on DNA replication.

    PubMed

    Fornsaglio, Jamie L; O'Brien, Travis J; Patierno, Steven R

    2005-11-01

    The reactive species produced by the reduction of Cr(VI), particularly Cr(III), can form both ionic and coordinate covalent complexes with DNA. These Cr(III)-DNA interactions consist of Cr-DNA monoadducts, Cr-DNA ternary adducts, and Cr-DNA interstrand cross-links (Cr-ICLs), the latter of which are DNA polymerase arresting lesions (PALs). We sought to determine the impact of Cr-DNA interactions on the formation of replication blocking lesions in S. cerevisiae using a PCR-based method. We found that target sequence (TS) amplification using DNA isolated from Cr(VI)-treated yeast actually increased as a function of Cr(VI) concentration. Moreover, the enhanced TS amplification was reproduced in vitro using Cr(III)-treated DNA. In contrast, PCR amplification of TS from DNA isolated from yeast exposed to equitoxic doses of the inorganic DNA cross-linking agent cisplatin (CDDP), was decreased in a concentration-dependent manner. This paradox suggested that a specific Cr-DNA interaction, such as an ionic Cr-DNA complex, was responsible for the enhanced TS amplification, thereby masking the replication-blocking effect of certain ternary Cr-DNA adducts (i.e. interstrand cross-links). To test this possibility, we removed ionically associated Cr from the DNA using salt extraction prior to PCR analysis. This procedure obviated the increased amplification and revealed a dose-dependent decrease in TS amplification and an increase in Cr-PALs. These data from DNA analyzed ex vivo after treatment of intact cells indicate that ionic interactions of Cr with DNA result in increased DNA amplification whereas coordinate-covalent Cr-DNA complexes lead to formation of Cr-PALs. Thus, these results suggest that treatment of living cells with Cr(VI) leads to two modes of Cr-binding, which may have conflicting effects on DNA replication.

  3. Mismatch repair balances leading and lagging strand DNA replication fidelity.

    PubMed

    Lujan, Scott A; Williams, Jessica S; Pursell, Zachary F; Abdulovic-Cui, Amy A; Clark, Alan B; Nick McElhinny, Stephanie A; Kunkel, Thomas A

    2012-01-01

    The two DNA strands of the nuclear genome are replicated asymmetrically using three DNA polymerases, α, δ, and ε. Current evidence suggests that DNA polymerase ε (Pol ε) is the primary leading strand replicase, whereas Pols α and δ primarily perform lagging strand replication. The fact that these polymerases differ in fidelity and error specificity is interesting in light of the fact that the stability of the nuclear genome depends in part on the ability of mismatch repair (MMR) to correct different mismatches generated in different contexts during replication. Here we provide the first comparison, to our knowledge, of the efficiency of MMR of leading and lagging strand replication errors. We first use the strand-biased ribonucleotide incorporation propensity of a Pol ε mutator variant to confirm that Pol ε is the primary leading strand replicase in Saccharomyces cerevisiae. We then use polymerase-specific error signatures to show that MMR efficiency in vivo strongly depends on the polymerase, the mismatch composition, and the location of the mismatch. An extreme case of variation by location is a T-T mismatch that is refractory to MMR. This mismatch is flanked by an AT-rich triplet repeat sequence that, when interrupted, restores MMR to > 95% efficiency. Thus this natural DNA sequence suppresses MMR, placing a nearby base pair at high risk of mutation due to leading strand replication infidelity. We find that, overall, MMR most efficiently corrects the most potentially deleterious errors (indels) and then the most common substitution mismatches. In combination with earlier studies, the results suggest that significant differences exist in the generation and repair of Pol α, δ, and ε replication errors, but in a generally complementary manner that results in high-fidelity replication of both DNA strands of the yeast nuclear genome.

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

    PubMed

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

    2014-12-01

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

  5. Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells

    SciTech Connect

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

    1984-10-01

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

  6. Differential Expression of DNA Double-Strand Break Repair Proteins in Breast Cells

    DTIC Science & Technology

    2001-07-01

    resting breast tissues from 10 different patients express both components of DNA-PK, DNAPKcs and Ku. These tissues also expressed XRCC4, DNA Ligase IV...DNA-PK in human breast tissues by immuno-histochemistry and extended these studies to two other components of the NHEJ repair pathway, XRCC4 and DNA ... ligase IV, as well as three other DNA repair components NBS1, MRE11, and PCNA. In contrast to the original report, 90% of the epithelial cells in normal

  7. Repair of DNA Lesions by a Reductive Electron Tansfer

    NASA Astrophysics Data System (ADS)

    Carell, Thomas

    2003-03-01

    Electron transfer phenomena in DNA are of fundamental importance for DNA damage[1] and DNA repair.[2] The movement of a positive charge (hole) through DNA[3-6] has been shown to proceed over significant distances. Two mechanisms, namely coherent superexchange for small transfer distances and hole, or polaron hopping for long range transfer are used to describe this phenomenon. In contrast to hole transfer, little is known about the transport of excess electrons (negative charges) through a DNA duplex. Such an excess electron transfer, however, is important in biology because DNA photolyase enzymes repair UV-induced cyclobutane pyrimidine dimer lesions (T=T) in the DNA duplex by an electron transfer from a reduced an deprotonated FADH-cofactor to the dimer lesion. The presentation covers recent results obtained in our group about the distance and sequence dependence of an excess electron transfer in a defined donor-DNA-acceptor system.[7-9] The prepared DNA double strands contain a reduced flavin electron donor and a thymine dimer acceptor, separated by adenine:thymine (A:T)n bridges of various lengths. The electron injection is initiated by irradiation of the DNA-double strand at 360 nm, which causes excitation of the reduced and deprotonated flavin donor. The injected electron, if captured by the dimer (T=T), triggers subsequently a cycloreversion, which is detectable by HPLC. A plot of the observed splitting yields against the distance between the flavin donor and the dimer gave a straight line with a small beta'-value of beta' = 0.1 Å-1. Such small beta'-values were determined for long range hole transfer as well. Our data show that excess electron transfer proceeds similarly efficient. Plotting of the yield data according to the hopping model ln(yield per minute) against ln(N) by assuming that every T between the flavin donor and the dimer acceptor can function as a discrete charge carrier (N), gives a straight line with a reasonable eta-value of close to 2

  8. Stable interactions between DNA polymerase δ catalytic and structural subunits are essential for efficient DNA repair.

    PubMed

    Brocas, Clémentine; Charbonnier, Jean-Baptiste; Dhérin, Claudine; Gangloff, Serge; Maloisel, Laurent

    2010-10-05

    Eukaryotic DNA polymerase δ (Pol δ) activity is crucial for chromosome replication and DNA repair and thus, plays an essential role in genome stability. In Saccharomyces cerevisiae, Pol δ is a heterotrimeric complex composed of the catalytic subunit Pol3, the structural B subunit Pol31, and Pol32, an additional auxiliary subunit. Pol3 interacts with Pol31 thanks to its C-terminal domain (CTD) and this interaction is of functional importance both in DNA replication and DNA repair. Interestingly, deletion of the last four C-terminal Pol3 residues, LSKW, in the pol3-ct mutant does not affect DNA replication but leads to defects in homologous recombination and in break-induced replication (BIR) repair pathways. The defect associated with pol3-ct could result from a defective interaction between Pol δ and a protein involved in recombination. However, we show that the LSKW motif is required for the interaction between Pol3 C-terminal end and Pol31. This loss of interaction is relevant in vivo since we found that pol3-ct confers HU sensitivity on its own and synthetic lethality with a POL32 deletion. Moreover, pol3-ct shows genetic interactions, both suppression and synthetic lethality, with POL31 mutant alleles. Structural analyses indicate that the B subunit of Pol δ displays a major conserved region at its surface and that pol31 alleles interacting with pol3-ct, correspond to substitutions of Pol31 amino acids that are situated in this particular region. Superimposition of our Pol31 model on the 3D architecture of the phylogenetically related DNA polymerase α (Pol α) suggests that Pol3 CTD interacts with the conserved region of Pol31, thus providing a molecular basis to understand the defects associated with pol3-ct. Taken together, our data highlight a stringent dependence on Pol δ complex stability in DNA repair.

  9. Repeat instability during DNA repair: Insights from model systems

    PubMed Central

    Usdin, Karen; House, Nealia C. M.; Freudenreich, Catherine H.

    2015-01-01

    The expansion of repeated sequences is the cause of over 30 inherited genetic diseases, including Huntington disease, myotonic dystrophy (types 1 and 2), fragile X syndrome, many spinocerebellar ataxias, and some cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat expansions are dynamic, and disease inheritance and progression are influenced by the size and the rate of expansion. Thus, an understanding of the various cellular mechanisms that cooperate to control or promote repeat expansions is of interest to human health. In addition, the study of repeat expansion and contraction mechanisms has provided insight into how repair pathways operate in the context of structure-forming DNA, as well as insights into non-canonical roles for repair proteins. Here we review the mechanisms of repeat instability, with a special emphasis on the knowledge gained from the various model systems that have been developed to study this topic. We cover the repair pathways and proteins that operate to maintain genome stability, or in some cases cause instability, and the cross-talk and interactions between them. PMID:25608779

  10. DNA-repair in mild cognitive impairment and Alzheimer's disease.

    PubMed

    Bucholtz, Nina; Demuth, Ilja

    2013-10-01

    While the pathogenesis of the sporadic form of Alzheimer disease (late onset Alzheimer disease, LOAD) is not fully understood, it seems to be clear that a combination of genetic and environmental factors are involved and influence the course of the disease. Among these factors, elevated levels of oxidative stress have been recognized and individual differences in the capacity to deal with DNA damage caused by its effects have been the subject of numerous studies. This review summarizes the research on DNA repair proteins and genes in the context of LOAD pathogenesis and its possible prodromal stage, mild cognitive impairment (MCI). The current status of the research in this field is discussed with respect to methodological issues which might have compromised the outcome of some studies and future directions of investigation on this subject are depicted.

  11. Sister chromatid exchange, DNA repair, and single-gene mutation

    SciTech Connect

    Carrano, A.V.; Thompson, L.H.

    1982-01-01

    Sister chromatid exchange (SCE) has been studied in cultured mammalian cells with regard to the nature of the inducing lesion, mutation induction, and factors that modify the observed frequency following mutagen exposure, SCEs can be induced by a wide spectrum of DNA lesions and, for nine agents examined, the frequency of induced SCE is linearly related to induced single-gene mutation. Further, a deficiency in DNA repair may alter the expression of both SCE and mutation in a qualitatively similar manner. The frequency of SCE induced by mitomycin-C is suppressed in heterochromatic relative to euchromatin and, in nondividing lymphocytes, the lesions leading to the formation of SCEs may persist for several months.

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

    SciTech Connect

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

    1994-04-26

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

  13. Genetic and environmental influence on DNA strand break repair: a twin study.

    PubMed

    Garm, Christian; Moreno-Villanueva, Maria; Bürkle, Alexander; Larsen, Lisbeth Aagaard; Bohr, Vilhelm A; Christensen, Kaare; Stevnsner, Tinna

    2013-07-01

    Accumulation of DNA damage deriving from exogenous and endogenous sources has significant consequences for cellular survival, and is implicated in aging, cancer, and neurological diseases. Different DNA repair pathways have evolved in order to maintain genomic stability. Genetic and environmental factors are likely to influence DNA repair capacity. In order to gain more insight into the genetic and environmental contribution to the molecular basis of DNA repair, we have performed a human twin study, where we focused on the consequences of some of the most abundant types of DNA damage (single-strand breaks), and some of the most hazardous lesions (DNA double-strand breaks). DNA damage signaling response (Gamma-H2AX signaling), relative amount of endogenous damage, and DNA-strand break repair capacities were studied in peripheral blood mononuclear cells from 198 twins (94 monozygotic and 104 dizygotic). We did not detect genetic effects on the DNA-strand break variables in our study.

  14. Inhibition of Topoisomerase (DNA) I (TOP1): DNA Damage Repair and Anticancer Therapy

    PubMed Central

    Xu, Yang; Her, Chengtao

    2015-01-01

    Most chemotherapy regimens contain at least one DNA-damaging agent that preferentially affects the growth of cancer cells. This strategy takes advantage of the differences in cell proliferation between normal and cancer cells. Chemotherapeutic drugs are usually designed to target rapid-dividing cells because sustained proliferation is a common feature of cancer [1,2]. Rapid DNA replication is essential for highly proliferative cells, thus blocking of DNA replication will create numerous mutations and/or chromosome rearrangements—ultimately triggering cell death [3]. Along these lines, DNA topoisomerase inhibitors are of great interest because they help to maintain strand breaks generated by topoisomerases during replication. In this article, we discuss the characteristics of topoisomerase (DNA) I (TOP1) and its inhibitors, as well as the underlying DNA repair pathways and the use of TOP1 inhibitors in cancer therapy. PMID:26287259

  15. Human DNA polymerase θ grasps the primer terminus to mediate DNA repair

    PubMed Central

    Zahn, Karl E.; Averill, April M.; Aller, Pierre; Wood, Richard D.; Doublié, Sylvie

    2015-01-01

    DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Breast, lung and oral cancers over-express polymerase θ, and reduction of its activity in mammalian cells increases sensitivity to double-strand break inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ employs a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction to the 3’-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions, or extend poorly annealed DNA termini to mediate end-joining. PMID:25775267

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

    PubMed Central

    Brown, JB; Akutsu, Tatsuya

    2009-01-01

    Background DNA repair is the general term for the collection of critical mechanisms which repair many forms of DNA damage such as methylation or ionizing radiation. DNA repair has mainly been studied in experimental and clinical situations, and relatively few information-based approaches to new extracting DNA repair knowledge exist. As a first step, automatic detection of DNA repair proteins in genomes via informatics techniques is desirable; however, there are many forms of DNA repair and it is not a straightforward process to identify and classify repair proteins with a single optimal method. We perform a study of the ability of homology and machine learning-based methods to identify and classify DNA repair proteins, as well as scan vertebrate genomes for the presence of novel repair proteins. Combinations of primary sequence polypeptide frequency, secondary structure, and homology information are used as feature information for input to a Support Vector Machine (SVM). Results We identify that SVM techniques are capable of identifying portions of DNA repair protein datasets without admitting false positives; at low levels of false positive tolerance, homology can also identify and classify proteins with good performance. Secondary structure information provides improved performance compared to using primary structure alone. Furthermore, we observe that machine learning methods incorporating homology information perform best when data is filtered by some clustering technique. Analysis by applying these methodologies to the scanning of multiple vertebrate genomes confirms a positive correlation between the size of a genome and the number of DNA repair protein transcripts it is likely to contain, and simultaneously suggests that all organisms have a non-zero minimum number of repair genes. In addition, the scan result clusters several organisms' repair abilities in an evolutionarily consistent fashion. Analysis also identifies several functionally unconfirmed

  17. DNA-PK is Involved in Repairing a Transient Surge of DNA BreaksInduced by Deceleration of DNA Replication.

    SciTech Connect

    Shimura, Tsutomu; Martin, Melvenia M.; Torres, Michael J.; Gu,Cory; Pluth, Janice M.; DiBernardi, Maria A.; McDonald, Jeffrey S.; Aladjem, Mirit I.

    2006-09-25

    ells that suffer substantial inhibition of DNA replication halt their cell cycle via a checkpoint response mediated by the PI3 kinases ATM and ATR. It is unclear how cells cope with milder replication insults, which are under the threshold for ATM and ATR activation. A third PI3 kinase, DNA-dependent protein kinase (DNA-PK), is also activated following replication inhibition, but the role DNA-PK might play in response to perturbed replication is unclear, since this kinase does not activate the signaling cascades involved in the S-phase checkpoint. Here we report that mild, transient drug-induced perturbation of DNA replication rapidly induced DNA breaks that promptly disappeared in cells that contained a functional DNA-PK whereas such breaks persisted in cells that were deficient in DNA-PK activity. After the initial transient burst of DNA breaks, cells with a functional DNA-PK did not halt replication and continued to synthesize DNA at a slow pace in the presence of replication inhibitors. In contrast, DNA-PK deficient cells subject to low levels of replication inhibition halted cell cycle progression via an ATR-mediated S-phase checkpoint. The ATM kinase was dispensable for the induction of the initial DNA breaks. These observations suggest that DNA-PK is involved in setting a high threshold for the ATR-Chkl-mediated S-phase checkpoint by promptly repairing DNA breaks that appear immediately following inhibition of DNA replication.

  18. Immunoglobulin variable region hypermutation is associated with a DNA repair deficit

    SciTech Connect

    Valles-Ayoub, Y.; Govan, H.L. III; Braun, J. )

    1991-03-11

    The molecular mechanism of Ig variable region hypermutation is unknown, but has been hypothesized to involve an error-prone DNA repair process. In this study, the authors used a novel PCR-based assay to compare repair of UV-induced DNA damage in mantle zone versus germinal center B lymphocytes. They observed that DNA repair activity within rearranged VDJ loci was sluggish in germinal center B lymphocytes compared to repair activity monitored in mantle zone B lymphocytes. In contrast, DNA repair times within the germline V{sub H}5 gene family, the variable region J{sub H}{endash}C{sub H} intron, and the N-ras gene was rapid and similar in both germinal center and mantle zone B cells. These results reflect a DNA repair deficit which, as expected for hypermutation, is selective for rearranged Ig VDG in germinal center cells. To directly measure the fidelity of DNA repair, the repaired PCR-amplified gene segments were analyzed for sequence changes by restriction enzyme digestion. In experiments thus far, repair of germline V{sub H}5 was error-free in both germinal center and mantle zone B cells. However, while rearranged V{sub H}5 segments were also error-free in mantle zone cells, they were highly mutated in germinal center cells. These findings provide direct biochemical evidence for the role of a sequence- and stage-specific error-prone DNA repair pathway in Ig V gene hypermutation.

  19. The production and repair of aflatoxin B sub 1 -induced DNA damage

    SciTech Connect

    Leadon, S.A.

    1990-05-01

    To investigate the influence of function or activity of a DNA sequence on its repair, we have studied excision repair of aflatoxin B{sub 1} (AFB{sub 1})-induced damage in the nontranscribed, heterochromatic alpha DNA of monkey cells and in the metallothionein genes of human cells. In confluent cells, AFB{sub 1} adducts are produced in similar frequencies in alpha and in the rest of the DNA, but removal from alpha DNA is severely deficient, however, removal of AFB{sub 1} adducts from alpha DNA is enhanced by small doses of UV. The repair deficiencies are not observed in actively growing cells. We have also shown that there is preferential repair of AFB{sub 1} damage in active genes. AFB{sub 1} damage is efficiently repaired in the active human metallothionein (hMT) genes, but deficiently repaired in inactive hMT genes. 51 refs., 3 tabs.

  20. A common element involved in transcriptional regulation of two DNA alkylation repair genes (MAG and MGT1) of Saccharomyces cerevisiae.

    PubMed Central

    Xiao, W; Singh, K K; Chen, B; Samson, L

    1993-01-01

    The Saccharomyces cerevisiae MAG gene encodes a 3-methyladenine DNA glycosylase that protects cells from killing by alkylating agents. MAG mRNA levels are induced not only by alkylating agents but also by DNA-damaging agents that do not produce alkylated DNA. We constructed a MAG-lacZ gene fusion to help identify the cis-acting promoter elements involved in regulating MAG expression. Deletion analysis defined the presence of one upstream activating sequence and one upstream repressing sequence (URS) and suggested the presence of a second URS. One of the MAG URS elements matches a decamer consensus sequence present in the promoters of 11 other S. cerevisiae DNA repair and metabolism genes, including the MGT1 gene, which encodes an O6-methylguanine DNA repair methyltransferase. Two proteins of 26 and 39 kDa bind specifically to the MAG and MGT1 URS elements. We suggest that the URS-binding proteins may play an important role in the coordinate regulation of these S. cerevisiae DNA repair genes. Images PMID:8246943

  1. Is thymidine glycol containing DNA a substrate of E. coli DNA mismatch repair system?

    PubMed

    Perevozchikova, Svetlana A; Trikin, Roman M; Heinze, Roger J; Romanova, Elena A; Oretskaya, Tatiana S; Friedhoff, Peter; Kubareva, Elena A

    2014-01-01

    The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active "sliding clamp" conformation on Tg-DNA is problematic.

  2. DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks.

    PubMed

    van Overbeek, Megan; Capurso, Daniel; Carter, Matthew M; Thompson, Matthew S; Frias, Elizabeth; Russ, Carsten; Reece-Hoyes, John S; Nye, Christopher; Gradia, Scott; Vidal, Bastien; Zheng, Jiashun; Hoffman, Gregory R; Fuller, Christopher K; May, Andrew P

    2016-08-18

    The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using "error-prone" DNA-repair machinery to generate precise edits.

  3. Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

    PubMed

    Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

    2010-07-07

    Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process.

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

    PubMed

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

    2013-10-01

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

  5. DNA damage, oxidative mutagen sensitivity, and repair of oxidative DNA damage in nonmelanoma skin cancer patients.

    PubMed

    Bendesky, Andrés; Michel, Alejandra; Sordo, Monserrat; Calderón-Aranda, Emma S; Acosta-Saavedra, Leonor C; Salazar, Ana M; Podoswa, Nancy; Ostrosky-Wegman, Patricia

    2006-08-01

    Nonmelanoma skin cancer (NMSC) is the most frequent type of cancer in humans. Exposure to UV radiation is a major risk factor for NMSC, and oxidative DNA damage, caused either by UV radiation itself or by other agents, may be involved in its induction. Increased sensitivity to oxidative damage and an altered DNA repair capacity (DRC) increase the risk of many types of cancer; however, sensitivity to oxidizing agents has not been evaluated for NMSC, and results regarding DRC in NMSC are inconclusive. In the present study, we evaluated DNA damage and repair in leukocytes from 41 NMSC patients and 45 controls. The Comet assay was used to measure basal and H(2)O(2)-induced DNA damage, as well as the DRC, while the cytokinesis-block micronucleus assay was used to measure the basal level of chromosome damage. Although basal DNA damage was higher for the controls than for the patients, this finding was mainly due to sampling more controls in the summer, which was associated with longer comet tails. In contrast, H(2)O(2)-induced DNA damage was significantly higher in cases than in controls, and this parameter was not influenced by the season of the year. The DRC for the H(2)O(2)-induced damage was similar for cases and controls and unrelated to seasonality. Finally, the frequency of binucleated lymphocytes with micronuclei was similar for cases and controls. The results of this study indicate that NMSC patients are distinguished from controls by an increased sensitivity to oxidative DNA damage.

  6. DNA lesions, inducible DNA repair, and cell division: Three key factors in mutagenesis and carcinogenesis

    SciTech Connect

    Ames, B.N.; Shigenaga, M.K.; Gold, L.S.

    1993-12-01

    DNA lesions that escape repair have a certain probability of giving rise to mutations when the cell divides. Endogenous DNA damage is high: 10{sup 6} oxidative lesions are present per rat cell. An exogenous mutagen produces an increment in lesions over the background rate of endogenous lesions. The effectiveness of a particular lesion depends on whether it is excised by a DNA repair system and the probability that it gives rise to a mutation when the cell divides. When the cell divides, an unrepaired DNA lesion has a certain probability of giving rise to a mutation. Thus, an important factor in the mutagenic effect of an exogenous agent whether it is genotoxic or non-genotoxic, is the increment it causes over the background cell division rate (mitogenesis) in cells that appear to matter most in cancer, the stem cells, which are not on their way to being discarded. Increasing their cell division rate increases by high doses of chemicals. If both the rate of DNA lesions and cell division are increased, then there will be a multiplicative effect on mutagenesis (and carcinogenesis), for example, by high doses of a mutagen that also increases mitogenesis through cell killing. The defense system against reactive electrophilic mutagens, such as the glutathione transferases, are also almost all inducible and buffer cells against increments in active forms of chemicals that can cause DNA lesions. A variety of DNA repair defense systems, almost all inducible, buffer the cell against any increment in DNA lesions. Therefore, the effect of a particular chemical insult depends on the level of each defense, which in turn depends on the past history of exposure. Exogenous agents can influence the induction and effectiveness of these defenses. Defenses can be partially disabled by lack of particular micronutrients in the diet (e.g., antioxidants).

