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Sample records for complex dna repair

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

    SciTech Connect

    Gao, Qi; Williams, L.D.; Egli, M.; Rabinovich, D.; Rich, A. ); Chen, Shunle; Quigley, G.J. )

    1991-03-15

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

  2. Stalled transcription complexes promote DNA repair at a distance

    PubMed Central

    Haines, Nia M.; Kim, Young-In T.; Smith, Abigail J.; Savery, Nigel J.

    2014-01-01

    Transcription-coupled nucleotide excision repair (TCR) accelerates the removal of noncoding lesions from the template strand of active genes, and hence contributes to genome-wide variations in mutation frequency. Current models for TCR suppose that a lesion must cause RNA polymerase (RNAP) to stall if it is to be a substrate for accelerated repair. We have examined the substrate requirements for TCR using a system in which transcription stalling and damage location can be uncoupled. We show that Mfd-dependent TCR in bacteria involves the formation of a damage search complex that can detect lesions downstream of a stalled RNAP, and that the strand specificity of the accelerated repair pathway is independent of the requirement for a lesion to stall RNAP. We also show that an ops (operon polarity suppressor) transcription pause site, which causes backtracking of RNAP, can promote the repair of downstream lesions when those lesions do not themselves cause the polymerase to stall. Our findings indicate that the transcription-repair coupling factor Mfd, which is an ATP-dependent superfamily 2 helicase that binds to RNAP, continues to translocate along DNA after RNAP has been displaced until a lesion in the template strand is located. The discovery that pause sites can promote the repair of nonstalling lesions suggests that TCR pathways may play a wider role in modulating mutation frequencies in different parts of the genome than has previously been suspected. PMID:24554077

  3. Recombinational DNA repair is regulated by compartmentalization of DNA lesions at the nuclear pore complex.

    PubMed

    Géli, Vincent; Lisby, Michael

    2015-12-01

    The nuclear pore complex (NPC) is emerging as a center for recruitment of a class of "difficult to repair" lesions such as double-strand breaks without a repair template and eroded telomeres in telomerase-deficient cells. In addition to such pathological situations, a recent study by Su and colleagues shows that also physiological threats to genome integrity such as DNA secondary structure-forming triplet repeat sequences relocalize to the NPC during DNA replication. Mutants that fail to reposition the triplet repeat locus to the NPC cause repeat instability. Here, we review the types of DNA lesions that relocalize to the NPC, the putative mechanisms of relocalization, and the types of recombinational repair that are stimulated by the NPC, and present a model for NPC-facilitated repair. PMID:26422820

  4. 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.

  5. Structure of the Rad50 Mre11 DNA Repair Complex from Saccharomyces cerevisiae by Electron Microscopy*

    E-print Network

    Erickson, Harold P.

    Structure of the Rad50 Mre11 DNA Repair Complex from Saccharomyces cerevisiae by Electron Microscopy* Received for publication, July 3, 2001 Published, JBC Papers in Press, July 24, 2001, DOI 10- tural analysis of full-length S. cerevisiae Rad50, alone and in a complex with yeast Mre11 by electron

  6. The Fanconi anemia DNA repair pathway: structural and functional insights into a complex disorder.

    PubMed

    Walden, Helen; Deans, Andrew J

    2014-01-01

    Mutations in any of at least sixteen FANC genes (FANCA-Q) cause Fanconi anemia, a disorder characterized by sensitivity to DNA interstrand crosslinking agents. The clinical features of cytopenia, developmental defects, and tumor predisposition are similar in each group, suggesting that the gene products participate in a common pathway. The Fanconi anemia DNA repair pathway consists of an anchor complex that recognizes damage caused by interstrand crosslinks, a multisubunit ubiquitin ligase that monoubiquitinates two substrates, and several downstream repair proteins including nucleases and homologous recombination enzymes. We review progress in the use of structural and biochemical approaches to understanding how each FANC protein functions in this pathway. PMID:24773018

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

    SciTech Connect

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

    2011-12-31

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

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

    SciTech Connect

    Joo, Woo; Xu, Guozhou; Persky, Nicole S.; Smogorzewska, Agata; Rudge, Derek G.; Buzovetsky, Olga; Elledge, Stephen J.; Pavletich, Nikola P.