  7. Defective DNA strand break repair after DNA damage in prostate cancer cells: implications for genetic instability and prostate cancer progression.

    PubMed

    Fan, Rong; Kumaravel, Tirukalikundram S; Jalali, Farid; Marrano, Paula; Squire, Jeremy A; Bristow, Robert G

    2004-12-01

    Together with cell cycle checkpoint control, DNA repair plays a pivotal role in protecting the genome from endogenous and exogenous DNA damage. Although increased genetic instability has been associated with prostate cancer progression, the relative role of DNA double-strand break repair in malignant versus normal prostate epithelial cells is not known. In this study, we determined the RNA and protein expression of a series of DNA double-strand break repair genes in both normal (PrEC-epithelial and PrSC-stromal) and malignant (LNCaP, DU-145, and PC-3) prostate cultures. Expression of genes downstream of ATM after ionizing radiation-induced DNA damage reflected the p53 status of the cell lines. In the malignant prostate cell lines, mRNA and protein levels of the Rad51, Xrcc3, Rad52, and Rad54 genes involved in homologous recombination were elevated approximately 2- to 5-fold in comparison to normal PrEC cells. The XRCC1, DNA polymerase-beta and -delta proteins were also elevated. There were no consistent differences in gene expression relating to the nonhomologous end-joining pathway. Despite increased expression of DNA repair genes, malignant prostate cancer cells had defective repair of DNA breaks, alkali-labile sites, and oxidative base damage. Furthermore, after ionizing radiation and mitomycin C treatment, chromosomal aberration assays confirmed that malignant prostate cells had defective DNA repair. This discordance between expression and function of DNA repair genes in malignant prostate cancer cells supports the hypothesis that prostate tumor progression may reflect aberrant DNA repair. Our findings support the development of novel treatment strategies designed to reinstate normal DNA repair in prostate cancer cells.

  8. Repair of uv damaged DNA: Genes and proteins of yeast and human

    SciTech Connect

    Prakash, L.

    1992-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, and to study the human homologs of yeast excision repair and postreplication repair proteins progress is described.

  9. Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells.

    PubMed

    Matt, Katja; Burger, Katharina; Gebhard, Daniel; Bergemann, Jörg

    2016-03-01

    Caloric restrictive feeding prolongs the lifespan of a variety of model organisms like rodents and invertebrates. It has been shown that caloric restriction reduces age-related as well as overall-mortality, reduces oxidative stress and influences DNA repair ability positively. There are numerous studies underlining this, but fewer studies involving humans exist. To contribute to a better understanding of the correlation of calorie reduction and DNA repair in humans, we adapted the host cell reactivation assay to an application with human peripheral blood mononuclear cells. Furthermore, we used this reliable and reproducible assay to research the influence of a special kind of calorie reduction, namely F. X. Mayr therapy, on DNA repair capacity. We found a positive effect in all persons with low pre-existing DNA repair capacity. In individuals with normal pre-existing DNA repair capacity, no effect on DNA repair capacity was detectable. Decline of DNA repair, accumulation of oxidative DNA damages, mitochondrial dysfunction, telomere shortening as well as caloric intake are widely thought to contribute to aging. With regard to that, our results can be considered as a strong indication that calorie reduction may support DNA repair processes and thus contribute to a healthier aging.

  10. The Impact of Hedgehog Signaling Pathway on DNA Repair Mechanisms in Human Cancer

    PubMed Central

    Meng, Erhong; Hanna, Ann; Samant, Rajeev S.; Shevde, Lalita A.

    2015-01-01

    Defined cellular mechanisms have evolved that recognize and repair DNA to protect the integrity of its structure and sequence when encountering assaults from endogenous and exogenous sources. There are five major DNA repair pathways: mismatch repair, nucleotide excision repair, direct repair, base excision repair and DNA double strand break repair (including non-homologous end joining and homologous recombination repair). Aberrant activation of the Hedgehog (Hh) signaling pathway is a feature of many cancer types. The Hh pathway has been documented to be indispensable for epithelial-mesenchymal transition, invasion and metastasis, cancer stemness, and chemoresistance. The functional transcription activators of the Hh pathway include the GLI proteins. Inhibition of the activity of GLI can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in enabling tumor cells to survive lethal types of DNA damage induced by chemotherapy and radiotherapy. Thus, Hh signaling presents an important therapeutic target to overcome DNA repair-enabled multi-drug resistance and consequently increase chemotherapeutic response in the treatment of cancer. PMID:26197339

  11. The retinoblastoma tumor suppressor modulates DNA repair and radioresponsiveness

    PubMed Central

    Thangavel, Chellappagounder; Liu, Yi; O’Neill, Raymond; Sharma, Ankur; McMahon, Steve B.; Mellert, Hestia; Addya, Sankar; Ertel, Adam; Birbe, Ruth; Fortina, Paolo; Dicker, Adam P; Knudsen, Karen E; Den, Robert B

    2014-01-01

    Purpose Perturbations in the RB pathway are overrepresented in advanced prostate cancer; RB loss promotes bypass of first line hormone therapy. Conversely, preliminary studies suggested that RB-deficient tumors may become sensitized to a subset of DNA damaging agents. Here, the molecular and in vivo consequence of RB status was analyzed in models of clinical relevance. Experimental Design Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAPC4 cells and hormone resistant C42, 22Rv1 cells; stable knockdown of RB using shRNA). Multiple mechanisms were interrogated including cell cycle, apoptosis, and DNA damage repair. Transcriptome analysis was performed, validated, and mechanisms discerned. Cell survival was measured using clonogenic cell survival assay and in vivo analysis was performed in nude mice with human derived tumor xenografts. Results Loss of RB enhanced the radioresponsiveness of both hormone sensitive and castrate resistant prostate cancer. Hypersensitivity to ionizing radiation was not mediated by cell cycle or p53. RB loss led to alteration in DNA damage repair and activation of the NFκB pathway and subsequent cellular apoptosis through PLK3. In vivo xenografts of RB deficient tumors exhibited diminished tumor mass, lower PSA kinetics and decreased tumor growth after treatment with ionizing radiation (p<0.05). Conclusions Loss of RB confers increased radiosensitivity in prostate cancer. This hypersensitization was mediated by alterations in apoptotic signaling. Combined, these not only provide insight into the molecular consequence of RB loss, but also credential RB status as a putative biomarker for predicting response to radiation therapy. PMID:25165096

  12. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    SciTech Connect

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D. )

    1991-07-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links.

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

    SciTech Connect

    Goldthwait, D.A.

    1984-02-01

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

  14. Coordinated nuclease activities counteract Ku at single-ended DNA double-strand breaks

    PubMed Central

    Chanut, Pauline; Britton, Sébastien; Coates, Julia; Jackson, Stephen P.; Calsou, Patrick

    2016-01-01

    Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 3′ single-strand DNA overhang by exonuclease activities in a process called DNA resection. However, it is anticipated that the highly abundant DNA end-binding protein Ku sequesters seDSBs and shields them from exonuclease activities. Despite pioneering works in yeast, it is unclear how mammalian cells counteract Ku at seDSBs to allow HR to proceed. Here we show that in human cells, ATM-dependent phosphorylation of CtIP and the epistatic and coordinated actions of MRE11 and CtIP nuclease activities are required to limit the stable loading of Ku on seDSBs. We also provide evidence for a hitherto unsuspected additional mechanism that contributes to prevent Ku accumulation at seDSBs, acting downstream of MRE11 endonuclease activity and in parallel with MRE11 exonuclease activity. Finally, we show that Ku persistence at seDSBs compromises Rad51 focus assembly but not DNA resection. PMID:27641979

  15. Recombinational DNA repair in a cellular context: a search for the homology search.

    PubMed

    Weiner, Allon; Zauberman, Nathan; Minsky, Abraham

    2009-10-01

    Double-strand DNA breaks (DSBs) are the most detrimental lesion that can be sustained by the genetic complement, and their inaccurate mending can be just as damaging. According to the consensual view, precise DSB repair relies on homologous recombination. Here, we review studies on DNA repair, chromatin diffusion and chromosome confinement, which collectively imply that a genome-wide search for a homologous template, generally thought to be a pivotal stage in all homologous DSB repair pathways, is improbable. The implications of this assertion for the scope and constraints of DSB repair pathways and for the ability of diverse organisms to cope with DNA damage are discussed.

  16. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium.

    PubMed

    Cooper, Karen L; Dashner, Erica J; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye; Hudson, Laurie G

    2016-01-15

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; <10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations.

  17. The role of DNA damage repair in aging of adult stem cells.

    PubMed

    Kenyon, Jonathan; Gerson, Stanton L

    2007-01-01

    DNA repair maintains genomic stability and the loss of DNA repair capacity results in genetic instability that may lead to a decline of cellular function. Adult stem cells are extremely important in the long-term maintenance of tissues throughout life. They regenerate and renew tissues in response to damage and replace senescent terminally differentiated cells that no longer function. Oxidative stress, toxic byproducts, reduced mitochondrial function and external exposures all damage DNA through base modification or mis-incorporation and result in DNA damage. As in most cells, this damage may limit the survival of the stem cell population affecting tissue regeneration and even longevity. This review examines the hypothesis that an age-related loss of DNA damage repair pathways poses a significant threat to stem cell survival and longevity. Normal stem cells appear to have strict control of gene expression and DNA replication whereas stem cells with loss of DNA repair may have altered patterns of proliferation, quiescence and differentiation. Furthermore, stem cells with loss of DNA repair may be susceptible to malignant transformation either directly or through the emergence of cancer-prone stem cells. Human diseases and animal models of loss of DNA repair provide longitudinal analysis of DNA repair processes in stem cell populations and may provide links to the physiology of aging.

  18. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium

    PubMed Central

    Cooper, Karen L.; Dashner, Erica J.; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye

    2015-01-01

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; <10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. PMID:26627003

  19. DNA repair of a single UV photoproduct in a designed nucleosome

    SciTech Connect

    Kosmoskil, Joseph V.; Ackerman, Eric J. ); Smerdon, Michael J.

    2001-08-28

    Eukaryotic DNA repair enzymes must interact with the architectural hierarchy of chromatin. The challenge of finding damaged DNA complexed with histone proteins in nucleosomes is complicated by the need to maintain local chromatin structures involved in regulating other DNA processing events. The heterogeneity of lesions induced by DNA-damaging agents has led us to design homogeneously damaged substrates to directly compare repair of naked DNA with that of nucleosomes. Here we report that nucleotide excision repair in Xenopus nuclear extracts can effectively repair a single UV radiation photoproduct located 5 bases from the dyad center of a positioned nucleosome, although the nucleosome is repaired at about half the rate at which the naked DNA fragment is. Extract repair within the nucleosome is > 50-fold more rapid than either enzymatic photoreversal or endonuclease cleavage of the lesion in vitro. Furthermore, nucleosome formation occurs (after repair) only on damaged naked DNA ( 165-bp fragments) during a 1-h incubation in these extracts, even in the presence of a large excess of undamaged DNA. This is an example of selective nucleosome assembly by Xenopus nuclear extracts on a short linear DNA fragment containing a DNA lesion.

  20. RAD54 forms DNA repair foci in response to DNA damage in living plant cells.

    PubMed

    Hirakawa, Takeshi; Hasegawa, Junko; White, Charles I; Matsunaga, Sachihiro

    2017-02-02

    Plants have various defense mechanisms against environmental stresses that induce DNA damage. Genetic and biochemical analyses have revealed the sensing and signaling of DNA damage, but little is known about subnuclear dynamics in response to DNA damage in living plant cells. Here, we observed that the chromatin remodeling factor RAD54, which is involved in DNA repair via the homologous recombination pathway, formed subnuclear foci (termed RAD54 foci) in Arabidopsis thaliana after induction of DNA double-strand breaks. The appearance of RAD54 foci was dependent on the ATAXIA-TELANGIECTASIA MUTATED-SUPPRESSOR OF GAMMA RESPONSE 1 pathway, and RAD54 foci were co-localized with γH2AX signals. Laser irradiation of a subnuclear area demonstrated that in living cells RAD54 was specifically accumulated at the damaged site. In addition, the formation of RAD54 foci showed specificity for cell type and region. We conclude that RAD54 foci correspond to DNA repair foci in A. thaliana.

  1. 53BP1 fosters fidelity of homology-directed DNA repair.

    PubMed

    Ochs, Fena; Somyajit, Kumar; Altmeyer, Matthias; Rask, Maj-Britt; Lukas, Jiri; Lukas, Claudia

    2016-08-01

    Repair of DNA double-strand breaks (DSBs) in mammals is coordinated by the ubiquitin-dependent accumulation of 53BP1 at DSB-flanking chromatin. Owing to its ability to limit DNA-end processing, 53BP1 is thought to promote nonhomologous end-joining (NHEJ) and to suppress homology-directed repair (HDR). Here, we show that silencing 53BP1 or exhausting its capacity to bind damaged chromatin changes limited DSB resection to hyper-resection and results in a switch from error-free gene conversion by RAD51 to mutagenic single-strand annealing by RAD52. Thus, rather than suppressing HDR, 53BP1 fosters its fidelity. These findings illuminate causes and consequences of synthetic viability acquired through 53BP1 silencing in cells lacking the BRCA1 tumor suppressor. We show that such cells survive DSB assaults at the cost of increasing reliance on RAD52-mediated HDR, which may fuel genome instability. However, our findings suggest that when challenged by DSBs, BRCA1- and 53BP1-deficient cells may become hypersensitive to, and be eliminated by, RAD52 inhibition.

  2. Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes

    PubMed Central

    Shaposhnikov, Mikhail; Proshkina, Ekaterina; Shilova, Lyubov; Zhavoronkov, Alex; Moskalev, Alexey

    2015-01-01

    DNA repair declines with age and correlates with longevity in many animal species. In this study, we investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster. PMID:26477511

  3. Comparison of rat and hamster hepatocyte primary culture/DNA repair assays

    SciTech Connect

    Kornbrust, D.J.; Barfknect, T.R.

    1984-01-01

    Previous studies have demonstrated marked differences in the capacity of hepatocytes from rats or hamsters to mediate the metabolic activation of chemical carcinogens to genotoxic (i.e., mutagenic) products. Thus far, very few investigations of species differences in DNA repair have been performed. Therefore, a comparison of the relative extent of DNA repair elicited by various genotoxic chemicals in rat and hamster hepatocyes was conducted, using the hepatocyte primary culture/DNA repair (HPC/DR) assay. Of the ll chemicals tested, eight were more potent in inducing DNA repair in hamster hepatocytes than in rat hepatocytes. Dimethylnitrosamine, diethylnitrosamine, 2-acetylaminofluorene, 9-aminoacridine, pararosaniline hydrochloride, 1-naphthylamine, benzidine and 1,2,3,4-diepoxybutane were all active in hamster hepatocytes at a concentration at least ten times less than the lowest effective concentration in rat hepatocytes. The direct-acting alkylating agent, methylmethane sulfonate, was equipotent inducing DNA repair in both rat and hamster hepatocytes, indicating that the differences in DNA repair observed for the other chemicals were probably not a result of species differences in DNA repair capacities. In contrast, 1-nitropyrene produced a greater DNA repair response in rat hepatocyes than hamster hepatocytes, while the bacterial mutagen 3-(chloromethyl)pyridine hydrochloride was inactive in both hepatocyte systems. These studies demonstrate the feasibility of using hamster hepatocytes in the HPC/DR assay and illustrate the utility of performing the assay with hepatocytes from more than one species.

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

    PubMed Central

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

    2015-01-01

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

  5. BRCA1-CtIP interaction in the repair of DNA double-strand breaks.

    PubMed

    Aparicio, Tomas; Gautier, Jean

    2016-07-01

    DNA termini at double-strand breaks are often chemically heterogeneous and require processing before initiation of repair. In a recent report, we demonstrated that CtIP and the MRE11-RAD50-NBS1 (MRN) nuclease complex cooperate with BRCA1 to specifically repair topoisomerase II-DNA adducted breaks. In contrast, BRCA1 is dispensable for repair of restriction endonuclease-generated double-strand breaks.

  6. A mediator methylation mystery: JMJD1C demethylates MDC1 to regulate DNA repair.

    PubMed

    Lu, Jian; Matunis, Michael J

    2013-12-01

    Mediator of DNA-damage checkpoint 1 (MDMDC1) has a central role in repair of DNA double-strand breaks (DSBs) by both homologous recombination and nonhomologous end joining, and its function is regulated by post-translational phosphorylation, ubiquitylation and sumoylation. In this issue, a new study by Watanabe et al. reveals that methylation of MDMDC1 is also critical for its function in DSB repair and specifically affects repair through BRCA1-dependent homologous recombination.

  7. A matter of life or death: modeling DNA damage and repair in bacteria.

    PubMed

    Karschau, Jens; de Almeida, Camila; Richard, Morgiane C; Miller, Samantha; Booth, Ian R; Grebogi, Celso; de Moura, Alessandro P S

    2011-02-16

    DNA damage is a hazard all cells must face, and evolution has created a number of mechanisms to repair damaged bases in the chromosome. Paradoxically, many of these repair mechanisms can create double-strand breaks in the DNA molecule which are fatal to the cell. This indicates that the connection between DNA repair and death is far from straightforward, and suggests that the repair mechanisms can be a double-edged sword. In this report, we formulate a mathematical model of the dynamics of DNA damage and repair, and we obtain analytical expressions for the death rate. We predict a counterintuitive relationship between survival and repair. We can discriminate between two phases: below a critical threshold in the number of repair enzymes, the half-life decreases with the number of repair enzymes, but becomes independent of the number of repair enzymes above the threshold. We are able to predict quantitatively the dependence of the death rate on the damage rate and other relevant parameters. We verify our analytical results by simulating the stochastic dynamics of DNA damage and repair. Finally, we also perform an experiment with Escherichia coli cells to test one of the predictions of our model.

  8. Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells

    PubMed Central

    1984-01-01

    The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior to their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. These results suggest that there may be a relationship between the sensitivity of xeroderma pigmentosum cells from each complementation group to specific DNA damaging agents and their inability to regulate nucleotide excision repair during cell stimulation. PMID:6480691

  9. Alu elements and DNA double-strand break repair.

    PubMed

    White, Travis B; Morales, Maria E; Deininger, Prescott L

    2015-01-01

    Alu elements represent one of the most common sources of homology and homeology in the human genome. Homeologous recombination between Alu elements represents a major form of genetic instability leading to deletions and duplications. Although these types of events have been studied extensively through genomic sequencing to assess the impact of Alu elements on disease mutations and genome evolution, the overall abundance of Alu elements in the genome often makes it difficult to assess the relevance of the Alu elements to specific recombination events. We recently reported a powerful new reporter gene system that allows the assessment of various cis and trans factors on the contribution of Alu elements to various forms of genetic instability. This allowed a quantitative measurement of the influence of mismatches on Alu elements and instability. It also confirmed that homeologous Alu elements are able to stimulate non-homologous end joining events in their vicinity. This appears to be dependent on portions of the mismatch repair pathway. We are now in a position to begin to unravel the complex influences of Alu density, mismatch and location with alterations of DNA repair processes in various tissues and tumors.

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

    NASA Astrophysics Data System (ADS)

    Altenberg, B.; Greulich, K. O.

    2011-10-01

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

  11. SIRT6 IN DNA REPAIR, METABOLISM, AND AGEING

    PubMed Central

    Lombard, David B.; Schwer, Bjoern; Alt, Frederick W.; Mostoslavsky, Raul

    2008-01-01

    Ageing, or increased mortality with time, coupled with physiologic decline, is a nearly universal yet poorly understood biological phenomenon. Studies in model organisms suggest that two conserved pathways modulate longevity: DNA damage repair and insulin/Igf1-like signaling. In addition, homologs of yeast Sir2 – the sirtuins – regulate lifespan in diverse organisms. Here, we focus on one particular sirtuin, SIRT6. Mice lacking SIRT6 develop a degenerative disorder that in some respects mimics models of accelerated ageing [1]. We discuss how sirtuins in general and SIRT6 specifically relate to other evolutionarily conserved pathways affecting ageing, and how SIRT6 might function to ensure organismal homeostasis and normal lifespan. PMID:18226091

  12. UV-inducible DNA repair in the cyanobacteria Anabaena spp

    SciTech Connect

    Levine, E.; Thiel, T.

    1987-09-01

    Strains of the filamentous cyanobacteria Anabaena spp. were capable of very efficient photoreactivation of UV irradiation-induced damage to DNA. Cells were resistant to several hundred joules of UV irradiation per square meter under conditions that allowed photoreactivation, and they also photoreactivated UV-damaged cyanophage efficiently. Reactivation of UV-irradiated cyanophage (Weigle reactivation) also occurred; UV irradiation of host cells greatly enhanced the plaque-forming ability of irradiated phage under nonphotoreactivating conditions. Postirradiation incubation of the host cells under conditions that allowed photoreactivation abolished the ability of the cells to perform Weigle reactivation of cyanophage N-1. Mitomycin C also induced Weigle reactivation of cyanophage N-1, but nalidixic acid did not. The inducible repair system (defined as the ability to perform Weigle reactivation of cyanophages) was relatively slow and inefficient compared with photoreactivation.

  13. SIRT6 stabilizes DNA-dependent Protein Kinase at chromatin for DNA double-strand break repair

    PubMed Central

    McCord, Ronald A.; Michishita, Eriko; Hong, Tao; Berber, Elisabeth; Boxer, Lisa D.; Kusumoto, Rika; Guan, Shenheng; Shi, Xiaobing; Gozani, Or; Burlingame, Alma L.; Bohr, Vilhelm A.; Chua, Katrin F.

    2009-01-01

    The Sir2 chromatin regulatory factor links maintenance of genomic stability to life span extension in yeast. The mammalian Sir2 family member SIRT6 has been proposed to have analogous functions, because SIRT6-deficiency leads to shortened life span and an aging-like degenerative phenotype in mice, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor with chromatin impacts on the efficiency of repair, and establish a link between chromatin regulation, DNA repair, and a mammalian Sir2 factor. PMID:20157594

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

    SciTech Connect

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

    2010-09-07

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

  15. Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

    PubMed Central

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

    2014-01-01

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

  16. Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair

    PubMed Central

    de Ory, Ana; Nagler, Katja; Carrasco, Begoña; Raguse, Marina; Zafra, Olga; Moeller, Ralf; de Vega, Miguel

    2016-01-01

    Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5′-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2′-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs. PMID:26826709

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

    PubMed Central

    Tudek, Barbara; Winczura, Alicja; Janik, Justyna; Siomek, Agnieszka; Foksinski, Marek; Oliński, Ryszard

    2010-01-01

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

  18. DNA repair and the evolution of transformation in Bacillus subtilis. 3. Sex with damaged DNA

    SciTech Connect

    Hoelzer, M.A.; Michod, R.E. )

    1991-06-01

    Natural genetic transformation in the bacterium Bacillus subtilis provides an experimental system for studying the evolutionary function of sexual recombination. The repair hypothesis proposes that during transformation the exogenous DNA taken up by cells is used as template for recombinational repair of damages in the recipient cell's genome. Earlier results demonstrated that the population density of transformed cells (i.e., sexual cells) increases, relative to nontransformed cells (primarily asexual cells), with increasing dosage of ultraviolet irradiation, provided that the cells are transformed with undamaged homologous DNA after they have become damaged. In nature, however, donor DNA for transformation is likely to come from cells that are as damaged as the recipient cells. In order to better simulate the effects of transformation in natural populations we conducted similar experiments as those just described using damaged donor DNA. The authors document in this report that transformants continue to increase in relative density even if they are transformed with damaged donor DNA. These results suggest that sites of transformation are often damaged sites in the recipient cell's genome.