    2011-08-29

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

  9. The NuRD chromatin–remodeling complex regulates signaling and repair of DNA damage

    PubMed Central

    Smeenk, Godelieve; Wiegant, Wouter W.; Vrolijk, Hans; Solari, Aldo P.; Pastink, Albert

    2010-01-01

    Cells respond to ionizing radiation (IR)–induced DNA double-strand breaks (DSBs) by orchestrating events that coordinate cell cycle progression and DNA repair. How cells signal and repair DSBs is not yet fully understood. A genome-wide RNA interference screen in Caenorhabditis elegans identified egr-1 as a factor that protects worm cells against IR. The human homologue of egr-1, MTA2 (metastasis-associated protein 2), is a subunit of the nucleosome-remodeling and histone deacetylation (NuRD) chromatin-remodeling complex. We show that knockdown of MTA2 and CHD4 (chromodomain helicase DNA-binding protein 4), the catalytic subunit (adenosine triphosphatase [ATPase]) of NuRD, leads to accumulation of spontaneous DNA damage and increased IR sensitivity. MTA2 and CHD4 accumulate in DSB-containing chromatin tracks generated by laser microirradiation. Directly at DSBs, CHD4 stimulates RNF8/RNF168-dependent formation of ubiquitin conjugates to facilitate the accrual of RNF168 and BRCA1. Finally, we show that CHD4 promotes DSB repair and checkpoint activation in response to IR. Thus, the NuRD chromatin–remodeling complex is a novel regulator of DNA damage responses that orchestrates proper signaling and repair of DSBs. PMID:20805320

  10. DNA conformation and repair of polymeric natural DNA damaged by antitumor azolato-bridged dinuclear Pt(II) complex.

    PubMed

    Mlcouskova, Jarmila; Kasparkova, Jana; Suchankova, Tereza; Komeda, Seiji; Brabec, Viktor

    2012-09-01

    Design of new antitumor Pt drugs is currently also focused on those new Pt complexes which form on DNA major adducts that can hardly be removed by DNA repair systems. An attempt of this kind has already been done by designing and synthesizing new antitumor azolato-bridged dinuclear Pt(II) complexes, such as [{cis-Pt(NH(3))(2)}(2)(?-OH)(?-pyrazolate)](2+) (AMPZ). This new Pt(II) complex exhibits markedly higher toxic effects in some tumor cell lines than conventional mononuclear cisplatin. The primary objective in the present study was to further delineate differences in the interactions of AMPZ and cisplatin with natural, high-molecular-mass DNA using a combination of biochemical and molecular biophysics techniques. The results demonstrate for the first time that little conformational distortions induced by AMPZ in highly polymeric DNA with a random nucleotide sequence represent a structural motif recognizable by DNA repair systems less efficiently than distortions induced by cisplatin. Thus, DNA adducts of azolato-bridged dinuclear Pt(II) complexes can escape repair mechanisms more easily than those of cisplatin, which may potentiate antitumor effects of these new metallodrugs in cancer cells. PMID:22687561

  11. 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 ? (Pol?), 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-Pol?, APE1-TDP1, APE1-PARP1 and Pol?-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 Pol?. Model DNA intermediates of BER are shown to induce significant rearrangement of the Pol? complexes with XRCC1 and PARP1, while having no detectable influence on the protein–protein binding affinities. The strength of APE1 interaction with Pol?, 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 Pol? 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

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

    PubMed Central

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

    2013-01-01

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

  13. 53BP1 and the LINC Complex Promote Microtubule-Dependent DSB Mobility and DNA Repair.

    PubMed

    Lottersberger, Francisca; Karssemeijer, Roos Anna; Dimitrova, Nadya; de Lange, Titia

    2015-11-01

    Increased mobility of chromatin surrounding double-strand breaks (DSBs) has been noted in yeast and mammalian cells but the underlying mechanism and its contribution to DSB repair remain unclear. Here, we use a telomere-based system to track DNA damage foci with high resolution in living cells. We find that the greater mobility of damaged chromatin requires 53BP1, SUN1/2 in the linker of the nucleoskeleton, and cytoskeleton (LINC) complex and dynamic microtubules. The data further demonstrate that the excursions promote non-homologous end joining of dysfunctional telomeres and implicated Nesprin-4 and kinesins in telomere fusion. 53BP1/LINC/microtubule-dependent mobility is also evident at irradiation-induced DSBs and contributes to the mis-rejoining of drug-induced DSBs in BRCA1-deficient cells showing that DSB mobility can be detrimental in cells with numerous DSBs. In contrast, under physiological conditions where cells have only one or a few lesions, DSB mobility is proposed to prevent errors in DNA repair. PAPERCLIP. PMID:26544937