  19. Inhibition of DNA replication and repair by anthralin or danthron in cultured human cells

    SciTech Connect

    Clark, J.M.; Hanawalt, P.C.

    1982-07-01

    The comparative effects of the tumor promoter anthralin and its analog, danthron, on semiconservative DNA replication and DNA repair synthesis were studied in cultured human cells. Bromodeoxyuridine was used as density label together with /sup 3/H-thymidine to distinguish replication from repair synthesis in isopycnic CsCl gradients. Anthralin at 1.1 microgram inhibited replication in T98G cells by 50%. In cells treated with 0.4 or 1.3 microM anthralin and additive effect was observed on the inhibition of replication by ultraviolet light (254 nm). In cells irradiated with 20 J/m2, 2.3 microM anthralin was required to inhibit repair synthesis by 50%. Thus there was no selective inhibitory effect of anthralin on repair synthesis. Danthron exhibited no detectable effect on either semiconservative replication or repair synthesis at concentrations below about 5.0 microM. Neither compound stimulated repair synthesis in the absence of ultraviolet irradiation. Thus, anthralin and danthron do not appear to react with DNA to form adducts that are subject to excision repair. Although both compounds appear to intercalate into supercoiled DNA in vitro to a limited extent, the degree of unwinding introduced by the respective drugs does not correlate with their relative effects on DNA synthesis in vivo. Therefore the inhibitory effect of anthralin on DNA replication and repair synthesis in T98G cells does not appear to result from the direct interaction of the drug with DNA.

  20. Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient

    SciTech Connect

    Stark, M.; Naiman, T.; Canaani, D. )

    1989-08-15

    In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced ({sup 3}H)thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line.

  1. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair.

    PubMed

    Gustafsson, Ann-Sofie; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-01

    Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80-95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure which is uncoupled from its essential function in DSB repair. This could have implications for the development of therapeutic strategies aiming to radiosensitize tumors by affecting the DNA-PKcs function.

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

    PubMed Central

    Luijsterburg, Martijn S.; von Bornstaedt, Gesa; Gourdin, Audrey M.; Politi, Antonio Z.; Moné, Martijn J.; Warmerdam, Daniël O.; Goedhart, Joachim; Vermeulen, Wim

    2010-01-01

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

  3. Oxygen-induced changes in mitochondrial DNA and DNA repair enzymes in aging rat lens.

    PubMed

    Zhang, Yi; Ouyang, Shan; Zhang, Lan; Tang, Xianling; Song, Zhen; Liu, Ping

    2010-01-01

    The treatment of patients with hyperbaric oxygen (HBO), vitrectomy and loss of vitreous gel during aging is associated with a high risk of subsequent development of nuclear cataract. Many studies proved that oxidation is the key reason of nuclear cataract. Reactive oxygen species (ROS) are formed in mitochondria as a by-product of normal metabolism and as a consequence of exposure to environmental compounds. Therefore, mitochondrial DNA (mtDNA) is at particularly high risk of ROS-induced damage. Oxidative damage to mtDNA has been implicated as a causative factor in a wide variety of degenerative diseases and aging. However, the effect of mtDNA damage to the lens has not been studied. The goals of the study were to identify if there was increased mtDNA damage in lens when the eye were exposed to hyperoxic or hypoxic conditions and also to evaluate the changes in gene expression of mtDNA base excision repair (mtBER) enzymes. Our data have shown that the damage of mtDNA, the expression of mtBER enzymes and the level of 8-OHdG in lens increased after inspired hyperoxia, which is likely associated with oxidative stress. However, there was no effect to mtDNA and mtBER enzymes in lens after inspired hypoxia. Nuclear cataract appeared rapidly at 14 month old rats in hyperoxia group, and lens kept transparency in other groups.

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

    PubMed

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

    2013-07-01

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

  5. DNA damage induced by boron neutron capture therapy is partially repaired by DNA ligase IV.

    PubMed

    Kondo, Natsuko; Sakurai, Yoshinori; Hirota, Yuki; Tanaka, Hiroki; Watanabe, Tsubasa; Nakagawa, Yosuke; Narabayashi, Masaru; Kinashi, Yuko; Miyatake, Shin-ichi; Hasegawa, Masatoshi; Suzuki, Minoru; Masunaga, Shin-ichiro; Ohnishi, Takeo; Ono, Koji

    2016-03-01

    Boron neutron capture therapy (BNCT) is a particle radiation therapy that involves the use of a thermal or epithermal neutron beam in combination with a boron ((10)B)-containing compound that specifically accumulates in tumor. (10)B captures neutrons and the resultant fission reaction produces an alpha ((4)He) particle and a recoiled lithium nucleus ((7)Li). These particles have the characteristics of high linear energy transfer (LET) radiation and therefore have marked biological effects. High-LET radiation is a potent inducer of DNA damage, specifically of DNA double-strand breaks (DSBs). The aim of the present study was to clarify the role of DNA ligase IV, a key player in the non-homologous end-joining repair pathway, in the repair of BNCT-induced DSBs. We analyzed the cellular sensitivity of the mouse embryonic fibroblast cell lines Lig4-/- p53-/- and Lig4+/+ p53-/- to irradiation using a thermal neutron beam in the presence or absence of (10)B-para-boronophenylalanine (BPA). The Lig4-/- p53-/- cell line had a higher sensitivity than the Lig4+/+ p53-/-cell line to irradiation with the beam alone or the beam in combination with BPA. In BNCT (with BPA), both cell lines exhibited a reduction of the 50 % survival dose (D 50) by a factor of 1.4 compared with gamma-ray and neutron mixed beam (without BPA). Although it was found that (10)B uptake was higher in the Lig4+/+ p53-/- than in the Lig4-/- p53-/- cell line, the latter showed higher sensitivity than the former, even when compared at an equivalent (10)B concentration. These results indicate that BNCT-induced DNA damage is partially repaired using DNA ligase IV.

  6. Germline mutations in DNA repair genes may predict neoadjuvant therapy response in triple negative breast patients.

    PubMed

    Spugnesi, Laura; Gabriele, Michele; Scarpitta, Rosa; Tancredi, Mariella; Maresca, Luisa; Gambino, Gaetana; Collavoli, Anita; Aretini, Paolo; Bertolini, Ilaria; Salvadori, Barbara; Landucci, Elisabetta; Fontana, Andrea; Rossetti, Elena; Roncella, Manuela; Naccarato, Giuseppe Antonio; Caligo, Maria Adelaide

    2016-12-01

    Triple negative breast cancers (TNBCs) represent about 15-20% of all breast cancer cases and are characterized by a complex molecular heterogeneity. Some TNBCs exhibit clinical and pathological properties similar to BRCA-mutated tumors, without actually bearing a mutation in BRCA genes. This "BRCAness" phenotype may be explained by germline mutations in other genes involved in DNA repair. Although respond to chemotherapy with alkylating agents, they have a high risk of recurrence and progression. Some studies have shown the efficacy of neoadjuvant therapy in TNBC patients with DNA repair defects, but proper biomarkers of DNA repair deficiency are still needed. Here, we investigated if mutations in DNA repair genes may be correlated with anthracyclines/taxanes neoadjuvant therapy response. DNA from 19 TNBC patients undergoing neoadjuvant therapy were subjected to next generation sequencing of a panel of 24 genes in DNA repair and breast cancer predisposition. In this study, 5 of 19 patients (26%) carried a pathogenic mutation in BRCA1, PALB2, RAD51C and two patients carried a probable pathogenic missense variant. Moreover, VUS (Variants of Unknown Significance) in other genes, predicted to be deleterious by in silico tools, were detected in five patients. Germline mutations in DNA repair genes were found to be associated with the group of TNBC patients who responded to therapy. We conclude that a subgroup of TNBC patients have defects in DNA repair genes, other than BRCA1, and such patients respond favourably to neoadjuvant anthracyclines/taxanes therapy. © 2016 Wiley Periodicals, Inc.

  7. DNA base excision repair of uracil residues in reconstituted nucleosome core particles

    PubMed Central

    Nilsen, Hilde; Lindahl, Tomas; Verreault, Alain

    2002-01-01

    The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase β and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase β on nucleosome cores. PMID:12411511

  8. Ku70 corrupts DNA repair in the absence of the Fanconi anemia pathway.

    PubMed

    Pace, Paul; Mosedale, Georgina; Hodskinson, Michael R; Rosado, Ivan V; Sivasubramaniam, Meera; Patel, Ketan J

    2010-07-09

    A conserved DNA repair response is defective in the human genetic illness Fanconi anemia (FA). Mutation of some FA genes impairs homologous recombination and error-prone DNA repair, rendering FA cells sensitive to DNA cross-linking agents. We found a genetic interaction between the FA gene FANCC and the nonhomologous end joining (NHEJ) factor Ku70. Disruption of both FANCC and Ku70 suppresses sensitivity to cross-linking agents, diminishes chromosome breaks, and reverses defective homologous recombination. Ku70 binds directly to free DNA ends, committing them to NHEJ repair. We show that purified FANCD2, a downstream effector of the FA pathway, might antagonize Ku70 activity by modifying such DNA substrates. These results reveal a function for the FA pathway in processing DNA ends, thereby diverting double-strand break repair away from abortive NHEJ and toward homologous recombination.

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

    SciTech Connect

    SUTHERLAND, B.M.

    2001-07-26

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

  10. Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice.

    PubMed

    Jimeno, Sonia; Fernández-Ávila, María Jesús; Cruz-García, Andrés; Cepeda-García, Cristina; Gómez-Cabello, Daniel; Huertas, Pablo

    2015-01-01

    DNA double strand breaks are the most cytotoxic lesions that can occur on the DNA. They can be repaired by different mechanisms and optimal survival requires a tight control between them. Here we uncover protein deneddylation as a major controller of repair pathway choice. Neddylation inhibition changes the normal repair profile toward an increase on homologous recombination. Indeed, RNF111/UBE2M-mediated neddylation acts as an inhibitor of BRCA1 and CtIP-mediated DNA end resection, a key process in repair pathway choice. By controlling the length of ssDNA produced during DNA resection, protein neddylation not only affects the choice between NHEJ and homologous recombination but also controls the balance between different recombination subpathways. Thus, protein neddylation status has a great impact in the way cells respond to DNA breaks.

  11. Understanding DNA Repair in Hyperthermophilic Archaea: Persistent Gaps and Other Reasons to Focus on the Fork.

    PubMed

    Grogan, Dennis W

    2015-01-01

    Although hyperthermophilic archaea arguably have a great need for efficient DNA repair, they lack members of several DNA repair protein families broadly conserved among bacteria and eukaryotes. Conversely, the putative DNA repair genes that do occur in these archaea often do not generate the expected phenotype when deleted. The prospect that hyperthermophilic archaea have some unique strategies for coping with DNA damage and replication errors has intellectual and technological appeal, but resolving this question will require alternative coping mechanisms to be proposed and tested experimentally. This review evaluates a combination of four enigmatic properties that distinguishes the hyperthermophilic archaea from all other organisms: DNA polymerase stalling at dU, apparent lack of conventional NER, lack of MutSL homologs, and apparent essentiality of homologous recombination proteins. Hypothetical damage-coping strategies that could explain this set of properties may provide new starting points for efforts to define how archaea differ from conventional models of DNA repair and replication fidelity.

  12. Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice

    PubMed Central

    Jimeno, Sonia; Fernández-Ávila, María Jesús; Cruz-García, Andrés; Cepeda-García, Cristina; Gómez-Cabello, Daniel; Huertas, Pablo

    2015-01-01

    DNA double strand breaks are the most cytotoxic lesions that can occur on the DNA. They can be repaired by different mechanisms and optimal survival requires a tight control between them. Here we uncover protein deneddylation as a major controller of repair pathway choice. Neddylation inhibition changes the normal repair profile toward an increase on homologous recombination. Indeed, RNF111/UBE2M-mediated neddylation acts as an inhibitor of BRCA1 and CtIP-mediated DNA end resection, a key process in repair pathway choice. By controlling the length of ssDNA produced during DNA resection, protein neddylation not only affects the choice between NHEJ and homologous recombination but also controls the balance between different recombination subpathways. Thus, protein neddylation status has a great impact in the way cells respond to DNA breaks. PMID:25567988

  13. Molecular epidemiology of DNA repair gene polymorphisms and head and neck cancer

    PubMed Central

    Wang, Meilin; Chu, Haiyan; Zhang, Zhengdong; Wei, Qingyi

    2013-01-01

    Although tobacco and alcohol consumption are two common risk factors of head and neck cancer (HNC), other specific etiologic causes, such as viral infection and genetic susceptibility factors, remain to be understood. Human DNA is often damaged by numerous endogenous and exogenous mutagens or carcinogens, and genetic variants in interaction with environmental exposure to these agents may explain interindividual differences in HNC risk. Single nucleotide polymorphisms (SNPs) in genes involved in the DNA damage-repair response are reported to be risk factors for various cancer types, including HNC. Here, we reviewed epidemiological studies that have assessed the associations between HNC risk and SNPs in DNA repair genes involved in base-excision repair, nucleotide-excision repair, mismatch repair, double-strand break repair and direct reversion repair pathways. We found, however, that only a few SNPs in DNA repair genes were found to be associated with significantly increased or decreased risk of HNC, and, in most cases, the effects were moderate, depending upon locus-locus interactions among the risk SNPs in the pathways. We believe that, in the presence of exposure, additional pathway-based analyses of DNA repair genes derived from genome-wide association studies (GWASs) in HNC are needed. PMID:23720673

  14. Repair of UVB-induced DNA damage is reduced in melanoma due to low XPC and global genome repair

    PubMed Central

    Budden, Timothy; Davey, Ryan J.; Vilain, Ricardo E.; Ashton, Katie A.; Braye, Stephen G.; Beveridge, Natalie J.; Bowden, Nikola A.

    2016-01-01

    UVB exposure leads to DNA damage, which when unrepaired induces C>T transitions. These mutations are found throughout the melanoma genome, particularly in non-transcribed regions. The global genome repair (GGR) branch of nucleotide excision repair (NER) is responsible for repairing UV-induced DNA damage across non-transcribed and silent regions of the genome. This study aimed to examine the relationship between UVB and GGR in melanoma. DNA repair capacity and relative expression of NER in melanocytes and melanoma cell lines before and after treatment with UVB was quantified. Transcript expression from 196 melanomas was compared to clinical parameters including solar elastosis and whole transcriptome data collected. Melanoma cell lines showed significantly reduced DNA repair when compared to melanocytes, most significantly in the S phase of the cell cycle. Expression of GGR components XPC, DDB1 and DDB2 was significantly lower in melanoma after UVB. In the melanoma tumours, XPC expression correlated with age of diagnosis and low XPC conferred significantly poorer survival. The same trend was seen in the TCGA melanoma dataset. Reduced GGR in melanoma may contribute to the UV mutation spectrum of the melanoma genome and adds further to the growing evidence of the link between UV, NER and melanoma. PMID:27487145

  15. DNA damage in Fabry patients: An investigation of oxidative damage and repair.

    PubMed

    Biancini, Giovana Brondani; Moura, Dinara Jaqueline; Manini, Paula Regina; Faverzani, Jéssica Lamberty; Netto, Cristina Brinckmann Oliveira; Deon, Marion; Giugliani, Roberto; Saffi, Jenifer; Vargas, Carmen Regla

    2015-06-01

    Fabry disease (FD) is a lysosomal storage disorder associated with loss of activity of the enzyme α-galactosidase A. In addition to accumulation of α-galactosidase A substrates, other mechanisms may be involved in FD pathophysiology, such as inflammation and oxidative stress. Higher levels of oxidative damage to proteins and lipids in Fabry patients were previously reported. However, DNA damage by oxidative species in FD has not yet been studied. We investigated basal DNA damage, oxidative DNA damage, DNA repair capacity, and reactive species generation in Fabry patients and controls. To measure oxidative damage to purines and pyrimidines, the alkaline version of the comet assay was used with two endonucleases, formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (EndoIII). To evaluate DNA repair, a challenge assay with hydrogen peroxide was performed. Patients presented significantly higher levels of basal DNA damage and oxidative damage to purines. Oxidative DNA damage was induced in both DNA bases by H2O2 in patients. Fabry patients presented efficient DNA repair in both assays (with and without endonucleases) as well as significantly higher levels of oxidative species (measured by dichlorofluorescein content). Even if DNA repair be induced in Fabry patients (as a consequence of continuous exposure to oxidative species), the repair is not sufficient to reduce DNA damage to control levels.

  16. Dynamic control of strand excision during human DNA mismatch repair.

    PubMed

    Jeon, Yongmoon; Kim, Daehyung; Martín-López, Juana V; Lee, Ryanggeun; Oh, Jungsic; Hanne, Jeungphill; Fishel, Richard; Lee, Jong-Bong

    2016-03-22

    Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3'- or 5'-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5'-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5' → 3' exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5'-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5' MMR.

  17. Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.

    PubMed Central

    Deng, W P; Nickoloff, J A

    1994-01-01

    Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication. Images PMID:8264607

  18. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links

    PubMed Central

    Räschle, Markus; Smeenk, Godelieve; Hansen, Rebecca K.; Temu, Tikira; Oka, Yasuyoshi; Hein, Marco Y.; Nagaraj, Nagarjuna; Long, David T.; Walter, Johannes C.; Hofmann, Kay; Storchova, Zuzana; Cox, Jürgen; Bekker-Jensen, Simon; Mailand, Niels; Mann, Matthias

    2017-01-01

    DNA interstrand cross-links (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. We devised a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we identified SLF1 and SLF2, which form a complex with RAD18 and together define a pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides a global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells. PMID:25931565

  19. 1999 Gordon Research Conference on Mammalian DNA Repair. Final Progress Report

    SciTech Connect

    1999-02-12

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

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

    PubMed Central

    Boulton, S J; Jackson, S P

    1996-01-01

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

  1. Thymine DNA Glycosylase Is Essential for Active DNA Demethylation by Linked Deamination-Base Excision Repair

    PubMed Central

    Cortellino, Salvatore; Xu, Jinfei; Sannai, Mara; Moore, Robert; Caretti, Elena; Cigliano, Antonio; Le Coz, Madeleine; Devarajan, Karthik; Wessels, Andy; Soprano, Dianne; Abramowitz, Lara K.; Bartolomei, Marisa S.; Rambow, Florian; Bassi, Maria Rosaria; Bruno, Tiziana; Fanciulli, Maurizio; Renner, Catherine; Klein-Szanto, Andres J.; Matsumoto, Yoshihiro; Kobi, Dominique; Davidson, Irwin; Alberti, Christophe; Larue, Lionel; Bellacosa, Alfonso

    2011-01-01

    Summary DNA methylation is a major epigenetic mechanism for gene silencing. While methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here we show that either knockout or catalytic inactivation of the DNA repair enzyme Thymine DNA Glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific, developmentally- and hormonally-regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage-response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair. PMID:21722948

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

    SciTech Connect

    Hadden, C.T.

    1981-01-01

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

  3. Structural and functional interaction between the human DNA repair proteins DNA ligase IV and XRCC4.

    PubMed

    Wu, Peï-Yu; Frit, Philippe; Meesala, SriLakshmi; Dauvillier, Stéphanie; Modesti, Mauro; Andres, Sara N; Huang, Ying; Sekiguchi, JoAnn; Calsou, Patrick; Salles, Bernard; Junop, Murray S

    2009-06-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  4. Chromatin Structure Following UV-Induced DNA Damage—Repair or Death?

    PubMed Central

    Farrell, Andrew W.; Halliday, Gary M.; Lyons, James Guy

    2011-01-01

    In eukaryotes, DNA is compacted into a complex structure known as chromatin. The unravelling of DNA is a crucial step in DNA repair, replication, transcription and recombination as this allows access to DNA for these processes. Failure to package DNA into the nucleosome, the individual unit of chromatin, can lead to genomic instability, driving a cell into apoptosis, senescence, or cellular proliferation. Ultraviolet (UV) radiation damage causes destabilisation of chromatin integrity. UV irradiation induces DNA damage such as photolesions and subjects the chromatin to substantial rearrangements, causing the arrest of transcription forks and cell cycle arrest. Highly conserved processes known as nucleotide and base excision repair (NER and BER) then begin to repair these lesions. However, if DNA repair fails, the cell may be forced into apoptosis. The modification of various histones as well as nucleosome remodelling via ATP-dependent chromatin remodelling complexes are required not only to repair these UV-induced DNA lesions, but also for apoptosis signalling. Histone modifications and nucleosome remodelling in response to UV also lead to the recruitment of various repair and pro-apoptotic proteins. Thus, the way in which a cell responds to UV irradiation via these modifications is important in determining its fate. Failure of these DNA damage response steps can lead to cellular proliferation and oncogenic development, causing skin cancer, hence these chromatin changes are critical for a proper response to UV-induced injury. PMID:22174650

  5. FEN1 participates in repair of the 5'-phosphotyrosyl terminus of DNA single-strand breaks.

    PubMed

    Kametani, Yukiko; Takahata, Chiaki; Narita, Takashi; Tanaka, Kiyoji; Iwai, Shigenori; Kuraoka, Isao

    2016-01-01

    Etoposide is a widely used anticancer drug and a DNA topoisomerase II (Top2) inhibitor. Etoposide produces Top2-attached single-strand breaks (Top2-SSB complex) and double-strand breaks (Top2-DSB complex) that are thought to induce cell death in tumor cells. The Top2-SSB complex is more abundant than the Top2-DSB complex. Human tyrosyl-DNA phosphodiesterase 2 (TDP2) is required for efficient repair of Top2-DSB complexes. However, the identities of the proteins involved in the repair of Top2-SSB complexes are unknown, although yeast genetic data indicate that 5' to 3' structure-specific DNA endonuclease activity is required for alternative repair of Top2 DNA damage. In this study, we purified a flap endonuclease 1 (FEN1) and xeroderma pigmentosum group G protein (XPG) in the 5' to 3' structure-specific DNA endonuclease family and synthesized single-strand break DNA substrates containing a 5'-phoshotyrosyl bond, mimicking the Top2-SSB complex. We found that FEN1 and XPG did not remove the 5'-phoshotyrosyl bond-containing DSB substrates but removed the 5'-phoshotyrosyl bond-containing SSB substrates. Under DNA repair conditions, FEN1 efficiently repaired the 5'-phoshotyrosyl bond-containing SSB substrates in the presence of DNA ligase and DNA polymerase. Therefore, FEN1 may play an important role in the repair of Top2-SSB complexes in etoposide-treated cells.

  6. Repair of DNA double strand breaks: in vivo biochemistry.

    PubMed

    Sugawara, Neal; Haber, James E

    2006-01-01

    Double strand breaks (DSBs) can cause damage to the genomic integrity of a cell as well as initiate genetic recombination processes. The HO and I-SceI endonucleases from budding yeast have provided a way to study these events by inducing a unique DSB in vivo under the control of a galactose-inducible promoter. The GAL::HO construct has been used extensively to study processes such as nonhomologous end joining, intra- and interchromosomal gene conversion, single strand annealing and break-induced recombination. Synchronously induced DSBs have also been important in the study of the DNA damage checkpoint, adaptation, and recovery pathways of yeast. This chapter describes methods of using GAL::HO to physically monitor the progression of events following a DSB, specifically the events leading to the switching of mating type by gene conversion of MAT using the silent donors at HML and HMR. Southern blot analysis can be used to follow the overall events in this process such as the formation of the DSB and product. Denaturing alkaline gels and slot blot techniques can be employed to follow the 5' to 3' resection of DNA starting at the DSB. After resection, the 3' tail initiates a homology search and then strand invades its homologous sequence at the donor cassette. Polymerase chain reaction is an important means to assay strand invasion and the priming of new DNA synthesis as well as the completion of gene conversion. Methods such as chromatin immunoprecipitation have provided a means to study many proteins that associate with a DSB, including not only recombination proteins, but also proteins involved in nonhomologous end joining, cell cycle arrest, chromatin remodeling, cohesin function, and mismatch repair.