  14. XRCC1 Coordinates Disparate Responses and Multiprotein Repair Complexes Depending on the Nature and Context of the DNA Damage

    PubMed Central

    Hanssen-Bauer, Audun; Solvang-Garten, Karin; Sundheim, Ottar; Peña-Diaz, Javier; Andersen, Sonja; Slupphaug, Geir; Krokan, Hans E; Wilson, David M; Akbari, Mansour; Otterlei, Marit

    2011-01-01

    XRCC1 is a scaffold protein capable of interacting with several DNA repair proteins. Here we provide evidence for the presence of XRCC1 in different complexes of sizes from 200 to 1500 kDa, and we show that immunoprecipitates using XRCC1 as bait are capable of complete repair of AP sites via both short patch (SP) and long patch (LP) base excision repair (BER). We show that POL? and PNK colocalize with XRCC1 in replication foci and that POL? and PNK, but not PCNA, colocalize with constitutively present XRCC1-foci as well as damage-induced foci when low doses of a DNA-damaging agent are applied. We demonstrate that the laser dose used for introducing DNA damage determines the repertoire of DNA repair proteins recruited. Furthermore, we demonstrate that recruitment of POL? and PNK to regions irradiated with low laser dose requires XRCC1 and that inhibition of PARylation by PARP-inhibitors only slightly reduces the recruitment of XRCC1, PNK, or POL? to sites of DNA damage. Recruitment of PCNA and FEN-1 requires higher doses of irradiation and is enhanced by XRCC1, as well as by accumulation of PARP-1 at the site of DNA damage. These data improve our understanding of recruitment of BER proteins to sites of DNA damage and provide evidence for a role of XRCC1 in the organization of BER into multiprotein complexes of different sizes. Environ. Mol. Mutagen. 2011. © 2011 Wiley-Liss, Inc. PMID:21786338

  15. Celebrating DNA's Repair Crew.

    PubMed

    Kunkel, Thomas A

    2015-12-01

    This year, the Nobel Prize in Chemistry has been awarded to Tomas Lindahl, Aziz Sancar, and Paul Modrich for their seminal studies of the mechanisms by which cells from bacteria to man repair DNA damage that is generated by normal cellular metabolism and stress from the environment. These studies beautifully illustrate the remarkable power of DNA repair to influence life from evolution through disease susceptibility. PMID:26638062

  16. The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks

    PubMed Central

    Nakamura, Asako J.; Rao, V. Ashutosh; Pommier, Yves; Bonner, William M.

    2011-01-01

    The maintenance of genome stability requires efficient DNA double-stranded break (DSB) repair mediated by the phosphorylation of multiple histone H2AX molecules near the break sites. The phosphorylated H2AX (?H2AX) molecules form foci covering many megabases of chromatin. the formation of ?-H2AX foci is critical for efficient DNA damage response (DDR) and for the maintenance of genome stability, however, the mechanisms of protein organization in foci is largely unknown. To investigate the nature of ?H2AX foci formation, we analyzed the distribution of ?H2AX and other DDR proteins at DSB sites using a variety of techniques to visualize, expand and partially disrupt chromatin. We report here that ?H2AX foci change composition during the cell cycle, with proteins 53BP1, NBS1 and MRE11 dissociating from foci in G2 and mitosis to return at the beginning of the following G1. In contrast, MDC1 remained colocalized with ?-H2AX during mitosis. In addition, while ?H2AX was found to span large domains flanking DSB sites, 53BP1 and NBS1 were more localized and MDC1 colocalized in doublets in foci. H2AX and MDC1 were found to be involved in chromatin relaxation after DSB formation. Our data demonstrates that the DSB repair focus is a heterogeneous and dynamic structure containing internal complexity. PMID:20046100