  7. Water extracts of tree Hypericum sps. protect DNA from oxidative and alkylating damage and enhance DNA repair in colon cells.

    PubMed

    Ramos, Alice A; Marques, Filipe; Fernandes-Ferreira, Manuel; Pereira-Wilson, Cristina

    2013-01-01

    Diet may induce colon carcinogenesis through oxidative or alkylating DNA damage. However, diet may also contain anticarcinogenic compounds that contribute to cancer prevention. DNA damage prevention and/or induction of repair are two important mechanisms involved in cancer chemoprevention by dietary compounds. Hypericum sps. are widely used in traditional medicine to prepare infusions due to their beneficial digestive and neurologic effects. In this study, we investigated the potential of water extracts from three Hypericum sps. and some of their main phenolic compounds to prevent and repair oxidative and alkylating DNA damage in colon cells. The results showed that water extracts of Hypericum perforatum, Hypericum androsaemum, Hypericum undulatum, quercetin and rutin have protective effect against oxidative DNA damage in HT29 cells. Protective effect was also observed against alkylating DNA damage induced by methyl-methanesulfonate, except for H. androsaemum. With regard to alkylating damage repair H. perforatum, H. androsaemum and chlorogenic acid increased repair of alkylating DNA damage by base excision repair pathway. No effect was observed on nucleotide excision repair pathway. Antigenotoxic effects of Hypericum sps. may contribute to colon cancer prevention and the high amount of phenolic compounds present in Hypericum sps. play an important role in DNA protective effects.

  8. Collaborative effects of Photobacterium CuZn superoxide dismutase (SODs) and human AP endonuclease in DNA repair and SOD-deficient Escherichia coli under oxidative stress.

    PubMed

    Kim, Young Gon

    2004-01-15

    The defenses against free radical damage include specialized repair enzymes that correct oxidative damage in DNA and detoxification systems such as superoxide dismutases (SODs). These defenses may be coordinated genetically as global responses. We hypothesized that the expression of SOD and DNA repair genes would inhibit DNA damage under oxidative stress. Therefore, protection of Escherichia coli mutants deficient in SOD and DNA repair genes (sod-, xth-, and nfo-) was demonstrated by transforming the mutant strain with a plasmid pYK9 that encoded Photobacterium leiognathi CuZnSOD and human AP endonuclease. The results show that survival rates were increased in sod+ xth- nfo+ cells compared with sod- xth- ape-, sod- xth- ape-, and sod+ xth- ape- cells under oxidative stress generated with 0.1 mM paraquat or 3 mM H2O2. The data suggest that, at the least, SOD and DNA repair enzymes may collaborate on protection and repair of damaged DNA. Additionally, both enzymes are required for protection against free radicals.

  9. Double strand breaks in DNA inhibit nucleotide excision repair in vitro.

    PubMed

    Calsou, P; Frit, P; Salles, B

    1996-11-01

    Nucleotide excision repair (NER) was measured in human cell extracts incubated with either supercoiled or linearized damaged plasmid DNA as repair substrate. NER, as quantified by the extent of repair synthesis activity, was reduced by up to 80% in the case of linearized plasmid DNA when compared with supercoiled DNA. An excess of undamaged linearized plasmid in the repair mixture did not interfere with DNA repair synthesis activity on a supercoiled damaged plasmid, indicating a cis-acting inhibiting effect. In contrast, gaps on circular or linearized plasmids were filled in identically by the DNA polymerases operating in the extracts. When the extent of damage-dependent incision activity was measured, a approximately 70% reduction of repair incision activity by human cell extract was observed on linearized damaged plasmids. Recessed, protruding, or blunt ends were similarly inhibitory. NER activity was partly restored when the extracts were preincubated with autoimmune human sera containing antibodies against the nuclear DNA end-binding heterodimer Ku. In addition, the inhibition of repair activity on linear damaged plasmids was released in extracts from rodent cells deficient in Ku activity but not in extracts from murine scid cells devoid of Ku-associated DNA-dependent kinase activity.

  10. DNA repair in cells sensitive and resistant to cis-diamminedichloroplatinum(II): Host cell reactivation of damaged plasmid DNA

    SciTech Connect

    Sheibani, N.; Jennerwein, M.M.; Eastman, A. )

    1989-04-04

    cis-Diamminedichloroplatinum(II) (cis-DDP) has a broad clinical application as an effective anticancer drug. However, development of resistance to the cytotoxic effects is a limiting factor. In an attempt to understand the mechanism of resistance, the authors have employed a host cell reactivation assay of DNA repair using a cis-DDP-damaged plasmid vector. The efficiency of DNA repair was assayed by measuring the activity of an enzyme coded for by the plasmid vector. The plasmid expression vector pRSV cat contains the bacterial gene coding for chloramphenicol acetyltransferase (CAT) in a configuration which permits expression in mammalian cells. The plasmid was transfected into repair-proficient and -deficient Chinese hamster ovary cells, and CAT activity was subsequently measured in cell lysates. In the repair-deficient cells, one cis-DDP adduct per cat gene was sufficient to eliminate expression. An equivalent inhibition of CAT expression in the repair-proficient cells did not occur until about 8 times the amount of damage was introduced into the plasmid. These results implicate DNA intrastrand cross-links as the lesions responsible for the inhibition of CAT expression. This assay was used to investigate the potential role of DNA repair in mediating cis-DDP resistance in murine leukemia L1210 cells. The assay readily detects the presence or absence of repair and confirms that these resistant L1210 cells have an enhanced capacity for repair of cis-DDP-induced intrastrand cross-links.

  11. Homologous recombination is a primary pathway to repair DNA double-strand breaks generated during DNA rereplication.

    PubMed

    Truong, Lan N; Li, Yongjiang; Sun, Emily; Ang, Katrina; Hwang, Patty Yi-Hwa; Wu, Xiaohua

    2014-10-17

    Re-initiation of DNA replication at origins within a given cell cycle would result in DNA rereplication, which can lead to genome instability and tumorigenesis. DNA rereplication can be induced by loss of licensing control at cellular replication origins, or by viral protein-driven multiple rounds of replication initiation at viral origins. DNA double-strand breaks (DSBs) are generated during rereplication, but the mechanisms of how these DSBs are repaired to maintain genome stability and cell viability are poorly understood in mammalian cells. We generated novel EGFP-based DSB repair substrates, which specifically monitor the repair of rereplication-associated DSBs. We demonstrated that homologous recombination (HR) is an important mechanism to repair rereplication-associated DSBs, and sister chromatids are used as templates for such HR-mediated DSB repair. Micro-homology-mediated non-homologous end joining (MMEJ) can also be used but to a lesser extent compared to HR, whereas Ku-dependent classical non-homologous end joining (C-NHEJ) has a minimal role to repair rereplication-associated DSBs. In addition, loss of HR activity leads to severe cell death when rereplication is induced. Therefore, our studies identify HR, the most conservative repair pathway, as the primary mechanism to repair DSBs upon rereplication.

  12. How to Relate Complex DNA Repair Genotypes to Pathway Function and, Ultimately, Health Risk

    SciTech Connect

    Jones, IM

    2002-01-09

    Exposure to ionizing radiation increases the incidence of cancer. However, predicting which individuals are at most risk from radiation exposure is a distant goal. Predictive ability is needed to guide policies that regulate radiation exposure and ensure that medical treatments have maximum benefit and minimum risk. Differences between people in susceptibility to radiation are largely based on their genotype, the genes inherited from their parents. Among the important genes are those that produce proteins that repair DNA damaged by radiation. Base Excision Repair (BER) proteins repair single strand breaks and oxidized bases in DNA. Double Strand Break Repair proteins repair broken chromosomes. Using technologies and information from the Human Genome Project, we have previously determined that the DNA sequence of DNA repair genes varies within the human population. An average of 3-4 different variants were found that affect the protein for each of 37 genes studied. The average frequency of these variants is 5%. Given the many genes in each DNA repair pathway and their many variants, technical ability to determine an individual's repair genotype greatly exceeds ability to interpret the information. A long-term goal is to relate DNA repair genotypes to health risk from radiation. This study focused on the BER pathway. The BER genes are known, variants of the genes have been identified at LLNL, and LLNL had recently developed an assay for BER function using white blood cells. The goal of this initial effort was to begin developing data that could be used to test the hypothesis that many different genotypes have similar DNA repair capacity phenotypes (function). Relationships between genotype and phenotype could then be used to group genotypes with similar function and ultimately test the association of groups of genotypes with health risk from radiation. Genotypes with reduced repair function are expected to increase risk of radiation-induced health effects. The goal

  13. Proteasome inhibition enhances resistance to DNA damage via upregulation of Rpn4-dependent DNA repair genes.

    PubMed

    Karpov, Dmitry S; Spasskaya, Daria S; Tutyaeva, Vera V; Mironov, Alexander S; Karpov, Vadim L

    2013-09-17

    The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.

  14. DNA-PKcs structure suggests an allosteric mechanism modulating DNA double-strand break repair.

    PubMed

    Sibanda, Bancinyane L; Chirgadze, Dimitri Y; Ascher, David B; Blundell, Tom L

    2017-02-03

    DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a central component of nonhomologous end joining (NHEJ), repairing DNA double-strand breaks that would otherwise lead to apoptosis or cancer. We have solved its structure in complex with the C-terminal peptide of Ku80 at 4.3 angstrom resolution using x-ray crystallography. We show that the 4128-amino acid structure comprises three large structural units: the N-terminal unit, the Circular Cradle, and the Head. Conformational differences between the two molecules in the asymmetric unit are correlated with changes in accessibility of the kinase active site, which are consistent with an allosteric mechanism to bring about kinase activation. The location of KU80ct194 in the vicinity of the breast cancer 1 (BRCA1) binding site suggests competition with BRCA1, leading to pathway selection between NHEJ and homologous recombination.

  15. Spatiotemporal dynamics of DNA repair proteins following laser microbeam induced DNA damage - when is a DSB not a DSB?

    PubMed

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

    2013-08-30

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

  16. Cdc14A and Cdc14B Redundantly Regulate DNA Double-Strand Break Repair

    PubMed Central

    Lin, Han; Ha, Kyungsoo; Lu, Guojun; Fang, Xiao; Cheng, Ranran; Zuo, Qiuhong

    2015-01-01

    Cdc14 is a phosphatase that controls mitotic exit and cytokinesis in budding yeast. In mammals, the two Cdc14 homologues, Cdc14A and Cdc14B, have been proposed to regulate DNA damage repair, whereas the mitotic exit and cytokinesis rely on another phosphatase, PP2A-B55α. It is unclear if the two Cdc14s work redundantly in DNA repair and which repair pathways they participate in. More importantly, their target(s) in DNA repair remains elusive. Here we report that Cdc14B knockout (Cdc14B−/−) mouse embryonic fibroblasts (MEFs) showed defects in repairing ionizing radiation (IR)-induced DNA double-strand breaks (DSBs), which occurred only at late passages when Cdc14A levels were low. This repair defect could occur at early passages if Cdc14A levels were also compromised. These results indicate redundancy between Cdc14B and Cdc14A in DSB repair. Further, we found that Cdc14B deficiency impaired both homologous recombination (HR) and nonhomologous end joining (NHEJ), the two major DSB repair pathways. We also provide evidence that Cdh1 is a downstream target of Cdc14B in DSB repair. PMID:26283732

  17. N-cadherin coordinates AMP kinase-mediated lung vascular repair.

    PubMed

    Jian, Ming-Yuan; Liu, Yanping; Li, Qian; Wolkowicz, Paul; Alexeyev, Mikhail; Zmijewski, Jaroslaw; Creighton, Judy

    2016-01-01

    Injury to the pulmonary circulation compromises endothelial barrier function and increases lung edema. Resolution of lung damage involves restoring barrier integrity, a process requiring reestablishment of endothelial cell-cell adhesions. However, mechanisms underlying repair in lung endothelium are poorly understood. In pulmonary microvascular endothelium, AMP kinase α1 (AMPKα1) stimulation enhances recovery of the endothelial barrier after LPS-induced vascular damage. AMPKα1 colocalizes to a discrete membrane compartment with the adhesion protein neuronal cadherin (N-cadherin). This study sought to determine N-cadherin's role in the repair process. Short-hairpin RNA against full-length N-cadherin or a C-terminally truncated N-cadherin, designed to disrupt the cadherin's interactions with intracellular proteins, were expressed in lung endothelium. Disruption of N-cadherin's intracellular domain caused translocation of AMPK away from the membrane and attenuated AMPK-mediated restoration of barrier function in LPS-treated endothelium. AMPK activity measurements indicated that lower basal AMPK activity in cells expressing the truncated N-cadherin compared with controls. Moreover, the AMPK stimulator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) failed to increase AMPK activity in cells expressing the modified N-cadherin, indicating uncoupling of a functional association between AMPK and the cadherin. Isolated lung studies confirmed a physiologic role for this pathway in vivo. AMPK activation reversed LPS-induced increase in permeability, whereas N-cadherin inhibition hindered AMPK-mediated repair. Thus N-cadherin coordinates the vascular protective actions of AMPK through a functional link with the kinase. This study provides insight into intrinsic repair mechanisms in the lung and supports AMPK stimulation as a modality for treating vascular disease.

  18. Influence of XRCC1 Genetic Polymorphisms on Ionizing Radiation-Induced DNA Damage and Repair

    PubMed Central

    Sterpone, Silvia; Cozzi, Renata

    2010-01-01

    It is well known that ionizing radiation (IR) can damage DNA through a direct action, producing single- and double-strand breaks on DNA double helix, as well as an indirect effect by generating oxygen reactive species in the cells. Mammals have evolved several and distinct DNA repair pathways in order to maintain genomic stability and avoid tumour cell transformation. This review reports important data showing a huge interindividual variability on sensitivity to IR and in susceptibility to developing cancer; this variability is principally represented by genetic polymorphisms, that is, DNA repair gene polymorphisms. In particular we have focussed on single nucleotide polymorphisms (SNPs) of XRCC1, a gene that encodes for a scaffold protein involved basically in Base Excision Repair (BER). In this paper we have reported and presented recent studies that show an influence of XRCC1 variants on DNA repair capacity and susceptibility to breast cancer. PMID:20798883

  19. Breast Cancer Susceptibility and DNA Damage/Repair

    DTIC Science & Technology

    2003-06-01

    greater breast cancer risk. Polymorphisms in base excision repair (xRCCl), nucleotide excision repair (XPD, XPC), double-strand break repair ( XRCC3 ...than those who were wild type. The XRCC3 Thr24l-Met substitution also showed a suggestive association with breast cancer risk.

  20. DNA Damage Signalling and Repair Inhibitors: The Long-Sought-After Achilles’ Heel of Cancer

    PubMed Central

    Velic, Denis; Couturier, Anthony M.; Ferreira, Maria Tedim; Rodrigue, Amélie; Poirier, Guy G.; Fleury, Fabrice; Masson, Jean-Yves

    2015-01-01

    For decades, radiotherapy and chemotherapy were the two only approaches exploiting DNA repair processes to fight against cancer. Nowadays, cancer therapeutics can be a major challenge when it comes to seeking personalized targeted medicine that is both effective and selective to the malignancy. Over the last decade, the discovery of new targeted therapies against DNA damage signalling and repair has offered the possibility of therapeutic improvements in oncology. In this review, we summarize the current knowledge of DNA damage signalling and repair inhibitors, their molecular and cellular effects, and future therapeutic use. PMID:26610585

  1. Recent progress with the DNA repair mutants of Chinese hamster ovary cells

    SciTech Connect

    Thompson, L.H.; Salazar, E.P.; Brookman, K.W.; Collins, C.C.; Stewart, S.A.; Busch, D.B.; Weber, C.A.

    1986-04-02

    Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs.

  2. Fanconi anaemia and the repair of Watson and Crick DNA crosslinks.

    PubMed

    Kottemann, Molly C; Smogorzewska, Agata

    2013-01-17

    The function of Fanconi anaemia proteins is to maintain genomic stability. Their main role is in the repair of DNA interstrand crosslinks, which, by covalently binding the Watson and the Crick strands of DNA, impede replication and transcription. Inappropriate repair of interstrand crosslinks causes genomic instability, leading to cancer; conversely, the toxicity of crosslinking agents makes them a powerful chemotherapeutic. Fanconi anaemia proteins can promote stem-cell function, prevent tumorigenesis, stabilize replication forks and inhibit inaccurate repair. Recent advances have identified endogenous aldehydes as possible culprits of DNA damage that may induce the phenotypes seen in patients with Fanconi anaemia.

  3. Nick translation - a new assay for monitoring DNA damage and repair in cultured human fibroblasts

    SciTech Connect

    Snyder, R.D.; Matheson, D.W.

    1985-01-01

    An in vitro assay has been developed to detect DNA damage and repair following chemical treatment of human diploid fibroblasts. DNA damage is measured by following the Escherichia coli DNA polymerase I-catalyzed incorporation of radiolabeled deoxycytidine triphosphate (dCTP) into the DNA of lysolecithin-permeabilized cells. DNA strand breaks with free 3' OH termini serve as template sites for incorporation, and decrease of this incorporation with time, following removal of the test chemical, indicates loss (repair) of initial damage. Inhibition of the DNA excision repair process by the addition of the repair inhibitors arabinofuranosyl cytosine (ara-C) and hydroxyurea (HU) during the incubation period gives rise to an increased number of template sites, manifesting itself in increased incorporation and indicating the induction of long-patch excision repair. Results presented demonstrate that all 14 direct-acting carcinogens tested and 8 of 14 carcinogens requiring metabolic activation give positive indication of DNA damage, repair, or both. Eleven of 14 noncarcinogens tested were scored as negative, the other 3 having previously been shown to interact with cellular DNA. This assay is shown to have predictive capability at least equal to that of UDS assays but to allow a broader spectrum of genotoxic effects to be analyzed.

  4. Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

    PubMed Central

    Kuzminov, Andrei

    1999-01-01

    Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation. PMID:10585965

  5. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

    SciTech Connect

    Ganesan, Shanthi Keating, Aileen F.

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

  6. Comet assays to assess DNA damage and repair in grass shrimp embryos exposed to phototoxicants.

    PubMed

    Lee, R; Kim, G B

    2002-01-01

    Exposure of grass shrimp (Palaemonetes pugio) embryos to four compounds (anthracene, pyrene, alpha-terthienyl, methylene blue) along with solar exposure resulted in extensive DNA strand damage using the comet assay. DNA tail moments of embryos exposed to these chemicals in the dark ranged from 1.8 to 4.3, while exposure to chemicals and solar resulted in tail moments of 14.3-15.3. Reduction of DNA tail moments when solar exposed embryos were transferred to the dark, suggested DNA repair systems were active. The comet assay can be used to follow both DNA damage and repair following exposure to phototoxic chemicals.

  7. Overview of DNA Repair in Trypanosoma cruzi, Trypanosoma brucei, and Leishmania major

    PubMed Central

    Passos-Silva, Danielle Gomes; Rajão, Matheus Andrade; Nascimento de Aguiar, Pedro Henrique; Vieira-da-Rocha, João Pedro; Machado, Carlos Renato; Furtado, Carolina

    2010-01-01

    A wide variety of DNA lesions arise due to environmental agents, normal cellular metabolism, or intrinsic weaknesses in the chemical bonds of DNA. Diverse cellular mechanisms have evolved to maintain genome stability, including mechanisms to repair damaged DNA, to avoid the incorporation of modified nucleotides, and to tolerate lesions (translesion synthesis). Studies of the mechanisms related to DNA metabolism in trypanosomatids have been very limited. Together with recent experimental studies, the genome sequencing of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, three related pathogens with different life cycles and disease pathology, has revealed interesting features of the DNA repair mechanism in these protozoan parasites, which will be reviewed here. PMID:20976268

  8. HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins

    PubMed Central

    Hrecka, Kasia; Hao, Caili; Shun, Ming-Chieh; Kaur, Sarabpreet; Swanson, Selene K.; Florens, Laurence; Washburn, Michael P.; Skowronski, Jacek

    2016-01-01

    HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4DCAF1 E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4DCAF1 E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4DCAF1 E3. Thus, separate functions of HIV-1 Vpr usurp CRL4DCAF1 E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host. PMID:27335459

  9. The DNA-dependent protein kinase: A multifunctional protein kinase with roles in DNA double strand break repair and mitosis.

    PubMed

    Jette, Nicholas; Lees-Miller, Susan P

    2015-03-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes.

  10. Aberrant DNA Double-strand Break Repair Threads in Breast Carcinoma: Orchestrating Genomic Insult Survival.

    PubMed

    Kumar, Azad; Purohit, Shruti; Sharma, Nilesh Kumar

    2016-12-01

    Breast carcinoma is a heterogeneous disease that has exhibited rapid resistance to treatment in the last decade. Depending genotype and phenotype of breast cancer, there are discernible differences in DNA repair protein responses including DNA double strand break repair. It is a fact that different molecular sub-types of breast carcinoma activate these dedicated protein pathways in a distinct manner. The DNA double-strand damage repair machinery is manipulated by breast carcinoma to selectively repair the damage or insults inflicted by the genotoxic effects of chemotherapy or radiation therapy. The two DNA double-strand break repair pathways employed by breast carcinoma are homologous recombination and non-homologous end joining. In recent decades, therapeutic interventions targeting one or more factors involved in repairing DNA double-strand breaks inflicted by chemo/radiation therapy have been widely studied. Herein, this review paper summarizes the recent evidence and ongoing clinical trials citing potential therapeutic combinatorial interventions targeting DNA double-strand break repair pathways in breast carcinoma.

  11. Dietary and genetic modulation of DNA repair in healthy human adults.

    PubMed

    Tyson, J; Mathers, J C

    2007-02-01

    The DNA in all cells of the human body is subject to damage continuously from exogenous agents, internal cellular processes and spontaneous decomposition. Failure to repair such damage is fundamental to the development of many diseases and to ageing. Fortunately, the vast majority of DNA damage is detected and repaired by one of five complementary DNA repair systems. However, recent studies have shown that even in healthy individuals there is a wide inter-individual variation in DNA repair capacity. Part of this variation can be accounted for by polymorphisms in the genes encoding DNA repair proteins. However, it is probable that environmental factors, including dietary exposure as well as diet-gene interactions, are also responsible for much of the difference in repair capacity between individuals. Whilst there is some evidence from human studies that generalised malnutrition or low intakes of specific nutrients may affect DNA repair, as yet there is limited understanding of the molecular mechanisms through which nutrients can modulate this key cellular process.

  12. Aberrant DNA Double-strand Break Repair Threads in Breast Carcinoma: Orchestrating Genomic Insult Survival

    PubMed Central

    Kumar, Azad; Purohit, Shruti; Sharma, Nilesh Kumar

    2016-01-01

    Breast carcinoma is a heterogeneous disease that has exhibited rapid resistance to treatment in the last decade. Depending genotype and phenotype of breast cancer, there are discernible differences in DNA repair protein responses including DNA double strand break repair. It is a fact that different molecular sub-types of breast carcinoma activate these dedicated protein pathways in a distinct manner. The DNA double-strand damage repair machinery is manipulated by breast carcinoma to selectively repair the damage or insults inflicted by the genotoxic effects of chemotherapy or radiation therapy. The two DNA double-strand break repair pathways employed by breast carcinoma are homologous recombination and non-homologous end joining. In recent decades, therapeutic interventions targeting one or more factors involved in repairing DNA double-strand breaks inflicted by chemo/radiation therapy have been widely studied. Herein, this review paper summarizes the recent evidence and ongoing clinical trials citing potential therapeutic combinatorial interventions targeting DNA double-strand break repair pathways in breast carcinoma. PMID:28053956

  13. Novel DNA mismatch-repair activity involving YB-1 in human mitochondria.

    PubMed

    de Souza-Pinto, Nadja C; Mason, Penelope A; Hashiguchi, Kazunari; Weissman, Lior; Tian, Jingyan; Guay, David; Lebel, Michel; Stevnsner, Tinna V; Rasmussen, Lene Juel; Bohr, Vilhelm A

    2009-06-04

    Maintenance of the mitochondrial genome (mtDNA) is essential for proper cellular function. The accumulation of damage and mutations in the mtDNA leads to diseases, cancer, and aging. Mammalian mitochondria have proficient base excision repair, but the existence of other DNA repair pathways is still unclear. Deficiencies in DNA mismatch repair (MMR), which corrects base mismatches and small loops, are associated with DNA microsatellite instability, accumulation of mutations, and cancer. MMR proteins have been identified in yeast and coral mitochondria; however, MMR proteins and function have not yet been detected in human mitochondria. Here we show that human mitochondria have a robust mismatch-repair activity, which is distinct from nuclear MMR. Key nuclear MMR factors were not detected in mitochondria, and similar mismatch-binding activity was observed in mitochondrial extracts from cells lacking MSH2, suggesting distinctive pathways for nuclear and mitochondrial MMR. We identified the repair factor YB-1 as a key candidate for a mitochondrial mismatch-binding protein. This protein localizes to mitochondria in human cells, and contributes significantly to the mismatch-binding and mismatch-repair activity detected in HeLa mitochondrial extracts, which are significantly decreased when the intracellular levels of YB-1 are diminished. Moreover, YB-1 depletion in cells increases mitochondrial DNA mutagenesis. Our results show that human mitochondria contain a functional MMR repair pathway in which YB-1 participates, likely in the mismatch-binding and recognition steps.

  14. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

  15. Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair.

    PubMed

    Davis, Luther; Maizels, Nancy

    2014-03-11

    DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9(D10A) nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors.

  16. DNA DAMAGE REPAIR AND CELL CYCLE CONTROL: A NATURAL BIO-DEFENSE MECHANISM

    EPA Science Inventory

    DNA DAMAGE REPAIR AND CELL CYCLE CONTROL: A natural bio-defense mechanism
    Anuradha Mudipalli.

    Maintenance of genetic information, including the correct sequence of nucleotides in DNA, is essential for replication, gene expression, and protein synthesis. DNA lesions onto...

  17. Mechanism of RAD51-dependent DNA interstrand cross-link repair.

    PubMed

    Long, David T; Räschle, Markus; Joukov, Vladimir; Walter, Johannes C

    2011-07-01

    DNA interstrand cross-links (ICLs) are toxic DNA lesions whose repair in S phase of eukaryotic cells is incompletely understood. In Xenopus egg extracts, ICL repair is initiated when two replication forks converge on the lesion. Dual incisions then create a DNA double-strand break (DSB) in one sister chromatid, whereas lesion bypass restores the other sister. We report that the broken sister chromatid is repaired via RAD51-dependent strand invasion into the regenerated sister. Recombination acts downstream of FANCI-FANCD2, yet RAD51 binds ICL-stalled replication forks independently of FANCI-FANCD2 and before DSB formation. Our results elucidate the functional link between the Fanconi anemia pathway and the recombination machinery during ICL repair. In addition, they demonstrate the complete repair of a DSB via homologous recombination in vitro.

  18. Repair of gamma-ray-induced DNA base damage in xeroderma pigmentosum cells

    SciTech Connect

    Fornace, A.J. Jr.; Dobson, P.P.; Kinsella, T.J.

    1986-04-01

    The repair of DNA damage produced by /sup 137/Cs gamma irradiation was measured with a preparation from Micrococcus luteus containing DNA damage-specific endonucleases in combination with alkaline elution. The frequency of these endonuclease sensitive sites (ESS) was determined after 54 or 110 Gy of oxic irradiation in normal and xeroderma pigmentosum (XP) fibroblasts from complementation groups A, C, D, and G. Repair was rapid in all cell strains with greater than 50% repair after 1.5 h of repair incubation. At later repair times, 12-17 h, more ESS remained in XP than in normal cells. The frequency of excess ESS in XP cells was approximately 0.04 per 10(9) Da of DNA per Gy which was equivalent to 10% of the initial ESS produced. The removal of ESS was comparable in XP cells with normal radiosensitivity and XP3BR cells which have been reported to be moderately radiosensitive.

  19. Triple Negative Breast Cancers Have a Reduced Expression of DNA Repair Genes

    PubMed Central

    Andreis, Daniele; Bertoni, Ramona; Giardini, Roberto; Fox, Stephen B.; Broggini, Massimo; Bottini, Alberto; Zanoni, Vanessa; Bazzola, Letizia; Foroni, Chiara; Generali, Daniele; Damia, Giovanna

    2013-01-01

    DNA repair is a key determinant in the cellular response to therapy and tumor repair status could play an important role in tailoring patient therapy. Our goal was to evaluate the mRNA of 13 genes involved in different DNA repair pathways (base excision, nucleotide excision, homologous recombination, and Fanconi anemia) in paraffin embedded samples of triple negative breast cancer (TNBC) compared to luminal A breast cancer (LABC). Most of the genes involved in nucleotide excision repair and Fanconi Anemia pathways, and CHK1 gene were significantly less expressed in TNBC than in LABC. PARP1 levels were higher in TNBC than in LABC. In univariate analysis high level of FANCA correlated with an increased overall survival and event free survival in TNBC; however multivariate analyses using Cox regression did not confirm FANCA as independent prognostic factor. These data support the evidence that TNBCs compared to LABCs harbour DNA repair defects. PMID:23825533

  20. Interspecies comparisons of tissues DNA damage, repair, fixation, and replication

    SciTech Connect

    Slaga, T.J.

    1988-04-01

    The many anatomical, physiological, and biochemical differences among various mammalian species make it difficult to extrapolate carcinogenic potency data from animals to humans. The process is further complicated by the multistep origin of most malignant tumors in animals and humans due to the interaction of target cells with both endogenous and exogenous factors. Species differences in these aspects of carcinogenesis must also be considered when attempting to evaluate the carcinogenic risks of chemicals to humans. Cancer development in animals involves at least three distinct stages: initiation, promotion, and progression. Intra- and interspecies differences in susceptibility to carcinogenesis may be related to any one or a combination of these stages. Variation in species susceptibility to tumor initiation may result from differences in the abilities of various species to metabolize a potential carcinogen to an ultimate carcinogenic form and/or to detoxify the carcinogen. Most comparative studies among species have only revealed subtle differences in metabolism. DNA adducts from several activated carcinogens have been found to be the same in a number of tissues from various species, including humans. Capacity for DNA repair is apparently a critical factor in the initiation of carcinogenesis in target cells of different species but is less critical among mice that differ in susceptibility to two-stage carcinogenesis of the skin and liver. Susceptibility variations among stocks and strains to such carcinogenesis appear to be related to alterations in tumor promotion. Additional comparative studies are critically needed on all aspects of carcinogenesis to permit effective extrapolation of carcinogenic potency data from animals to humans.

  1. Interspecies comparisons of tissue DNA damage, repair, fixation, and replication.

    PubMed Central

    Slaga, T J

    1988-01-01

    The many anatomical, physiological, and biochemical differences among various mammalian species make it difficult to extrapolate carcinogenic potency data from animals to humans. The process is further complicated by the multistep origin of most malignant tumors in animals and humans due to the interaction of target cells with both endogenous and exogenous factors. Species differences in these aspects of carcinogenesis must also be considered when attempting to evaluate the carcinogenic risks of chemicals to humans. Cancer development in animals involves at least three distinct stages: initiation, promotion, and progression. Intra- and interspecies differences in susceptibility to carcinogenesis may be related to any one or a combination of these stages. Variation in species susceptibility to tumor initiation may result from differences in the abilities of various species to metabolize a potential carcinogen to an ultimate carcinogenic form and/or to detoxify the carcinogen. Most comparative studies among species have only revealed subtle differences in metabolism. DNA adducts from several activated carcinogens have been found to be the same in a number of tissues from various species, including humans. Capacity for DNA repair is apparently a critical factor in the initiation of carcinogenesis in target cells of different species but is less critical among mice that differ in susceptibility to two-stage carcinogenesis of the skin and liver. Susceptibility variations among stocks and strains to such carcinogenesis appear to be related to alterations in tumor promotion. Additional comparative studies are critically needed on all aspects of carcinogenesis to permit effective extrapolation of carcinogenic potency data from animals to humans. PMID:3289910

  2. DNA repair in human fibroblasts treated with a combination of chemicals

    SciTech Connect

    Ahmed, F.E.; Setlow, R.B.

    1981-07-01

    Excision repair of DNA damage was measured by the photolysis of bromodeoxyuridine incorporated during repair in normal human and xeroderma pigmentosum group C fibroblasts (XP C) treated with a combination of the carcinogens N-acetoxy-2-acetylamino fluorene (AAAF), and 4-nitroquinoline 1-oxide (4NQO). Repair was additive in normal and XP C cells treated with AAAF plus 4NQO, indicating that there are different rate limiting steps for removal of 4NQO and AAAF lesions.

  3. Mouse models of DNA mismatch repair in cancer research

    PubMed Central

    Lee, Kyeryoung; Tosti, Elena; Edelmann, Winfried

    2016-01-01

    Germline mutations in DNA mismatch repair (MMR) genes are the cause of hereditary non-polyposis colorectal cancer/Lynch syndrome (HNPCC/LS) one of the most common cancer predisposition syndromes, and defects in MMR are also prevalent in sporadic colorectal cancers. In the past, the generation and analysis of mouse lines with knockout mutations in all of the known MMR genes has provided insight into how loss of individual MMR genes affects genome stability and contributes to cancer susceptibility. These studies also revealed essential functions for some of the MMR genes in B cell maturation and fertility. In this review, we will provide a brief overview of the cancer predisposition phenotypes of recently developed mouse models with targeted mutations in MutS and MutL homologs (Msh and Mlh, respectively) and their utility as preclinical models. The focus will be on mouse lines with conditional MMR mutations that have allowed more accurate modeling of human cancer syndromes in mice and that together with new technologies in gene targeting, hold great promise for the analysis of MMR-deficient intestinal tumors and other cancers which will drive the development of preventive and therapeutic treatment strategies. PMID:26708047

  4. Mouse models of DNA mismatch repair in cancer research.

    PubMed

    Lee, Kyeryoung; Tosti, Elena; Edelmann, Winfried

    2016-02-01

    Germline mutations in DNA mismatch repair (MMR) genes are the cause of hereditary non-polyposis colorectal cancer/Lynch syndrome (HNPCC/LS) one of the most common cancer predisposition syndromes, and defects in MMR are also prevalent in sporadic colorectal cancers. In the past, the generation and analysis of mouse lines with knockout mutations in all of the known MMR genes has provided insight into how loss of individual MMR genes affects genome stability and contributes to cancer susceptibility. These studies also revealed essential functions for some of the MMR genes in B cell maturation and fertility. In this review, we will provide a brief overview of the cancer predisposition phenotypes of recently developed mouse models with targeted mutations in MutS and MutL homologs (Msh and Mlh, respectively) and their utility as preclinical models. The focus will be on mouse lines with conditional MMR mutations that have allowed more accurate modeling of human cancer syndromes in mice and that together with new technologies in gene targeting, hold great promise for the analysis of MMR-deficient intestinal tumors and other cancers which will drive the development of preventive and therapeutic treatment strategies.

  5. The role of DNA repair in the pluripotency and differentiation of human stem cells.

    PubMed

    Rocha, Clarissa Ribeiro Reily; Lerner, Leticia Koch; Okamoto, Oswaldo Keith; Marchetto, Maria Carolina; Menck, Carlos Frederico Martins

    2013-01-01

    All living cells utilize intricate DNA repair mechanisms to address numerous types of DNA lesions and to preserve genomic integrity, and pluripotent stem cells have specific needs due to their remarkable ability of self-renewal and differentiation into different functional cell types. Not surprisingly, human stem cells possess a highly efficient DNA repair network that becomes less efficient upon differentiation. Moreover, these cells also have an anaerobic metabolism, which reduces the mitochondria number and the likelihood of oxidative stress, which is highly related to genomic instability. If DNA lesions are not repaired, human stem cells easily undergo senescence, cell death or differentiation, as part of their DNA damage response, avoiding the propagation of stem cells carrying mutations and genomic alterations. Interestingly, cancer stem cells and typical stem cells share not only the differentiation potential but also their capacity to respond to DNA damage, with important implications for cancer therapy using genotoxic agents. On the other hand, the preservation of the adult stem cell pool, and the ability of cells to deal with DNA damage, is essential for normal development, reducing processes of neurodegeneration and premature aging, as one can observe on clinical phenotypes of many human genetic diseases with defects in DNA repair processes. Finally, several recent findings suggest that DNA repair also plays a fundamental role in maintaining the pluripotency and differentiation potential of embryonic stem cells, as well as that of induced pluripotent stem (iPS) cells. DNA repair processes also seem to be necessary for the reprogramming of human cells when iPS cells are produced. Thus, the understanding of how cultured pluripotent stem cells ensure the genetic stability are highly relevant for their safe therapeutic application, at the same time that cellular therapy is a hope for DNA repair deficient patients.

  6. Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: the molecular choreography.

    PubMed

    Thompson, Larry H

    2012-01-01

    The faithful maintenance of chromosome continuity in human cells during DNA replication and repair is critical for preventing the conversion of normal diploid cells to an oncogenic state. The evolution of higher eukaryotic cells endowed them with a large genetic investment in the molecular machinery that ensures chromosome stability. In mammalian and other vertebrate cells, the elimination of double-strand breaks with minimal nucleotide sequence change involves the spatiotemporal orchestration of a seemingly endless number of proteins ranging in their action from the nucleotide level to nucleosome organization and chromosome architecture. DNA DSBs trigger a myriad of post-translational modifications that alter catalytic activities and the specificity of protein interactions: phosphorylation, acetylation, methylation, ubiquitylation, and SUMOylation, followed by the reversal of these changes as repair is completed. "Superfluous" protein recruitment to damage sites, functional redundancy, and alternative pathways ensure that DSB repair is extremely efficient, both quantitatively and qualitatively. This review strives to integrate the information about the molecular mechanisms of DSB repair that has emerged over the last two decades with a focus on DSBs produced by the prototype agent ionizing radiation (IR). The exponential growth of molecular studies, heavily driven by RNA knockdown technology, now reveals an outline of how many key protein players in genome stability and cancer biology perform their interwoven tasks, e.g. ATM, ATR, DNA-PK, Chk1, Chk2, PARP1/2/3, 53BP1, BRCA1, BRCA2, BLM, RAD51, and the MRE11-RAD50-NBS1 complex. Thus, the nature of the intricate coordination of repair processes with cell cycle progression is becoming apparent. This review also links molecular abnormalities to cellular pathology as much a possible and provides a framework of temporal relationships.

  7. Differential requirement for SUB1 in chromosomal and plasmid double-strand DNA break repair.

    PubMed

    Yu, Lijian; Volkert, Michael R

    2013-01-01

    Non homologous end joining (NHEJ) is an important process that repairs double strand DNA breaks (DSBs) in eukaryotic cells. Cells defective in NHEJ are unable to join chromosomal breaks. Two different NHEJ assays are typically used to determine the efficiency of NHEJ. One requires NHEJ of linearized plasmid DNA transformed into the test organism; the other requires NHEJ of a single chromosomal break induced either by HO endonuclease or the I-SceI restriction enzyme. These two assays are generally considered equivalent and rely on the same set of NHEJ genes. PC4 is an abundant DNA binding protein that has been suggested to stimulate NHEJ. Here we tested the role of PC4's yeast homolog SUB1 in repair of DNA double strand breaks using different assays. We found SUB1 is required for NHEJ repair of DSBs in plasmid DNA, but not in chromosomal DNA. Our results suggest that these two assays, while similar are not equivalent and that repair of plasmid DNA requires additional factor(s) that are not required for NHEJ repair of chromosomal double-strand DNA breaks. Possible roles for Sub1 proteins in NHEJ of plasmid DNA are discussed.

  8. Formation and Repair of Tobacco Carcinogen-Derived Bulky DNA Adducts

    PubMed Central

    Hang, Bo

    2010-01-01

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

  9. A requirement for polymerized actin in DNA double-strand break repair.

    PubMed

    Andrin, Christi; McDonald, Darin; Attwood, Kathleen M; Rodrigue, Amélie; Ghosh, Sunita; Mirzayans, Razmik; Masson, Jean-Yves; Dellaire, Graham; Hendzel, Michael J

    2012-07-01

    Nuclear actin is involved in several nuclear processes from chromatin remodeling to transcription. Here we examined the requirement for actin polymerization in DNA double-strand break repair. Double-strand breaks are considered the most dangerous type of DNA lesion. Double-strand break repair consists of a complex set of events that are tightly regulated. Failure at any step can have catastrophic consequences such as genomic instability, oncogenesis or cell death. Many proteins involved in this repair process have been identified and their roles characterized. We discovered that some DNA double-strand break repair factors are capable of associating with polymeric actin in vitro and specifically, that purified Ku70/80 interacts with polymerized actin under these conditions. We find that the disruption of polymeric actin inhibits DNA double strand break repair both in vitro and in vivo. Introduction of nuclear targeted mutant actin that cannot polymerize, or the depolymerization of endogenous actin filaments by the addition of cytochalasin D, alters the retention of Ku80 at sites of DNA damage in live cells. Our results suggest that polymeric actin is required for proper DNA double-strand break repair and may function through the stabilization of the Ku heterodimer at the DNA damage site.

  10. Formation and Repair of Tobacco Carcinogen-Derived Bulky DNA Adducts

    DOE PAGES

    Hang, Bo

    2010-01-01

    DNA adducts play a central role in chemical carcinogenesis. The analysis of formation and repair of smoking-related DNA adducts remains particularly challenging as both smokers and nonsmokers exposed to smoke are repetitively under attack from complex mixtures of carcinogens such as polycyclic aromatic hydrocarbons and N -nitrosamines. The bulky DNA adducts, which usually have complex structure, are particularly important because of their biological relevance. Several known cellular DNA repair pathways have been known to operate in human cells on specific types of bulky DNA adducts, for example, nucleotide excision repair, base excision repair, and direct reversal involving O 6more » -alkylguanine DNA alkyltransferase or AlkB homologs. Understanding the mechanisms of adduct formation and repair processes is critical for the assessment of cancer risk resulting from exposure to cigarette smoke, and ultimately for developing strategies of cancer prevention. This paper highlights the recent progress made in the areas concerning formation and repair of bulky DNA adducts in the context of tobacco carcinogen-associated genotoxic and carcinogenic effects.« less

  11. Cell resistance to the Cytolethal Distending Toxin involves an association of DNA repair mechanisms

    PubMed Central

    Bezine, Elisabeth; Malaisé, Yann; Loeuillet, Aurore; Chevalier, Marianne; Boutet-Robinet, Elisa; Salles, Bernard; Mirey, Gladys; Vignard, Julien

    2016-01-01

    The Cytolethal Distending Toxin (CDT), produced by many bacteria, has been associated with various diseases including cancer. CDT induces DNA double-strand breaks (DSBs), leading to cell death or mutagenesis if misrepaired. At low doses of CDT, other DNA lesions precede replication-dependent DSB formation, implying that non-DSB repair mechanisms may contribute to CDT cell resistance. To address this question, we developed a proliferation assay using human cell lines specifically depleted in each of the main DNA repair pathways. Here, we validate the involvement of the two major DSB repair mechanisms, Homologous Recombination and Non Homologous End Joining, in the management of CDT-induced lesions. We show that impairment of single-strand break repair (SSBR), but not nucleotide excision repair, sensitizes cells to CDT, and we explore the interplay of SSBR with the DSB repair mechanisms. Finally, we document the role of the replicative stress response and demonstrate the involvement of the Fanconi Anemia repair pathway in response to CDT. In conclusion, our work indicates that cellular survival to CDT-induced DNA damage involves different repair pathways, in particular SSBR. This reinforces a model where CDT-related genotoxicity primarily involves SSBs rather than DSBs, underlining the importance of cell proliferation during CDT intoxication and pathogenicity. PMID:27775089

  12. Deletion-bias in DNA double-strand break repair differentially contributes to plant genome shrinkage.

    PubMed

    Vu, Giang T H; Cao, Hieu X; Reiss, Bernd; Schubert, Ingo

    2017-02-28

    In order to prevent genome instability, cells need to be protected by a number of repair mechanisms, including DNA double-strand break (DSB) repair. The extent to which DSB repair, biased towards deletions or insertions, contributes to evolutionary diversification of genome size is still under debate. We analyzed mutation spectra in Arabidopsis thaliana and in barley (Hordeum vulgare) by PacBio sequencing of three DSB-targeted loci each, uncovering repair via gene conversion, single strand annealing (SSA) or nonhomologous end-joining (NHEJ). Furthermore, phylogenomic comparisons between A. thaliana and two related species were used to detect naturally occurring deletions during Arabidopsis evolution. Arabidopsis thaliana revealed significantly more and larger deletions after DSB repair than barley, and barley displayed more and larger insertions. Arabidopsis displayed a clear net loss of DNA after DSB repair, mainly via SSA and NHEJ. Barley revealed a very weak net loss of DNA, apparently due to less active break-end resection and easier copying of template sequences into breaks. Comparative phylogenomics revealed several footprints of SSA in the A. thaliana genome. Quantitative assessment of DNA gain and loss through DSB repair processes suggests deletion-biased DSB repair causing ongoing genome shrinking in A. thaliana, whereas genome size in barley remains nearly constant.

  13. Chromatin Dynamics during DNA Repair Revealed by Pair Correlation Analysis of Molecular Flow in the Nucleus

    PubMed Central

    Hinde, Elizabeth; Kong, Xiangduo; Yokomori, Kyoko; Gratton, Enrico

    2014-01-01

    Chromatin dynamics modulate DNA repair factor accessibility throughout the DNA damage response. The spatiotemporal scale upon which these dynamics occur render them invisible to live cell imaging. Here we present a believed novel assay to monitor the in vivo structural rearrangements of chromatin during DNA repair. By pair correlation analysis of EGFP molecular flow into chromatin before and after damage, this assay measures millisecond variations in chromatin compaction with submicron resolution. Combined with laser microirradiation we employ this assay to monitor the real-time accessibility of DNA at the damage site. We find from comparison of EGFP molecular flow with a molecule that has an affinity toward double-strand breaks (Ku-EGFP) that DNA damage induces a transient decrease in chromatin compaction at the damage site and an increase in compaction to adjacent regions, which together facilitate DNA repair factor recruitment to the lesion with high spatiotemporal control. PMID:24988341

  14. Impact of ribonucleotide incorporation by DNA polymerases β and λ on oxidative base excision repair

    PubMed Central

    Crespan, Emmanuele; Furrer, Antonia; Rösinger, Marcel; Bertoletti, Federica; Mentegari, Elisa; Chiapparini, Giulia; Imhof, Ralph; Ziegler, Nathalie; Sturla, Shana J.; Hübscher, Ulrich; van Loon, Barbara; Maga, Giovanni

    2016-01-01

    Oxidative stress is a very frequent source of DNA damage. Many cellular DNA polymerases (Pols) can incorporate ribonucleotides (rNMPs) during DNA synthesis. However, whether oxidative stress-triggered DNA repair synthesis contributes to genomic rNMPs incorporation is so far not fully understood. Human specialized Pols β and λ are the important enzymes involved in the oxidative stress tolerance, acting both in base excision repair and in translesion synthesis past the very frequent oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxo-G). We found that Pol β, to a greater extent than Pol λ can incorporate rNMPs opposite normal bases or 8-oxo-G, and with a different fidelity. Further, the incorporation of rNMPs opposite 8-oxo-G delays repair by DNA glycosylases. Studies in Pol β- and λ-deficient cell extracts suggest that Pol β levels can greatly affect rNMP incorporation opposite oxidative DNA lesions. PMID:26917111

  15. MRN, CtIP, and BRCA1 mediate repair of topoisomerase II-DNA adducts.

    PubMed

    Aparicio, Tomas; Baer, Richard; Gottesman, Max; Gautier, Jean

    2016-02-15

    Repair of DNA double-strand breaks (DSBs) with complex ends poses a special challenge, as additional processing is required before DNA ligation. For example, protein-DNA adducts must be removed to allow repair by either nonhomologous end joining or homology-directed repair. Here, we investigated the processing of topoisomerase II (Top2)-DNA adducts induced by treatment with the chemotherapeutic agent etoposide. Through biochemical analysis in Xenopus laevis egg extracts, we establish that the MRN (Mre11, Rad50, and Nbs1) complex, CtIP, and BRCA1 are required for both the removal of Top2-DNA adducts and the subsequent resection of Top2-adducted DSB ends. Moreover, the interaction between CtIP and BRCA1, although dispensable for resection of endonuclease-generated DSB ends, is required for resection of Top2-adducted DSBs, as well as for cellular resistance to etoposide during genomic DNA replication.