  17. Identification and Dissection of a Complex DNA Repair Sensitivity Phenotype in Baker's Yeast

    PubMed Central

    Demogines, Ann; Smith, Erin; Kruglyak, Leonid; Alani, Eric

    2008-01-01

    Complex traits typically involve the contribution of multiple gene variants. In this study, we took advantage of a high-density genotyping analysis of the BY (S288c) and RM strains of Saccharomyces cerevisiae and of 123 derived spore progeny to identify the genetic loci that underlie a complex DNA repair sensitivity phenotype. This was accomplished by screening hybrid yeast progeny for sensitivity to a variety of DNA damaging agents. Both the BY and RM strains are resistant to the ultraviolet light–mimetic agent 4-nitroquinoline 1-oxide (4-NQO); however, hybrid progeny from a BY×RM cross displayed varying sensitivities to the drug. We mapped a major quantitative trait locus (QTL), RAD5, and identified the exact polymorphism within this locus responsible for 4-NQO sensitivity. By using a backcrossing strategy along with array-assisted bulk segregant analysis, we identified one other locus, MKT1, and a QTL on Chromosome VII that also link to the hybrid 4-NQO–sensitive phenotype but confer more minor effects. This work suggests an additive model for sensitivity to 4-NQO and provides a strategy for mapping both major and minor QTL that confer background-specific phenotypes. It also provides tools for understanding the effect of genetic background on sensitivity to genotoxic agents. PMID:18617998

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

    PubMed Central

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

    2011-01-01

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

  19. Protein methylation and DNA repair.

    PubMed

    Lake, Aimee N; Bedford, Mark T

    2007-05-01

    DNA is under constant attack from intracellular and external mutagens. Sites of DNA damage need to be pinpointed so that the DNA repair machinery can be mobilized to the proper location. The identification of damaged sites, recruitment of repair factors, and assembly of repair "factories" is orchestrated by posttranslational modifications (PTMs). These PTMs include phosphorylation, ubiquitination, sumoylation, acetylation, and methylation. Here we discuss recent data surrounding the roles of arginine and lysine methylation in DNA repair processes. PMID:17306845

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

    NASA Astrophysics Data System (ADS)

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

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

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

    PubMed Central

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

    2014-01-01

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

  2. Preferential DNA repair in expressed genes

    SciTech Connect

    Hanawalt, P.C.

    1987-12-01

    Potentially deleterious alterations to DNA occur nonrandomly within the mammalian genome. These alterations include the adducts produced by many chemical carcinogens, but not the UV-induced cyclobutane pyrimidine dimer, which may be an exception. Recent studies in our laboratory have shown that the excision repair of pyrimidine dimers and certain other lesions is nonrandom in the mammalian genome, exhibiting a distinct preference for actively transcribed DNA sequences. An important consequence of this fact is that mutagenesis and carcinogenesis may be determined in part by the activities of the relevant genes. Repair may also be processive, and a model is proposed in which excision repair is coupled to transcription at the nuclear matrix. Similar but freely diffusing repair complexes may account for the lower overall repair efficiencies in the silent domains of the genome. Risk assessment in relations to chemical carcinogenesis requires assays that determine effective levels of DNA damage for producing malignancy. The existence of nonrandom repair in the genome casts into doubt the reliability of overall indicators of DNA binding and lesion repair for such determinations. Furthermore, some apparent differences between the intragenomic repair heterogeneity in rodent cells and that in human cells mandate a reevaluation of rodent test systems for human risk assessment. Tissue-specific and cell-specific differences in the coordinate regulation of gene expression and DNA repair may account for corresponding differences in the carcinogenic response.

  3. HSP90 regulates DNA repair via the interaction between XRCC1 and DNA polymerase ?

    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 ? (Pol?) and XRCC1 is thought to facilitate repair by recruiting Pol? 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 Pol?. 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 Pol? 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

  4. 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. PMID:26422132

  5. Structure of p15PAF-PCNA complex and implications for clamp sliding during DNA replication and repair

    NASA Astrophysics Data System (ADS)

    de Biasio, Alfredo; de Opakua, Alain Ibáñez; Mortuza, Gulnahar B.; Molina, Rafael; Cordeiro, Tiago N.; Castillo, Francisco; Villate, Maider; Merino, Nekane; Delgado, Sandra; Gil-Cartón, David; Luque, Irene; Diercks, Tammo; Bernadó, Pau; Montoya, Guillermo; Blanco, Francisco J.

    2015-03-01

    The intrinsically disordered protein p15PAF regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15PAF-PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15PAF tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15PAF also contacts the inside of, and passes through, the PCNA ring. The disordered p15PAF termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15PAF binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15PAF acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.

  6. 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.

  7. Structural biology of disease-associated repetitive DNA sequences and protein-DNA complexes involved in DNA damage and repair

    SciTech Connect

    Gupta, G.; Santhana Mariappan, S.V.; Chen, X.; Catasti, P.; Silks, L.A. III; Moyzis, R.K.; Bradbury, E.M.; Garcia, A.E.