  16. Structural basis for a novel mechanism of DNA bridging and alignment in eukaryotic DSB DNA repair.

    PubMed

    Gouge, Jérôme; Rosario, Sandrine; Romain, Félix; Poitevin, Frédéric; Béguin, Pierre; Delarue, Marc

    2015-04-15

    Eukaryotic DNA polymerase mu of the PolX family can promote the association of the two 3'-protruding ends of a DNA double-strand break (DSB) being repaired (DNA synapsis) even in the absence of the core non-homologous end-joining (NHEJ) machinery. Here, we show that terminal deoxynucleotidyltransferase (TdT), a closely related PolX involved in V(D)J recombination, has the same property. We solved its crystal structure with an annealed DNA synapsis containing one micro-homology (MH) base pair and one nascent base pair. This structure reveals how the N-terminal domain and Loop 1 of Tdt cooperate for bridging the two DNA ends, providing a templating base in trans and limiting the MH search region to only two base pairs. A network of ordered water molecules is proposed to assist the incorporation of any nucleotide independently of the in trans templating base. These data are consistent with a recent model that explains the statistics of sequences synthesized in vivo by Tdt based solely on this dinucleotide step. Site-directed mutagenesis and functional tests suggest that this structural model is also valid for Pol mu during NHEJ.

  17. Repair and replication of DNA in hereditary (bilateral) retinoblastoma cells after X-irradiation

    SciTech Connect

    Cleaver, J.E.; Char, D.; Charles, W.C.; Rand, N.

    1982-04-01

    Fibroblasts from patients with hereditary retinoblastoma reportedly exhibit increased sensitivity to killing by X-rays. Although some human syndromes with similar or greater hypersensitivity to DNA-damaging agents (e.g., X-rays, ultraviolet light, and chemical carcinogens), such as xeroderma pigmentosum, are deficient in DNA repair, most do not have such clearly demonstrable defects in repair. Retinoblastoma cells appear to be normal in repairing single-strand breaks and performing repair replication after X-irradiation and also in synthesizing poly(adenosine diphosphoribose). Semiconservative DNA replication in these cells, however, is slightly more resistant than normal after X-irradiation, suggesting that continued replication of damaged parental DNA could contribute to the pathogenesis of the disease. This effect is small, however, and may be a consequence rather than a cause of the fundamental enzymatic abnormality in retinoblastoma that causes the tumorigenesis.

  18. ATP-dependent chromatin remodeling and DNA double-strand break repair.

    PubMed

    van Attikum, Haico; Gasser, Susan M

    2005-08-01

    The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic stability. Two pathways for the repair of DBSs, nonhomologous end-joining (NHEJ) and homologous recombination (HR), have evolved in eukaryotes. These pathways, like processes such as transcription and replication, act on DNA that is embedded in nucleosomes. Recent studies have shown that DNA repair, like transcription, is facilitated both by histone tail modification and by ATP-dependent chromatin remodeling. This review emphasizes recent reports that demonstrate a function for the ATP-dependent chromatin remodeling complexes INO80 and RSC in NHEJ and HR. We also discuss the possible role of SWR1- and TIP60-mediated nucleosomal histone exchange in DNA repair.

  19. Nanotechnology in cancer therapy: targeting the inhibition of key DNA repair pathways.

    PubMed

    Aziz, K; Nowsheen, S; Georgakilas, A G

    2010-10-01

    Cancer therapy has been changing over the decades as we move away from the administration of broad spectrum cytotoxic drugs and towards the use of therapy targeted for each tumor type. After the induction of DNA damage through chemotherapeutic agents, tumor cells can survive due to their proficient DNA repair pathways, some of which are dysregulated in cancer. Latest improvements in nanotechnology and drug discovery has led to the discovery of some very unique, highly specific and innovative drugs as inhibitors of various DNA repair pathways like base excision repair and double strand break repair. In this review we look at the efficacy and potency of these small chemical molecules to target the processing of DNA damage induced by standard therapeutic agents. Emphasis is given to those drugs currently under clinical trials. We also discuss the future directions of using this nanotechnology to increase the therapeutic ratio in cancer treatment.

  20. Bisdemethoxycurcumin induces DNA damage and inhibits DNA repair associated protein expressions in NCI-H460 human lung cancer cells.

    PubMed

    Yu, Chien-Chih; Yang, Su-Tso; Huang, Wen-Wen; Peng, Shu-Fen; Huang, An-Cheng; Tang, Nou-Ying; Liu, Hsin-Chung; Yang, Mei-Due; Lai, Kuang-Chi; Chung, Jing-Gung

    2015-08-30

    Nonsmall cell lung carcinoma (NSCLC) is a devastating primary lung tumor resistant to conventional therapies. Bisdemethoxycurcumin (BDMC) is one of curcumin derivate from Turmeric and has been shown to induce NSCLC cell death. Although there is one report to show BDMC induced DNA double strand breaks, however, no available information to show BDMC induced DNA damage action with inhibited DNA repair protein in lung cancer cells in detail. In this study, we tested BDMC-induced DNA damage and condensation in NCI-H460 cells by using Comet assay and DAPI staining examinations, respectively and we found BDMC induced DNA damage and condension. Western blotting was used to examine the effects of BDMC on protein expression associated with DNA damage and repair and results indicated that BDMC suppressed the protein levels associated with DNA damage and repair, such as 14-3-3σ (an important checkpoint keeper of DDR), O6-methylguanine-DNA methyltransferase, DNA repair proteins breast cancer 1, early onset, mediator of DNA damage checkpoint 1 but activate phosphorylated p53 and p-H2A.X (phospho Ser140) in NCI-H460 cells. Confocal laser systems microscopy was used for examining the protein translocation and results show that BDMC increased the translocation of p-p53 and p-H2A.X (phospho Ser140) from cytosol to nuclei in NCI-H460 cells. In conclusion, BDMC induced DNA damage and condension and affect DNA repair proteins in NCI-H460 cells in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol, 2015.

  1. Biological significance of domain-oriented DNA repair in xeroderma pigmentosum cells

    SciTech Connect

    Kantor, G.J.; Elking, C.F.

    1988-02-15

    The patterns (domain oriented versus a random location) and amounts of DNA excision repair, determined by standard density gradient techniques and sedimentation properties of partially repaired and UV-endonuclease-digested DNA in alkaline sucrose gradients, are reported for UV (254 nm)-irradiated nondividing xeroderma pigmentosum complementation group C or A (XP-C, XP-A) and normal cells. Repair synthesis in relatively UV-resistant XP-C (XP4RO) cells is domain oriented and limited (10% of normal values) while it is randomly located and not as limited in more sensitive XP-A (XP8LO) cells. Thus, greater UV resistance is associated with a very limited but domain-oriented pattern of repair. In XP-C cells, both total and domain-oriented repair syntheses, while limited, increase with UV dose and plateau at about 15-20 J/m2, as observed for normal cells. We suggest that repair in XP-C is limited at the lower UV doses (less than 15-20 J/m2) by substrate levels in specific chromatin domains and not by availability of essential enzymes for domain-oriented repair. In contrast, the XP-A strain XP8LO exhibits normal repair activities for doses up to 5 J/m2 and limited repair at higher doses, indicating that repair occurs through normal pathways that are limited by reduced availability of an essential enzyme.

  2. DNA Repair Pathway Choice Is Influenced by the Health of Drosophila melanogaster

    PubMed Central

    Wang, Alethea D.; Agrawal, Aneil F.

    2012-01-01

    In nature, individuals vary tremendously in condition and this may be an important source of variation in mutation rate. Condition is likely to affect cell state and thereby impact the amount of DNA damage sustained and/or the way it is repaired. Here, we focus on DNA repair. If low-condition individuals are less capable of devoting the same level of resources to accurate repair, they may suffer higher mutation rates. However, repair decisions are also governed by various aspects of cell physiology, which may render the prediction that “higher-condition individuals use better repair mechanisms” too simplistic. We use a larval diet manipulation in Drosophila melanogaster to create high- and low-condition individuals and then contrast their relative usage of three repair pathways [homologous recombination (HR), single-strand annealing (SSA), and nonhomologous end joining (NHEJ)] that differ in their mechanistic requirements and their mutational consequences. We find that low-condition flies are more likely than high-condition flies to use the most conservative of these three repair pathways, suggesting that physiological constraints on repair pathway usage may be more important than energetic costs. We also show that the repair differences between high- and low-condition flies resemble those between young and old flies, suggesting the underlying mechanisms may be similar. Finally, we observe that the effect of larval diet on adult repair increases as flies age, indicating that developmental differences early in life can have long-lasting consequences. PMID:22813892

  3. Splicing controls the ubiquitin response during DNA double-strand break repair

    PubMed Central

    Pederiva, C; Böhm, S; Julner, A; Farnebo, M

    2016-01-01

    Although evidence that splicing regulates DNA repair is accumulating, the underlying mechanism(s) remain unclear. Here, we report that short-term inhibition of pre-mRNA splicing by spliceosomal inhibitors impairs cellular repair of DNA double-strand breaks. Indeed, interference with splicing as little as 1 h prior to irradiation reduced ubiquitylation of damaged chromatin and impaired recruitment of the repair factors WRAP53β, RNF168, 53BP1, BRCA1 and RAD51 to sites of DNA damage. Consequently, splicing-deficient cells exhibited significant numbers of residual γH2AX foci, as would be expected if DNA repair is defective. Furthermore, we show that this is due to downregulation of the E3 ubiquitin ligase RNF8 and that re-introduction of this protein into splicing-deficient cells restores ubiquitylation at sites of DNA damage, accumulation of downstream factors and subsequent repair. Moreover, downregulation of RNF8 explains the defective repair associated with knockdown of various splicing factors in recent genome-wide siRNA screens and, significantly, overexpression of RNF8 counteracts this defect. These discoveries reveal a mechanism that may not only explain how splicing regulates repair of double-strand breaks, but also may underlie various diseases caused by deregulation of splicing factors, including cancer. PMID:27315300

  4. Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair

    PubMed Central

    Tsabar, Michael; Waterman, David P.; Aguilar, Fiona; Katsnelson, Lizabeth; Eapen, Vinay V.; Memisoglu, Gonen; Haber, James E.

    2016-01-01

    To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair. PMID:27222517

  5. Repair synthesis by human cell extracts in DNA damaged by cis- and trans-diamminedichloroplatinum(II).

    PubMed Central

    Hansson, J; Wood, R D

    1989-01-01

    DNA damage was induced in closed circular plasmid DNA by treatment with cis- or trans-diamminedichloroplatinum(II). These plasmids were used as substrates in reactions to give quantitative measurements of DNA repair synthesis mediated by cell free extracts from human lymphoid cell lines. Adducts induced by both drugs stimulated repair synthesis in a dose dependent manner by an ATP-requiring process. Measurements by an isopycnic gradient sedimentation method gave an upper limit for the average patch sizes in this in vitro system of around 140 nucleotides. It was estimated that up to 3% of the drug adducts induce the synthesis of a repair patch. The repair synthesis is due to repair of a small fraction of frequent drug adducts, rather than extensive repair of a rare subclass of lesions. Nonspecific DNA synthesis in undamaged plasmids, caused by exonucleolytic degradation and resynthesis, was reduced by repeated purification of intact circular forms. An extract made from cells belonging to xeroderma pigmentosum complementation group A was deficient in repair synthesis in response to the presence of cis- or trans-diamminedichloroplatinum(II) adducts in DNA. Images PMID:2554251

  6. Differential repair of DNA damage in specific nucleotide sequences in monkey cells.

    PubMed Central

    Leadon, S A

    1986-01-01

    An immunological method was developed that isolates DNA fragments containing bromouracil in repair patches from unrepaired DNA using a monoclonal antibody that recognizes bromouracil. Cultured monkey cells were exposed to either UV light or the activated carcinogen aflatoxin B1 and excision repair of damage in DNA fragments containing the integrated and transcribed E. coli gpt gene was compared to that in the genome overall. A more rapid repair, of both UV and AFB1 damage was observed in the DNA fragments containing the E. coli gpt genes. The more efficient repair of UV damage was not due to a difference in the initial level of pyrimidine dimers as determined with a specific UV endonuclease. Consistent with previous observations using different methodology, repair of UV damage in the alpha sequences was found to occur at the same rate as that in the genome overall, while repair of AFB1 damage was deficient in alpha DNA. The preferential repair of damage in the gpt gene may be related to the functional state of the sequence and/or to alterations produced in the chromatin conformation by the integration of plasmid sequences carrying the gene. Images PMID:3786142

  7. Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair.

    PubMed

    Tsabar, Michael; Waterman, David P; Aguilar, Fiona; Katsnelson, Lizabeth; Eapen, Vinay V; Memisoglu, Gonen; Haber, James E

    2016-05-15

    To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair.

  8. Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis

    SciTech Connect

    Dong, Hui; Shi, Qiong; Song, Xiufang; Fu, Juanli; Hu, Lihua; Xu, Demei; Su, Chuanyang; Xia, Xiaomin; Song, Erqun; Song, Yang

    2015-07-01

    Our previous studies demonstrated that polychlorinated biphenyl (PCB) quinone induced oxidative DNA damage in HepG2 cells. To promote genomic integrity, DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair and apoptosis. PCB quinone-induced cell cycle arrest and apoptosis have been documented, however, whether PCB quinone insult induce DNA repair signaling is still unknown. In this study, we identified the activation of DDR and corresponding signaling events in HepG2 cells upon the exposure to a synthetic PCB quinone, PCB29-pQ. Our data illustrated that PCB29-pQ induces the phosphorylation of p53, which was mediated by ataxia telangiectasia mutated (ATM) protein kinase. The observed phosphorylated histone H2AX (γ-H2AX) foci and the elevation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) indicated that DDR was stimulated by PCB29-pQ treatment. Additionally, we found PCB29-pQ activates non-homologous end joining (NHEJ), base excision repair (BER) and nucleotide excision repair (NER) signalings. However, these repair pathways are not error-free processes and aberrant repair of DNA damage may cause the potential risk of carcinogenesis and mutagenesis. - Highlights: • Polychlorinated biphenyl quinone induces oxidative DNA damage in HepG2 cells. • The elevation of γ-H2AX and 8-OHdG indicates the activation of DNA damage response. • ATM-p53 signaling acts as the DNA damage sensor and effector. • Polychlorinated biphenyl quinone activates NHEJ, BER and NER signalings.

  9. Radiation-Induced Survivin Nuclear Accumulation is Linked to DNA Damage Repair

    SciTech Connect

    Capalbo, Gianni; Weiss, Christian; Reichert, Sebastian; Roedel, Claus

    2010-05-01

    Purpose: Increased expression of survivin has been identified as a negative prognostic marker in a variety of human cancers. We have previously shown that survivin is a radiation-resistance factor and that the therapeutic effect of survivin knock-down might result from an impaired DNA repair capacity. In this study, we aimed to elucidate an interrelationship between survivin's cellular localization and DNA double-strand break repair. Methods and Materials: Survivin's cellular distribution and nuclear complex formation were assayed by Western blotting of subcellular fractions, by immunofluorescence staining, and co-immunoprecipitation in SW480 colorectal cancer cells. DNA repair capacity was analyzed by kinetics of gamma-H2AX foci formation, and by DNA-dependent protein kinase (DNA-PKcs) assays in the presence of survivin-specific or nonspecific control siRNA. Results: Following irradiation, we observed a rapid nuclear accumulation of survivin and subsequent phosphorylation of the protein in the nucleus. Co-immunoprecipitation analyses from nuclear extracts revealed an interaction among survivin, Ku70, gamma-H2AX, MDC1, and DNA-PKcs that was confirmed by immunofluorescence co-localization in nuclear foci. Survivin knock down by siRNA resulted in an impaired DNA double strand break repair, as demonstrated by an increased detection of gamma-H2AX foci/nucleus at 60 min and a higher amount of residual gamma-H2AX foci at 24 hr postirradiation. Furthermore, we detected in survivin-depleted cells a hampered S2056 autophosphorylation of DNA-PKcs and a significantly decreased DNA-PKcs kinase activity. Conclusion: These data indicate that nuclear survivin is linked to DNA double-strand break repair by interaction with members of the DNA double-strand breaks repair machinery, thus regulating DNA-PKcs activity.

  10. Mutagen Sensitivity, Apoptosis, and Polymorphism in DNA Repair as Measures of Prostate Cancer Risk

    DTIC Science & Technology

    2006-02-01

    Cancer 1-1-1998;82(1):168-75. 8. Wood, R. D., Mitchell, M., Sgouros, J., and Lindahl, T. Human DNA Repair Genes. Science 2001;291:1284-1289. 9. Shen...Professor of Oncology and Medicine, Director of Cancer Genetics and Epidemiology Division, and Associate Director for Population Sciences . Dr. Shields...Recurrence After Surgery. Cancer 1-1-1998;82(1):168-75. 8. Wood, R. D., Mitchell, M., Sgouros, J., and Lindahl, T. Human DNA Repair Genes. Science

  11. A mathematical model for intracellular effects of toxins on DNA adduction and repair

    SciTech Connect

    Gaver, D.P.; Jacobs, P.A.; Carpenter, R.L.; Burkhart, J.G.

    1997-01-01

    The processes by which certain classes of toxic compounds or their metabolites may react with DNA to alter the genetic information contained in subsequent generations of cells or organisms are a major component of hazard associated with exposure to chemicals in the environment. Many classes of chemicals may form DNA adducts and there may or may not be a defined mechanism to remove a particular adduct from DNA independent of replication. Many compounds and metabolites that bind DNA also readily bind existing proteins; some classes of toxins and DNA adducts have the capacity to inactive a repair enzyme and divert the repair process competitively. This paper formulates an intracellular dynamic model for one aspect of the action of toxins that form DNA adducts, recognizing a capacity for removal of those adducts by a repair enzyme combined with reaction of the toxin and/or the DNA adduct to inactive the repair enzyme. This particular model illustrates the possible saturation of repair enzyme capacity by the toxin dosage and shows that bistable behavior can occur, with the potential to induce abrupt shifts away from steady-state equilibria. The model suggests that bistable behavior, dose and variation between individuals or tissues may combine under certain conditions to amplify the biological effect of dose observed as DNA adduction and its consequences as mutation. A model recognizing stochastic phenomena also indicates that variation in within-cell toxin concentration may promote jumps between stable equilibria.

  12. Enhanced Genotoxicity of Silver Nanoparticles in DNA Repair Deficient Mammalian Cells

    PubMed Central

    Lim, Hui Kheng; Asharani, P. V.; Hande, M. Prakash

    2012-01-01

    Silver nanoparticles (Ag-np) have been used in medicine and commercially due to their anti-microbial properties. Therapeutic potentials of these nanoparticles are being explored extensively despite the lack of information on their mechanism of action at molecular and cellular level. Here, we have investigated the DNA damage response and repair following Ag-np treatment in mammalian cells. Studies have shown that Ag-np exerts genotoxicity through double-strand breaks (DSBs). DNA-PKcs, the catalytic subunit of DNA dependent protein kinase, is an important caretaker of the genome which is known to be the main player mediating Non-homologous End-Joining (NHEJ) repair pathway. We hypothesize that DNA-PKcs is responsible for the repair of Ag-np induced DNA damage. In vitro studies have been carried out to investigate both cytotoxicity and genotoxicity induced by Ag-np in normal human cells, DNA-PKcs proficient, and deficient mammalian cells. Chemical inhibition of DNA-PKcs activity with NU7026, an ATP-competitive inhibitor of DNA-PKcs, has been performed to further validate the role of DNA-PKcs in this model. Our results suggest that Ag-np induced more prominent dose-dependent decrease in cell viability in DNA-PKcs deficient or inhibited cells. The deficiency or inhibition of DNA-PKcs renders the cells with higher susceptibility to DNA damage and genome instability which in turn contributed to greater cell cycle arrest/cell death. These findings support the fact that DNA-PKcs is involved in the repair of Ag-np induced genotoxicity and NHEJ repair pathway and DNA-PKcs particularly is activated to safeguard the genome upon Ag-np exposure. PMID:22707954

  13. Enhanced genotoxicity of silver nanoparticles in DNA repair deficient Mammalian cells.

    PubMed

    Lim, Hui Kheng; Asharani, P V; Hande, M Prakash

    2012-01-01

    Silver nanoparticles (Ag-np) have been used in medicine and commercially due to their anti-microbial properties. Therapeutic potentials of these nanoparticles are being explored extensively despite the lack of information on their mechanism of action at molecular and cellular level. Here, we have investigated the DNA damage response and repair following Ag-np treatment in mammalian cells. Studies have shown that Ag-np exerts genotoxicity through double-strand breaks (DSBs). DNA-PKcs, the catalytic subunit of DNA dependent protein kinase, is an important caretaker of the genome which is known to be the main player mediating Non-homologous End-Joining (NHEJ) repair pathway. We hypothesize that DNA-PKcs is responsible for the repair of Ag-np induced DNA damage. In vitro studies have been carried out to investigate both cytotoxicity and genotoxicity induced by Ag-np in normal human cells, DNA-PKcs proficient, and deficient mammalian cells. Chemical inhibition of DNA-PKcs activity with NU7026, an ATP-competitive inhibitor of DNA-PKcs, has been performed to further validate the role of DNA-PKcs in this model. Our results suggest that Ag-np induced more prominent dose-dependent decrease in cell viability in DNA-PKcs deficient or inhibited cells. The deficiency or inhibition of DNA-PKcs renders the cells with higher susceptibility to DNA damage and genome instability which in turn contributed to greater cell cycle arrest/cell death. These findings support the fact that DNA-PKcs is involved in the repair of Ag-np induced genotoxicity and NHEJ repair pathway and DNA-PKcs particularly is activated to safeguard the genome upon Ag-np exposure.

  14. Repair of nonreplicating UV-irradiated DNA: cooperative dark repair by Escherichia coli uvr and phr functions

    SciTech Connect

    Hays, J.B.; Martin, S.J.; Bhatia, K.

    1985-02-01

    The system previously used to study recombination of nonreplicating UV-irradiated phage lambda DNA was adapted to study UV repair. Irradiated phages infected undamaged homoimmune lysogens. Pyrimidine dimer content (by treatment with Micrococcus luteus UV endonuclease and alkaline sucrose sedimentation) and a biological activity endpoint (infectivity in transfection of uvrB recA recB spheroplasts) were followed. Unless room light was excluded during DNA extraction procedures, photoreactivation (Phr function) was significant. In uvr ..delta..phr bacteria, repair, by both assays, was very low but not zero. Even when light was totally excluded, Phr function appeared to play a role in Uvr-mediated excision repair: both dimer removal and restoration of infectivity were two to five times as efficient in uvr/sup +/ phr/sup +/ bacteria as in uvr/sup +/ ..delta..phr bacteria. Similarly, UV-irradiated phages plated with higher efficiencies on phr/sup +/ than ..delta..phr bacteria even under totally dark conditions. In uvr phr/sup +/ repressed infections, removal of dimers from nonreplicating DNA did not increase infectivity as much as in uvr2= infections, suggesting a requirement for repair of nondimer photoproducts by the uvrABC system.

  15. DSB (Im)mobility and DNA repair compartmentalization in mammalian cells.

    PubMed

    Lemaître, Charlène; Soutoglou, Evi

    2015-02-13

    Chromosomal translocations are considered as causal in approximately 20% of cancers. Therefore, understanding their mechanisms of formation is crucial in the prevention of carcinogenesis. The first step of translocation formation is the concomitant occurrence of double-strand DNA breaks (DSBs) in two different chromosomes. DSBs can be repaired by different repair mechanisms, including error-free homologous recombination (HR), potentially error-prone non-homologous end joining (NHEJ) and the highly mutagenic alternative end joining (alt-EJ) pathways. Regulation of DNA repair pathway choice is crucial to avoid genomic instability. In yeast, DSBs are mobile and can scan the entire nucleus to be repaired in specialized DNA repair centers or if they are persistent, in order to associate with the nuclear pores or the nuclear envelope where they can be repaired by specialized repair pathways. DSB mobility is limited in mammals; therefore, raising the question of whether the position at which a DSB occurs influences its repair. Here, we review the recent literature addressing this question. We first present the reports describing the extent of DSB mobility in mammalian cells. In a second part, we discuss the consequences of non-random gene positioning on chromosomal translocations formation. In the third part, we discuss the mobility of heterochromatic DSBs in light of our recent data on DSB repair at the nuclear lamina, and finally, we show that DSB repair compartmentalization at the nuclear periphery is conserved from yeast to mammals, further pointing to a role for gene positioning in the outcome of DSB repair. When regarded as a whole, the different studies reviewed here demonstrate the importance of nuclear architecture on DSB repair and reveal gene positioning as an important parameter in the study of tumorigenesis.

  16. Network-based characterization and prediction of human DNA repair genes and pathways

    PubMed Central

    Li, Yan-Hui; Zhang, Gai-Gai

    2017-01-01

    Network biology is a useful strategy to understand cell’s functional organization. In this study, for the first time, we successfully introduced network approaches to study properties of human DNA repair genes. Compared with non-DNA repair genes, we found distinguishing features for DNA repair genes: (i) they tend to have higher degrees; (ii) they tend to be located at global network center; (iii) they tend to interact directly with each other. Based on these features, we developed the first algorithm to predict new DNA repair genes. We tested several machine-learning models and found that support vector machine with kernel function of radial basis function (RBF) achieve the best performance, with precision = 0.74 and area under curve (AUC) = 0.96. In the end, we applied the algorithm to predict new DNA repair genes and got 32 new candidates. Literature supporting four of the predictions was found. We believe the network approaches introduced here might open a new avenue to understand DNA repair genes and pathways. The suggested algorithm and the predicted genes might be helpful for scientists in the field. PMID:28368026

  17. Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase

    PubMed Central

    Hellman, Lance M.; Spear, Tyler J.; Koontz, Colton J.; Melikishvili, Manana; Fried, Michael G.

    2014-01-01

    O6-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O6-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O6-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the ∼5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs. PMID:25080506

  18. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair.

    PubMed

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott; Scheibye-Knudsen, Morten; Desler, Claus; Hickson, Ian D; Bohr, Vilhelm A

    2014-04-01

    Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy.

  19. Kaempferol induces DNA damage and inhibits DNA repair associated protein expressions in human promyelocytic leukemia HL-60 cells.

    PubMed

    Wu, Lung-Yuan; Lu, Hsu-Feng; Chou, Yu-Cheng; Shih, Yung-Luen; Bau, Da-Tian; Chen, Jaw-Chyun; Hsu, Shu-Chun; Chung, Jing-Gung

    2015-01-01

    Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60

  20. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease.

    PubMed

    Dupuy, Aurélie; Sarasin, Alain

    2015-06-01

    Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

  1. Mechanistic Modelling of DNA Repair and Cellular Survival Following Radiation-Induced DNA Damage

    NASA Astrophysics Data System (ADS)

    McMahon, Stephen J.; Schuemann, Jan; Paganetti, Harald; Prise, Kevin M.

    2016-09-01

    Characterising and predicting the effects of ionising radiation on cells remains challenging, with the lack of robust models of the underlying mechanism of radiation responses providing a significant limitation to the development of personalised radiotherapy. In this paper we present a mechanistic model of cellular response to radiation that incorporates the kinetics of different DNA repair processes, the spatial distribution of double strand breaks and the resulting probability and severity of misrepair. This model enables predictions to be made of a range of key biological endpoints (DNA repair kinetics, chromosome aberration and mutation formation, survival) across a range of cell types based on a set of 11 mechanistic fitting parameters that are common across all cells. Applying this model to cellular survival showed its capacity to stratify the radiosensitivity of cells based on aspects of their phenotype and experimental conditions such as cell cycle phase and plating delay (correlation between modelled and observed Mean Inactivation Doses R2 > 0.9). By explicitly incorporating underlying mechanistic factors, this model can integrate knowledge from a wide range of biological studies to provide robust predictions and may act as a foundation for future calculations of individualised radiosensitivity.

  2. DNA damage, repair monitoring and epigenetic DNA methylation changes in seedlings of Chernobyl soybeans.

    PubMed

    Georgieva, Mariyana; Rashydov, Namik M; Hajduch, Martin

    2017-02-01

    This pilot study was carried out to assess the effect of radio-contaminated Chernobyl environment on plant genome integrity 27 years after the accident. For this purpose, nuclei were isolated from root tips of the soybean seedlings harvested from plants grown in the Chernobyl area for seven generations. Neutral, neutral-alkaline, and methylation-sensitive comet assays were performed to evaluate the induction and repair of primary DNA damage and the epigenetic contribution to stress adaptation mechanisms. An increased level of single and double strand breaks in the radio-contaminated Chernobyl seedlings at the stage of primary root development was detected in comparison to the controls. However, the kinetics of the recovery of DNA breaks of radio-contaminated Chernobyl samples revealed that lesions were efficiently repaired at the stage of cotyledon. Methylation-sensitive comet assay revealed comparable levels in the CCGG methylation pattern between control and radio-contaminated samples with a slight increase of approximately 10% in the latter ones. The obtained preliminary data allow us to speculate about the onset of mechanisms providing an adaptation potential to the accumulated internal irradiation after the Chernobyl accident. Despite the limitations of this study, we showed that comet assay is a sensitive and flexible technique which can be efficiently used for genotoxic screening of plant specimens in natural and human-made radio-contaminated areas, as well as for safety monitoring of agricultural products.

  3. Real-time observation of DNA repair: 2-aminopurine as a molecular probe

    NASA Astrophysics Data System (ADS)

    Krishnan, Rajagopal; Butcher, Christina E.; Oh, Dennis H.

    2008-02-01

    Triplex forming oligos (TFOs) that target psoralen photoadducts to specific DNA sequences have generated interest as a potential agent in gene therapy. TFOs also offer an opportunity to study the mechanism of DNA repair in detail. In an effort to understand the mechanism of DNA repair at a specific DNA sequence in real-time, we have designed a plasmid containing a psoralen reaction site adjacent to a TFO binding site corresponding to a sequence within the human interstitial collagenase gene. Two 2-aminopurine residues incorporated into the purine-rich strand of the TFO binding site and located within six nucleotides of the psoralen reaction site serve as molecular probes for excision repair events involving the psoralen photoadducts on that DNA strand. In duplex DNA, the 2-aminopurine fluorescence is quenched. However, upon thermal or formamide-induced denaturation of duplex DNA to single stranded DNA, the 2-aminopurine fluorescence increases by eight fold. These results suggest that monitoring 2-aminopurine fluorescence from plasmids damaged by psoralen TFOs may be a method for measuring excision of single-stranded damaged DNA from the plasmid in cells. A fluorescence-based molecular probe to the plasmid may significantly simplify the real-time observation of DNA repair in both populations of cells as well as single cells.

  4. Psoralen plus near-ultraviolet light: a possible new method for measuring DNA repair synthesis

    SciTech Connect

    Heimer, Y.M.; Kol, R.; Shiloh, Y.; Riklis, E.

    1983-09-01

    A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with ..gamma.. rays and methyl methanesulfonate. The advantages of the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited.

  5. Psoralen plus near-ultraviolet light: a possible new method for measuring DNA repair synthesis

    SciTech Connect

    Heimer, Y.M.; Kol, R.; Shiloh, Y.; Riklis, E.

    1983-09-01

    A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with gamma rays and methyl methanesulfonate. The advantages of the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited.

  6. Genetic analysis of DNA repair in the hyperthermophilic archaeon, Thermococcus kodakaraensis.

    PubMed

    Fujikane, Ryosuke; Ishino, Sonoko; Ishino, Yoshizumi; Forterre, Patrick

    2010-01-01

    Extensive biochemical and structural analyses have been performed on the putative DNA repair proteins of hyperthermophilic archaea, in contrast to the few genetic analyses of the genes encoding these proteins. Accordingly, little is known about the repair pathways used by archaeal cells at high temperature. Here, we attempted to disrupt the genes encoding the potential repair proteins in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis. We succeeded in isolating null mutants of the hjc, hef, hjm, xpb, and xpd genes, but not the radA, rad50, mre11, herA, nurA, and xpg/fen1 genes. Phenotypic analyses of the gene-disrupted strains showed that the xpb and xpd null mutants are only slightly sensitive to ultraviolet (UV) irradiation, methyl methanesulfonate (MMS) and mitomycin C (MMC), as compared with the wild-type strain. The hjm null mutant showed sensitivity specifically to mitomycin C. On the other hand, the null mutants of the hjc gene lacked increasing sensitivity to any type of DNA damage. The Hef protein is particularly important for maintaining genome homeostasis, by functioning in the repair of a wide variety of DNA damage in T. kodakaraensis cells. Deletion of the entire hef gene or of the segments encoding either its nuclease or helicase domain produced similar phenotypes. The high sensitivity of the Δhef mutants to MMC suggests that Hef performs a critical function in the repair process of DNA interstrand cross-links. These damage-sensitivity profiles suggest that the archaeal DNA repair system has processes depending on repair-related proteins different from those of eukaryotic and bacterial DNA repair systems using homologous repair proteins analyzed here.

  7. Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia.

    PubMed

    Marmignon, Antoine; Bischerour, Julien; Silve, Aude; Fojcik, Clémentine; Dubois, Emeline; Arnaiz, Olivier; Kapusta, Aurélie; Malinsky, Sophie; Bétermier, Mireille

    2014-08-01

    During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage

  8. Ku-Mediated Coupling of DNA Cleavage and Repair during Programmed Genome Rearrangements in the Ciliate Paramecium tetraurelia

    PubMed Central

    Marmignon, Antoine; Bischerour, Julien; Silve, Aude; Fojcik, Clémentine; Dubois, Emeline; Arnaiz, Olivier; Kapusta, Aurélie; Malinsky, Sophie; Bétermier, Mireille

    2014-01-01

    During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage

  9. Genetic characterization of cells of homocystinuria patients with disrupted DNA repair system

    SciTech Connect

    Sinel'shchikova, T.A.; L'vova, G.N.; Shoniya, N.N.; Zasukhina, G.D.

    1986-08-01

    Fibroblasts obtained from biopsy material and lymphocytes of patients with homocystinuria were investigated for repair activity according to the following criteria: rejoined DNA breaks, induced by 4-nitroquinoline-1-oxide and ..gamma..-radiation; indices of reactivation and induced mutagenesis of smallpox vaccine virus treated with these mutagens. In lymphocytes a defect of DNA repair was observed according to all criteria investigated. During passage of fibroblast cultures, inhibition of repair activity of cells was preserved according to ..gamma..-type. Increase in the number of spontaneous and ..gamma..-induced mutations of virus was noted according to degree of passage of fibroblasts.

  10. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

    PubMed Central

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø.; Rizzo, Carmelo J.; Guengerich, F. Peter; Tudek, Barbara

    2015-01-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno (ε)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ε-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ε-adducts, 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC) and 1,N2-ethenoguanine (1,N2-εG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ε-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed εA and εC from ds and ssDNA but were inactive toward 1,N2-εG. SC-1A repaired only εA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ε-adducts in dsDNA, while only εA and εC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only εC in ssDNA Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N2-εG and that ALKBH3 removes only εC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. PMID:25797601

  11. Human immunodeficiency virus type 1 Vpr protein binds to the uracil DNA glycosylase DNA repair enzyme.

    PubMed Central

    Bouhamdan, M; Benichou, S; Rey, F; Navarro, J M; Agostini, I; Spire, B; Camonis, J; Slupphaug, G; Vigne, R; Benarous, R; Sire, J

    1996-01-01

    The role of the accessory gene product Vpr during human immunodeficiency virus type 1 infection remains unclear. We have used the yeast two-hybrid system to identify cellular proteins that interact with Vpr and could be involved in its function. A cDNA clone which encodes the human uracil DNA glycosylase (UNG), a DNA repair enzyme involved in removal of uracil in DNA, has been isolated. Interaction between Vpr and UNG has been demonstrated by in vitro protein-protein binding assays using translated, radiolabeled Vpr and UNG recombinant proteins expressed as a glutathione S-transferase fusion protein. Conversely, purified UNG has been demonstrated to interact with Vpr recombinant protein expressed as a glutathione S-transferase fusion protein. Coimmunoprecipitation experiments confirmed that Vpr and UNG are associated within cells expressing Vpr. By using a panel of C- and N-terminally deleted Vpr mutants, we have determined that the core protein of Vpr, spanning amino acids 15 to 77, is involved in the interaction with UNG. We also demonstrate by in vitro experiments that the enzymatic activity of UNG is retained upon interaction with Vpr. PMID:8551605

  12. STRUCTURE OF THE DNA REPAIR HELICASE HEL308 REVEALS DNA BINDING AND AUTOINHIBITORY DOMAINS

    PubMed Central

    Richards, Jodi; Johnson, Ken; Liu, Huanting; Oke, Stephen McMahon. Muse; Carter, Lester; Naismith, James H; White, Malcolm F

    2012-01-01

    Hel308 is a superfamily 2 helicase conserved in eukaryotes and archaea. It is thought to function in the early stages of recombination following replication fork arrest, and has a specificity for removal of the lagging strand in model replication forks. A homologous helicase constitutes the N-terminal domain of human DNA polymerase Q. The Drosophila homologue mus301 is implicated in double strand break repair and meiotic recombination. We have solved the high-resolution crystal structure of Hel308 from the crenarchaeon Sulfolobus solfataricus, revealing a five-domain structure with a central pore lined with essential DNA binding residues. The fifth domain is shown to act as a molecular brake, clamping the ssDNA extruded through the central pore of the helicase structure to limit the enzyme’s helicase activity. This provides an elegant mechanism to tune the enzyme’s processivity to its functional role. Hel308 can displace streptavidin from a biotinylated DNA molecule, suggesting that one function of the enzyme may be in the removal of bound proteins at stalled replication forks and recombination intermediates. PMID:18056710

  13. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    PubMed

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1.

  14. The molecular origin of high DNA-repair efficiency by photolyase

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

    The primary dynamics in photomachinery such as charge separation in photosynthesis and bond isomerization in sensory photoreceptor are typically ultrafast to accelerate functional dynamics and avoid energy dissipation. The same is also true for the DNA repair enzyme, photolyase. However, it is not known how the photoinduced step is optimized in photolyase to attain maximum efficiency. Here, we analyse the primary reaction steps of repair of ultraviolet-damaged DNA by photolyase using femtosecond spectroscopy. With systematic mutations of the amino acids involved in binding of the flavin cofactor and the cyclobutane pyrimidine dimer substrate, we report our direct deconvolution of the catalytic dynamics with three electron-transfer and two bond-breaking elementary steps and thus the fine tuning of the biological repair function for optimal efficiency. We found that the maximum repair efficiency is not enhanced by the ultrafast photoinduced process but achieved by the synergistic optimization of all steps in the complex repair reaction.

  15. Regulation of DNA-end resection by hnRNPU-like proteins promotes DNA double-strand break signaling and repair.

    PubMed

    Polo, Sophie E; Blackford, Andrew N; Chapman, J Ross; Baskcomb, Linda; Gravel, Serge; Rusch, Andre; Thomas, Anoushka; Blundred, Rachel; Smith, Philippa; Kzhyshkowska, Julia; Dobner, Thomas; Taylor, A Malcolm R; Turnell, Andrew S; Stewart, Grant S; Grand, Roger J; Jackson, Stephen P

    2012-02-24

    DNA double-strand break (DSB) signaling and repair are critical for cell viability, and rely on highly coordinated pathways whose molecular organization is still incompletely understood. Here, we show that heterogeneous nuclear ribonucleoprotein U-like (hnRNPUL) proteins 1 and 2 play key roles in cellular responses to DSBs. We identify human hnRNPUL1 and -2 as binding partners for the DSB sensor complex MRE11-RAD50-NBS1 (MRN) and demonstrate that hnRNPUL1 and -2 are recruited to DNA damage in an interdependent manner that requires MRN. Moreover, we show that hnRNPUL1 and -2 stimulate DNA-end resection and promote ATR-dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents. Finally, we establish that hnRNPUL1 and -2 function downstream of MRN and CtBP-interacting protein (CtIP) to promote recruitment of the BLM helicase to DNA breaks. Collectively, these results provide insights into how mammalian cells respond to DSBs.

  16. Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines.

    PubMed

    Yamamoto, Kimiyo N; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P; Witt, Kristine L; Tice, Raymond R

    2011-08-01

    Included among the quantitative high throughput screens (qHTS) conducted in support of the US Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in seven isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis.

  17. BCL10 is recruited to sites of DNA damage to facilitate DNA double-strand break repair

    PubMed Central

    Ismail, Ismail Hassan; Dronyk, Ashley; Hu, Xiuying; Hendzel, Michael J.; Shaw, Andrew R.

    2016-01-01

    ABSTRACT Recent studies have found BCL10 can localize to the nucleus and that this is linked to tumor aggression and poorer prognosis. These studies suggest that BCL10 localization plays a novel role in the nucleus that may contribute to cellular transformation and carcinogenesis. In this study, we show that BCL10 functions as part of the DNA damage response (DDR). We found that BCL10 facilitates the rapid recruitment of RPA, BRCA1 and RAD51 to sites of DNA damage. Furthermore, we also found that ATM phosphorylates BCL10 in response to DNA damage. Functionally, BCL10 promoted DNA double-strand breaks repair, enhancing cell survival after DNA damage. Taken together our results suggest a novel role for BCL10 in the repair of DNA lesions. PMID:26771713

  18. Estimating the effect of human base excision repair protein variants on the repair of oxidative DNA base damage.

    PubMed

    Sokhansanj, Bahrad A; Wilson, David M

    2006-05-01

    Epidemiologic studies have revealed a complex association between human genetic variance and cancer risk. Quantitative biological modeling based on experimental data can play a critical role in interpreting the effect of genetic variation on biochemical pathways relevant to cancer development and progression. Defects in human DNA base excision repair (BER) proteins can reduce cellular tolerance to oxidative DNA base damage caused by endogenous and exogenous sources, such as exposure to toxins and ionizing radiation. If not repaired, DNA base damage leads to cell dysfunction and mutagenesis, consequently leading to cancer, disease, and aging. Population screens have identified numerous single-nucleotide polymorphism variants in many BER proteins and some have been purified and found to exhibit mild kinetic defects. Epidemiologic studies have led to conflicting conclusions on the association between single-nucleotide polymorphism variants in BER proteins and cancer risk. Using experimental data for cellular concentration and the kinetics of normal and variant BER proteins, we apply a previously developed and tested human BER pathway model to (i) estimate the effect of mild variants on BER of abasic sites and 8-oxoguanine, a prominent oxidative DNA base modification, (ii) identify ranges of variation associated with substantial BER capacity loss, and (iii) reveal nonintuitive consequences of multiple simultaneous variants. Our findings support previous work suggesting that mild BER variants have a minimal effect on pathway capacity whereas more severe defects and simultaneous variation in several BER proteins can lead to inefficient repair and potentially deleterious consequences of cellular damage.

  19. DNA Damage and Repair in Schizophrenia and Autism: Implications for Cancer Comorbidity and Beyond

    PubMed Central

    Markkanen, Enni; Meyer, Urs; Dianov, Grigory L.

    2016-01-01

    Schizophrenia and autism spectrum disorder (ASD) are multi-factorial and multi-symptomatic psychiatric disorders, each affecting 0.5%–1% of the population worldwide. Both are characterized by impairments in cognitive functions, emotions and behaviour, and they undermine basic human processes of perception and judgment. Despite decades of extensive research, the aetiologies of schizophrenia and ASD are still poorly understood and remain a significant challenge to clinicians and scientists alike. Adding to this unsatisfactory situation, patients with schizophrenia or ASD often develop a variety of peripheral and systemic disturbances, one prominent example of which is cancer, which shows a direct (but sometimes inverse) comorbidity in people affected with schizophrenia and ASD. Cancer is a disease characterized by uncontrolled proliferation of cells, the molecular origin of which derives from mutations of a cell’s DNA sequence. To counteract such mutations and repair damaged DNA, cells are equipped with intricate DNA repair pathways. Oxidative stress, oxidative DNA damage, and deficient repair of oxidative DNA lesions repair have been proposed to contribute to the development of schizophrenia and ASD. In this article, we summarize the current evidence of cancer comorbidity in these brain disorders and discuss the putative roles of oxidative stress, DNA damage and DNA repair in the aetiopathology of schizophrenia and ASD. PMID:27258260

  20. DNA Damage and Repair in Schizophrenia and Autism: Implications for Cancer Comorbidity and Beyond.

    PubMed

    Markkanen, Enni; Meyer, Urs; Dianov, Grigory L

    2016-06-01

    Schizophrenia and autism spectrum disorder (ASD) are multi-factorial and multi-symptomatic psychiatric disorders, each affecting 0.5%-1% of the population worldwide. Both are characterized by impairments in cognitive functions, emotions and behaviour, and they undermine basic human processes of perception and judgment. Despite decades of extensive research, the aetiologies of schizophrenia and ASD are still poorly understood and remain a significant challenge to clinicians and scientists alike. Adding to this unsatisfactory situation, patients with schizophrenia or ASD often develop a variety of peripheral and systemic disturbances, one prominent example of which is cancer, which shows a direct (but sometimes inverse) comorbidity in people affected with schizophrenia and ASD. Cancer is a disease characterized by uncontrolled proliferation of cells, the molecular origin of which derives from mutations of a cell's DNA sequence. To counteract such mutations and repair damaged DNA, cells are equipped with intricate DNA repair pathways. Oxidative stress, oxidative DNA damage, and deficient repair of oxidative DNA lesions repair have been proposed to contribute to the development of schizophrenia and ASD. In this article, we summarize the current evidence of cancer comorbidity in these brain disorders and discuss the putative roles of oxidative stress, DNA damage and DNA repair in the aetiopathology of schizophrenia and ASD.

  1. Transient RNA-DNA Hybrids Are Required for Efficient Double-Strand Break Repair.

    PubMed

    Ohle, Corina; Tesorero, Rafael; Schermann, Géza; Dobrev, Nikolay; Sinning, Irmgard; Fischer, Tamás

    2016-11-03

    RNA-DNA hybrids are a major internal cause of DNA damage within cells, and their degradation by RNase H enzymes is important for maintaining genomic stability. Here, we identified an unexpected role for RNA-DNA hybrids and RNase H enzymes in DNA repair. Using a site-specific DNA double-strand break (DSB) system in Schizosaccharomyces pombe, we showed that RNA-DNA hybrids form as part of the homologous-recombination (HR)-mediated DSB repair process and that RNase H enzymes are essential for their degradation and efficient completion of DNA repair. Deleting RNase H stabilizes RNA-DNA hybrids around DSB sites and strongly impairs recruitment of the ssDNA-binding RPA complex. In contrast, overexpressing RNase H1 destabilizes these hybrids, leading to excessive strand resection and RPA recruitment and to severe loss of repeat regions around DSBs. Our study challenges the existing model of HR-mediated DSB repair and reveals a surprising role for RNA-DNA hybrids in maintaining genomic stability.