    1997-07-01

    This project is aimed at formulating the sequence-structure-function correlations of various microsatellites in the human (and other eukaryotic) genomes. Here the authors have been able to develop and apply structure biology tools to understand the following: the molecular mechanism of length polymorphism microsatellites; the molecular mechanism by which the microsatellites in the noncoding regions alter the regulation of the associated gene; and finally, the molecular mechanism by which the expansion of these microsatellites impairs gene expression and causes the disease. Their multidisciplinary structural biology approach is quantitative and can be applied to all coding and noncoding DNA sequences associated with any gene. Both NIH and DOE are interested in developing quantitative tools for understanding the function of various human genes for prevention against diseases caused by genetic and environmental effects.

  8. 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.

  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. DNA Repair Deficiency in Neurodegeneration

    PubMed Central

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

    2011-01-01

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

  11. DNA demethylation by DNA repair

    E-print Network

    Gehring, Mary

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

  12. Novel essential DNA repair proteins Nse1 and Nse2 are subunits of the fission yeast Smc5-Smc6 complex.

    PubMed

    McDonald, W Hayes; Pavlova, Yelena; Yates, John R; Boddy, Michael N

    2003-11-14

    The structural maintenance of chromosomes (SMC) family of proteins play essential roles in genomic stability. SMC heterodimers are required for sister-chromatid cohesion (Cohesin: Smc1 & Smc3), chromatin condensation (Condensin: Smc2 & Smc4), and DNA repair (Smc5 & Smc6). The SMC heterodimers do not function alone and must associate with essential non-SMC subunits. To gain further insight into the essential and DNA repair roles of the Smc5-6 complex, we have purified fission yeast Smc5 and identified by mass spectrometry the co-precipitating proteins, Nse1 and Nse2. We show that both Nse1 and Nse2 interact with Smc5 in vivo, as part of the Smc5-6 complex. Nse1 and Nse2 are essential proteins and conserved from yeast to man. Loss of Nse1 and Nse2 function leads to strikingly similar terminal phenotypes to those observed for Smc5-6 inactivation. In addition, cells expressing hypomorphic alleles of Nse1 and Nse2 are, like Smc5-6 mutants, hypersensitive to DNA damage. Epistasis analysis suggests that like Smc5-6, Nse1, and Nse2 function together with Rhp51 in the homologous recombination repair of DNA double strand breaks. The results of this study strongly suggest that Nse1 and Nse2 are novel non-SMC subunits of the fission yeast Smc5-6 DNA repair complex. PMID:12966087

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

    SciTech Connect

    Noda, Taichi; Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 ; Takahashi, Akihisa; Kondo, Natsuko; Mori, Eiichiro; Okamoto, Noritomo; Nakagawa, Yosuke; Ohnishi, Ken; Zdzienicka, Malgorzata Z.; Thompson, Larry H.; Helleday, Thomas; Department of Genetics, Microbiology and Toxicology Stockholm University, SE-106 91 Stockholm ; Asada, Hideo; and others

    2011-01-07

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

  14. ZTF-8 Interacts with the 9-1-1 Complex and Is Required for DNA Damage Response and Double-Strand Break Repair in the C. elegans Germline

    PubMed Central

    Kim, Hyun-Min; Colaiácovo, Monica P.

    2014-01-01

    Germline mutations in DNA repair genes are linked to tumor progression. Furthermore, failure in either activating a DNA damage checkpoint or repairing programmed meiotic double-strand breaks (DSBs) can impair chromosome segregation. Therefore, understanding the molecular basis for DNA damage response (DDR) and DSB repair (DSBR) within the germline is highly important. Here we define ZTF-8, a previously uncharacterized protein conserved from worms to humans, as a novel factor involved in the repair of both mitotic and meiotic DSBs as well as in meiotic DNA damage checkpoint activation in the C. elegans germline. ztf-8 mutants exhibit specific sensitivity to ?-irradiation and hydroxyurea, mitotic nuclear arrest at S-phase accompanied by activation of the ATL-1 and CHK-1 DNA damage checkpoint kinases, as well as accumulation of both mitotic and meiotic recombination intermediates, indicating that ZTF-8 functions in DSBR. However, impaired meiotic DSBR progression partially fails to trigger the CEP-1/p53-dependent DNA damage checkpoint in late pachytene, also supporting a role for ZTF-8 in meiotic DDR. ZTF-8 partially co-localizes with the 9-1-1 DDR complex and interacts with MRT-2/Rad1, a component of this complex. The human RHINO protein rescues the phenotypes observed in ztf-8 mutants, suggesting functional conservation across species. We propose that ZTF-8 is involved in promoting repair at stalled replication forks and meiotic DSBs by transducing DNA damage checkpoint signaling via the 9-1-1 pathway. Our findings define a conserved function for ZTF-8/RHINO in promoting genomic stability in the germline. PMID:25329393