  2. Protein phosphatase 5 is necessary for ATR-mediated DNA repair

    SciTech Connect

    Kang, Yoonsung; Cheong, Hyang-Min; Lee, Jung-Hee; Song, Peter I.; Lee, Kwang-Ho; Kim, Sang-Yong; Jun, Jae Yeoul; You, Ho Jin

    2011-01-07

    Research highlights: {yields} Serine/threonine protein phosphatase 5 (PP5) has been shown to participate in ataxia telangiectasia-mutated (ATM)- and ATR (ATM- and Rad3-related)-mediated checkpoint pathways, which plays an important role in the DNA damage response and maintenance of genomic stability. {yields} However, it is not clear exactly how PP5 participates in this process. {yields} Our results indicate that PP5 is more closely related with ATR-mediated pathway than ATM-mediated pathway in DNA damage repair. -- Abstract: Several recent studies have shown that protein phosphatase 5 (PP5) participates in cell cycle arrest after DNA damage, but its roles in DNA repair have not yet been fully characterized. We investigated the roles of PP5 in the repair of ultraviolet (UV)- and neocarzinostatin (NCS)-induced DNA damage. The results of comet assays revealed different repair patterns in UV- and NCS-exposed U2OS-PS cells. PP5 is only essential for Rad3-related (ATR)-mediated DNA repair. Furthermore, the phosphorylation of 53BP1 and BRCA1, important mediators of DNA damage repair, and substrates of ATR and ATM decreased in U2OS-PS cells exposed to UV radiation. In contrast, the cell cycle arrest proteins p53, CHK1, and CHK2 were normally phosphorylated in U2OS and U2OS-PS cells exposed to UV radiation or treated with NCS. In view of these results, we suggest that PP5 plays a crucial role in ATR-mediated repair of UV-induced DNA damage.

  3. Germline Stem Cell Gene PIWIL2 Mediates DNA Repair through Relaxation of Chromatin

    PubMed Central

    Yin, De-Tao; Wang, Qien; Chen, Li; Liu, Meng-Yao; Han, Chunhua; Yan, Qingtao; Shen, Rulong; He, Gang; Duan, Wenrui; Li, Jian-Jian; Wani, Altaf; Gao, Jian-Xin

    2011-01-01

    DNA damage response (DDR) is an intrinsic barrier of cell to tumorigenesis initiated by genotoxic agents. However, the mechanisms underlying the DDR are not completely understood despite of extensive investigation. Recently, we have reported that ectopic expression of germline stem cell gene PIWIL2 is associated with tumor stem cell development, although the underlying mechanisms are largely unknown. Here we show that PIWIL2 is required for the repair of DNA-damage induced by various types of genotoxic agents. Upon ultraviolet (UV) irradiation, silenced PIWIL2 gene in normal human fibroblasts was transiently activated after treatment with UV light. This activation was associated with DNA repair, because Piwil2-deficienct mouse embryonic fibroblasts (mili-/- MEFs) were defective in cyclobutane pyrimidine dimers (CPD) repair after UV treatment. As a result, the UV-treated mili-/- MEFs were more susceptible to apoptosis, as characterized by increased levels of DNA damage-associated apoptotic proteins, such as active caspase-3, cleaved Poly (ADP-ribose) polymerase (PARP) and Bik. The impaired DNA repair in the mili-/- MEFs was associated with the reductions of histone H3 acetylation and chromatin relaxation, although the DDR pathway downstream chromatin relaxation appeared not to be directly affected by Piwil2. Moreover, guanine–guanine (Pt-[GG]) and double strand break (DSB) repair were also defective in the mili-/- MEFs treated by genotoxic chemicals Cisplatin and ionizing radiation (IR), respectively. The results indicate that Piwil2 can mediate DNA repair through an axis of Piwil2 → histone acetylation → chromatin relaxation upstream DDR pathways. The findings reveal a new role for Piwil2 in DNA repair and suggest that Piwil2 may act as a gatekeeper against DNA damage-mediated tumorigenesis. PMID:22110608

  4. Fibroblast growth factor type 2 signaling is critical for DNA repair in human keratinocyte stem cells.

    PubMed

    Harfouche, Ghida; Vaigot, Pierre; Rachidi, Walid; Rigaud, Odile; Moratille, Sandra; Marie, Mélanie; Lemaitre, Gilles; Fortunel, Nicolas O; Martin, Michèle T

    2010-09-01

    Tissue stem cells must be endowed with superior maintenance and repair systems to ensure genomic stability over multiple generations, which would be less necessary in more differentiated cells. We previously reported that human keratinocyte stem cells were more resistant to ionizing radiation toxicity than their direct progeny, the keratinocyte progenitor cells. In the present study we addressed the mechanisms underlying this difference. Investigations of DNA repair showed that both single and double DNA strand breaks were repaired more rapidly and more efficiently in stem cells than in progenitors. As cell signaling is a key regulatory step in the management of DNA damage, a gene profiling study was performed. Data revealed that several genes of the fibroblast growth factor type 2 (FGF2) signaling pathway were induced by DNA damage in stem cells and not in progenitors. Furthermore, an increased content of the FGF2 protein was found in irradiated stem cells, both for the secreted and the cellular forms of the protein. To examine the role of endogenous FGF2 in DNA repair, stem cells were exposed to FGF2 pathway inhibitors. Blocking the FGF2 receptor (FGF receptor 1) or the kinase (Ras-mitogen-activated protein kinase 1) resulted in a inhibition of single and double DNA strand-break repair in the keratinocyte stem cells. Moreover, supplementing the progenitor cells with exogenous FGF2 activated their DNA repair. We propose that, apart from its well-known role as a strong mitogen and prosurvival factor, FGF2 helps to maintain genomic integrity in stem cells by activating stress-induced DNA repair.

  5. Role of ATP in UV-induced DNA excision repair in human cells

    SciTech Connect

    Dresler, S.L.

    1986-05-01

    In permeable human fibroblasts, UV-induced DNA excision repair is dependent on ATP, with a K/sub m/ of approximately 1 mM. Omission of ATP from the reaction mix completely inhibits damage-specific incision of DNA, but has little effect on repair patch synthesis proceeding from previously incised sites. UV-induced excision repair in permeable xeroderma pigmentosum (XP) cells complemented with T4 UV endonuclease is also totally dependent on ATP. Because the T4 enzyme is not ATP-dependent, ATP must be required for an endogenous activity other than the incision of damaged DNA. Alkaline elution reveals that, in the absence of ATP, T4 UV endonuclease does incise the DNA of permeable UV-irradiated XP cells, but that the incision rate is stimulated approximately 2-fold by the addition of ATP. This 2-fold stimulation of incision can not, however, be responsible for the absolute ATP dependence of excision repair in UV endonuclease-complemented XP cells. Apparently, although T4 UV endonuclease can incise damaged nuclear DNA in the absence of ATP, the incised sites must also be altered in an ATP-dependent reaction before subsequent steps of the repair process can proceed. This conclusion, coupled with the fact that ATP stimulates incision of damaged nuclear DNA by T4 UV endonuclease and is absolutely required for incision of damaged nuclear DNA by the endogenous human UV endonuclease, suggests that an important function of the early ATP-dependent step in UV-induced excision repair is to make damaged sites in DNA accessible to repair enzymes.

  6. Genetic variants of the DNA repair genes from Exome Aggregation Consortium (EXAC) database: significance in cancer.

    PubMed

    Das, Raima; Ghosh, Sankar Kumar

    2017-04-01

    DNA repair pathway is a primary defense system that eliminates wide varieties of DNA damage. Any deficiencies in them are likely to cause the chromosomal instability that leads to cell malfunctioning and tumorigenesis. Genetic polymorphisms in DNA repair genes have demonstrated a significant association with cancer risk. Our study attempts to give a glimpse of the overall scenario of the germline polymorphisms in the DNA repair genes by taking into account of the Exome Aggregation Consortium (ExAC) database as well as the Human Gene Mutation Database (HGMD) for evaluating the disease link, particularly in cancer. It has been found that ExAC DNA repair dataset (which consists of 228 DNA repair genes) comprises 30.4% missense, 12.5% dbSNP reported and 3.2% ClinVar significant variants. 27% of all the missense variants has the deleterious SIFT score of 0.00 and 6% variants carrying the most damaging Polyphen-2 score of 1.00, thus affecting the protein structure and function. However, as per HGMD, only a fraction (1.2%) of ExAC DNA repair variants was found to be cancer-related, indicating remaining variants reported in both the databases to be further analyzed. This, in turn, may provide an increased spectrum of the reported cancer linked variants in the DNA repair genes present in ExAC database. Moreover, further in silico functional assay of the identified vital cancer-associated variants, which is essential to get their actual biological significance, may shed some lights in the field of targeted drug development in near future.

  7. Cell transcriptional state alters genomic patterns of DNA double-strand break repair in human astrocytes.

    PubMed

    Yong, Raymund L; Yang, Chunzhang; Lu, Jie; Wang, Huaien; Schlaff, Cody D; Tandle, Anita; Graves, Christian A; Elkahloun, Abdel G; Chen, Xiaoyuan; Zhuang, Zhengping; Lonser, Russell R

    2014-12-17

    The misrepair of DNA double-strand breaks in close spatial proximity within the nucleus can result in chromosomal rearrangements that are important in the pathogenesis of haematopoietic and solid malignancies. It is unknown why certain epigenetic states, such as those found in stem or progenitor cells, appear to facilitate neoplastic transformation. Here we show that altering the transcriptional state of human astrocytes alters patterns of DNA damage repair from ionizing radiation at a gene locus-specific and genome-wide level. Astrocytes induced into a reactive state exhibit increased DNA repair, compared with non-reactive cells, in actively transcribed chromatin after irradiation. In mapping these repair sites, we identify misrepair events and repair hotspots that are unique to each state. The precise characterization of genomic regions susceptible to mutation in specific transcriptional states provides new opportunities for addressing clonal evolution in solid cancers, in particular those where double-strand break induction is a cornerstone of clinical intervention.

  8. Pulsed-field gel electrophoresis analysis of multicellular DNA double-strand break damage and repair.

    PubMed

    Joshi, Nina; Grant, Stephen G

    2014-01-01

    This assay quantifies the extent of double-strand break (DSB) DNA damage in cell populations embedded in agarose and analyzed for migratory DNA using pulsed-field gel electrophoresis with ethidium bromide staining. The assay can measure preexisting damage as well as induction of DSB by chemical (e.g., bleomycin), physical (e.g., X-irradiation), or biological (e.g., restriction enzymes) agents. By incubating the cells under physiological conditions prior to processing, the cells can be allowed to repair DSB, primarily via the process of nonhomologous end joining. The amount of repair, corresponding to the repair capacity of the treated cells, is then quantified by determining the ratio of the fractions of activity released in the lanes in comparison to the total amount of DNA fragmentation following determination of an optimal exposure for maximum initial fragmentation. Repair kinetics can also be analyzed through a time-course regimen.

  9. Germline Mutations in FAN1 Cause Hereditary Colorectal Cancer by Impairing DNA Repair.

    PubMed

    Seguí, Nuria; Mina, Leonardo B; Lázaro, Conxi; Sanz-Pamplona, Rebeca; Pons, Tirso; Navarro, Matilde; Bellido, Fernando; López-Doriga, Adriana; Valdés-Mas, Rafael; Pineda, Marta; Guinó, Elisabet; Vidal, August; Soto, José Luís; Caldés, Trinidad; Durán, Mercedes; Urioste, Miguel; Rueda, Daniel; Brunet, Joan; Balbín, Milagros; Blay, Pilar; Iglesias, Silvia; Garré, Pilar; Lastra, Enrique; Sánchez-Heras, Ana Beatriz; Valencia, Alfonso; Moreno, Victor; Pujana, Miguel Ángel; Villanueva, Alberto; Blanco, Ignacio; Capellá, Gabriel; Surrallés, Jordi; Puente, Xose S; Valle, Laura

    2015-09-01

    Identification of genes associated with hereditary cancers facilitates management of patients with family histories of cancer. We performed exome sequencing of DNA from 3 individuals from a family with colorectal cancer who met the Amsterdam criteria for risk of hereditary nonpolyposis colorectal cancer. These individuals had mismatch repair-proficient tumors and each carried nonsense variant in the FANCD2/FANCI-associated nuclease 1 gene (FAN1), which encodes a nuclease involved in DNA inter-strand cross-link repair. We sequenced FAN1 in 176 additional families with histories of colorectal cancer and performed in vitro functional analyses of the mutant forms of FAN1 identified. We detected FAN1 mutations in approximately 3% of families who met the Amsterdam criteria and had mismatch repair-proficient cancers with no previously associated mutations. These findings link colorectal cancer predisposition to the Fanconi anemia DNA repair pathway, supporting the connection between genome integrity and cancer risk.

  10. Making Ends Meet: Repairing Breaks in Bacterial DNA by Non-Homologous End-Joining

    PubMed Central

    Bowater, Richard; Doherty, Aidan J

    2006-01-01

    DNA double-strand breaks (DSBs) are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ). Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHEJ apparatus has now been identified and characterized in the prokarya. Here we review the recent discoveries concerning bacterial NHEJ and discuss the possible origins of this repair system. We also examine the insights gained from the recent cellular and biochemical studies of this DSB-repair process and discuss the possible cellular roles of an NHEJ pathway in the life-cycle of prokaryotes and phages. PMID:16518468

  11. The effect of acute dose charge particle radiation on expression of DNA repair genes in mice.

    PubMed

    Tariq, Muhammad Akram; Soedipe, Ayodotun; Ramesh, Govindarajan; Wu, Honglu; Zhang, Ye; Shishodia, Shishir; Gridley, Daila S; Pourmand, Nader; Jejelowo, Olufisayo

    2011-03-01

    The space radiation environment consists of trapped particle radiation, solar particle radiation, and galactic cosmic radiation (GCR), in which protons are the most abundant particle type. During missions to the moon or to Mars, the constant exposure to GCR and occasional exposure to particles emitted from solar particle events (SPE) are major health concerns for astronauts. Therefore, in order to determine health risks during space missions, an understanding of cellular responses to proton exposure is of primary importance. The expression of DNA repair genes in response to ionizing radiation (X-rays and gamma rays) has been studied, but data on DNA repair in response to protons is lacking. Using qPCR analysis, we investigated changes in gene expression induced by positively charged particles (protons) in four categories (0, 0.1, 1.0, and 2.0 Gy) in nine different DNA repair genes isolated from the testes of irradiated mice. DNA repair genes were selected on the basis of their known functions. These genes include ERCC1 (5' incision subunit, DNA strand break repair), ERCC2/NER (opening DNA around the damage, Nucleotide Excision Repair), XRCC1 (5' incision subunit, DNA strand break repair), XRCC3 (DNA break and cross-link repair), XPA (binds damaged DNA in preincision complex), XPC (damage recognition), ATA or ATM (activates checkpoint signaling upon double strand breaks), MLH1 (post-replicative DNA mismatch repair), and PARP1 (base excision repair). Our results demonstrate that ERCC1, PARP1, and XPA genes showed no change at 0.1 Gy radiation, up-regulation at 1.0 Gy radiation (1.09 fold, 7.32 fold, 0.75 fold, respectively), and a remarkable increase in gene expression at 2.0 Gy radiation (4.83 fold, 57.58 fold and 87.58 fold, respectively). Expression of other genes, including ATM and XRCC3, was unchanged at 0.1 and 1.0 Gy radiation but showed up-regulation at 2.0 Gy radiation (2.64 fold and 2.86 fold, respectively). We were unable to detect gene expression for the

  12. Radiation- and drug-induced DNA repair in mammalian oocytes and embryos

    SciTech Connect

    Pedersen, R.A.; Brandriff, B.

    1980-01-01

    A review of studies showing ultraviolet- or drug-induced unscheduled DNA synthesis in mammalian oocytes and embryos suggests that the female gamete has an excision repair capacity from the earliest stages of oocyte growth. The oocyte's demonstrable excision repair capacity decreases at the time of meiotic maturation for unknown reasons, but the fully mature oocyte maintains a repair capacity, in contrast to the mature sperm, and contributes this to the zygote. Early embryo cells maintain relatively constant levels of excision repair until late fetal stages, when they lose their capacity for excision repair. These apparent changes in excision repair capacity do not have a simple relationship to known differences in radiation sensitivity of germ cells and embryos.

  13. Repair of transposable phage Mu DNA insertions begins only when the E. coli replisome collides with the transpososome.

    PubMed

    Jang, Sooin; Harshey, Rasika M

    2015-08-01

    We report a new cellular interaction between the infecting transposable phage Mu and the host Escherichia coli replication machinery during repair of Mu insertions, which involves filling-in of short target gaps on either side of the insertion, concomitant with degradation of extraneous long flanking DNA (FD) linked to Mu. Using the FD as a marker to follow repair, we find that after transposition into the chromosome, the unrepaired Mu is indefinitely stable until the replication fork arrives at the insertion site, whereupon the FD is rapidly degraded. When the fork runs into a Mu target gap, a double strand end (DSE) will result; we demonstrate fork-dependent DSEs proximal to Mu. These findings suggest that Pol III stalled at the transpososome is exploited for co-ordinated repair of both target gaps flanking Mu without replicating the intervening 37 kb of Mu, disassembling the stable transpososome in the process. This work is relevant to all transposable elements, including retroviral elements like HIV-1, which share with Mu the common problem of repair of their flanking target gaps.

  14. Repair of oxidatively induced DNA damage by DNA glycosylases: Mechanisms of action, substrate specificities and excision kinetics.

    PubMed

    Dizdaroglu, Miral; Coskun, Erdem; Jaruga, Pawel

    Endogenous and exogenous reactive species cause oxidatively induced DNA damage in living organisms by a variety of mechanisms. As a result, a plethora of mutagenic and/or cytotoxic products are formed in cellular DNA. This type of DNA damage is repaired by base excision repair, although nucleotide excision repair also plays a limited role. DNA glycosylases remove modified DNA bases from DNA by hydrolyzing the glycosidic bond leaving behind an apurinic/apyrimidinic (AP) site. Some of them also possess an accompanying AP-lyase activity that cleaves the sugar-phosphate chain of DNA. Since the first discovery of a DNA glycosylase, many studies have elucidated the mechanisms of action, substrate specificities and excision kinetics of these enzymes present in all living organisms. For this purpose, most studies used single- or double-stranded oligodeoxynucleotides with a single DNA lesion embedded at a defined position. High-molecular weight DNA with multiple base lesions has been used in other studies with the advantage of the simultaneous investigation of many DNA base lesions as substrates. Differences between the substrate specificities and excision kinetics of DNA glycosylases have been found when these two different substrates were used. Some DNA glycosylases possess varying substrate specificities for either purine-derived lesions or pyrimidine-derived lesions, whereas others exhibit cross-activity for both types of lesions. Laboratory animals with knockouts of the genes of DNA glycosylases have also been used to provide unequivocal evidence for the substrates, which had previously been found in in vitro studies, to be the actual substrates in vivo as well. On the basis of the knowledge gained from the past studies, efforts are being made to discover small molecule inhibitors of DNA glycosylases that may be used as potential drugs in cancer therapy.

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

    SciTech Connect

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

    1999-07-12

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

  16. Crystal structure of the DNA nucleotide excision repair enzyme UvrB from Thermus thermophilus

    PubMed Central

    Machius, Mischa; Henry, Lisa; Palnitkar, Maya; Deisenhofer, Johann

    1999-01-01

    Nucleotide excision repair (NER) is the most important DNA-repair mechanism in living organisms. In prokaryotes, three enzymes forming the UvrABC system initiate NER of a variety of structurally different DNA lesions. UvrB, the central component of this system, is responsible for the ultimate DNA damage recognition and participates in the incision of the damaged DNA strand. The crystal structure of Thermus thermophilus UvrB reveals a core that is structurally similar to core regions found in helicases, where they constitute molecular motors. Additional domains implicated in binding to DNA and various components of the NER system are attached to this central core. The architecture and distribution of DNA binding sites suggest a possible model for the DNA damage recognition process. PMID:10518516

  17. DNA replication meets genetic exchange: chromosomal damage and its repair by homologous recombination.

    PubMed

    Kuzminov, A

    2001-07-17

    Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair.

  18. Characterization of DNA binding and pairing activities associated with the native SFPQ·NONO DNA repair protein complex.

    PubMed

    Udayakumar, Durga; Dynan, William S

    2015-08-07

    Nonhomologous end joining (NHEJ) is a major pathway for repair of DNA double-strand breaks. We have previously shown that a complex of SFPQ (PSF) and NONO (p54(nrb)) cooperates with Ku protein at an early step of NHEJ, forming a committed preligation complex and stimulating end-joining activity by 10-fold or more. SFPQ and NONO show no resemblance to other repair factors, and their mechanism of action is uncertain. Here, we use an optimized microwell-based assay to characterize the in vitro DNA binding behavior of the native SFPQ·NONO complex purified from human (HeLa) cells. SFPQ·NONO and Ku protein bind independently to DNA, with little evidence of cooperativity and only slight mutual interference at high concentration. Whereas Ku protein requires free DNA ends for binding, SFPQ·NONO does not. Both Ku and SFPQ·NONO have pairing activity, as measured by the ability of DNA-bound protein to capture a second DNA fragment in a microwell-based assay. Additionally, SFPQ·NONO stimulates DNA-dependent protein kinase autophosphorylation, consistent with the ability to promote formation of a synaptic complex formation without occluding the DNA termini proper. These findings suggest that SFPQ·NONO promotes end joining by binding to internal DNA sequences and cooperating with other repair proteins to stabilize a synaptic pre-ligation complex.

  19. Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

    PubMed

    Martins-Pinheiro, Marinalva; Schons-Fonseca, Luciane; da Silva, Josefa B; Domingos, Renan H; Momo, Leonardo Hiroyuki Santos; Simões, Ana Carolina Quirino; Ho, Paulo Lee; da Costa, Renata M A

    2016-04-01

    Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen.

  20. Nucleotide excision repair and photolyase repair of UV photoproducts in nucleosomes: assessing the existence of nucleosome and non-nucleosome rDNA chromatin in vivo.

    PubMed

    Tremblay, Maxime; Toussaint, Martin; D'Amours, Annie; Conconi, Antonio

    2009-02-01

    The genome is organized into nuclear domains, which create microenvironments that favor distinct chromatin structures and functions (e.g., highly repetitive sequences, centromeres, telomeres, noncoding sequences, inactive genes, RNA polymerase II and III transcribed genes, and the nucleolus). Correlations have been drawn between gene silencing and proximity to a heterochromatic compartment. At the other end of the scale are ribosomal genes, which are transcribed at a very high rate by RNA polymerase I (~60% of total transcription), have a loose chromatin structure, and are clustered in the nucleolus. The rDNA sequences have 2 distinct structures: active rRNA genes, which have no nucleosomes; and inactive rRNA genes, which have nucleosomes. Like DNA transcription and replication, DNA repair is modulated by the structure of chromatin, and the kinetics of DNA repair vary among the nuclear domains. Although research on DNA repair in all chromosomal contexts is important to understand the mechanisms of genome maintenance, this review focuses on nucleotide excision repair and photolyase repair of UV photoproducts in the first-order packing of DNA in chromatin: the nucleosome. In addition, it summarizes the studies that have demonstrated the existence of the 2 rDNA chromatins, and the way this feature of the rDNA locus allows for direct comparison of DNA repair in 2 very different structures: nucleosome and non-nucleosome DNA.