  15. Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability

    PubMed Central

    Querol-Audí, Jordi; Yan, Chunli; Xu, Xiaojun; Tsutakawa, Susan E.; Tsai, Miaw-Sheue; Tainer, John A.; Cooper, Priscilla K.; Nogales, Eva; Ivanov, Ivaylo

    2012-01-01

    Processivity clamps such as proliferating cell nuclear antigen (PCNA) and the checkpoint sliding clamp Rad9/Rad1/Hus1 (9-1-1) act as versatile scaffolds in the coordinated recruitment of proteins involved in DNA replication, cell-cycle control, and DNA repair. Association and handoff of DNA-editing enzymes, such as flap endonuclease 1 (FEN1), with sliding clamps are key processes in biology, which are incompletely understood from a mechanistic point of view. We have used an integrative computational and experimental approach to define the assemblies of FEN1 with double-flap DNA substrates and either proliferating cell nuclear antigen or the checkpoint sliding clamp 9-1-1. Fully atomistic models of these two ternary complexes were developed and refined through extensive molecular dynamics simulations to expose their conformational dynamics. Clustering analysis revealed the most dominant conformations accessible to the complexes. The cluster centroids were subsequently used in conjunction with single-particle electron microscopy data to obtain a 3D EM reconstruction of the human 9-1-1/FEN1/DNA assembly at 18-Å resolution. Comparing the structures of the complexes revealed key differences in the orientation and interactions of FEN1 and double-flap DNA with the two clamps that are consistent with their respective functions in providing inherent flexibility for lagging strand DNA replication or inherent stability for DNA repair. PMID:22586102

  16. Scrunching During DNA Repair Synthesis

    PubMed Central

    Garcia-Diaz, Miguel; Bebenek, Katarzyna; Larrea, Andres A.; Havener, Jody M.; Perera, Lalith; Krahn, Joseph M.; Pedersen, Lars C.; Ramsden, Dale A.; Kunkel, Thomas A.

    2009-01-01

    Family X polymerases like DNA polymerase ? (pol ?) are well suited for filling short gaps during DNA repair because they simultaneously bind both the 5? and 3? ends of short gaps. DNA binding and gap filling are well characterized for one nucleotide gaps, but the location of yet-to-be-copied template nucleotides in longer gaps is unknown. Here we present crystal structures revealing that when bound to a two-nucleotide gap, pol ? scrunches the template strand and binds the additional uncopied template base in an extrahelical position within a binding pocket comprised of three conserved amino acids. Replacing these amino acids with alanine results in less processive gap filling and less efficient NHEJ involving two nucleotide gaps. Thus, akin to scrunching by RNA polymerase during transcription initiation, scrunching occurs during gap filling DNA synthesis associated with DNA repair. PMID:19701199

  17. Sirtuins, Metabolism, and DNA repair

    PubMed Central

    Choi, Jee-Eun; Mostoslavsky, Raul

    2014-01-01

    Cells evolve to actively coordinate nutrient availability with cellular activity in order to maintain metabolic homeostasis. In addition, active pathways to repair DNA damage are crucial to avoid deleterious genomic instability. In recent years, it has become increasingly clear that availability of intermediate metabolites may play an important role in DNA repair, suggesting that these two seemingly distant cellular activities may be highly coordinated. The sirtuin family of proteins now described as deacylases (they can also remove acyl groups other than acetyl moieties), it appears to have evolved to control both metabolism and DNA repair. In this review, we discuss recent advances that lay the foundation to understanding the role of sirtuins in these two biological processes, and the potential crosstalk to coordinate them. PMID:25005742

  18. Chromatin Disassembly and Reassembly During DNA Repair

    PubMed Central

    Linger, Jeffrey G.; Tyler, Jessica K.

    2008-01-01

    Current research is demonstrating that the packaging of the eukaryotic genome together with histone proteins into chromatin is playing a fundamental role in DNA repair and the maintenance of genomic integrity. As is well established to be the case for transcription, the chromatin structure dynamically changes during DNA repair. Recent studies indicate that the complete removal of histones from DNA and their subsequent reassembly onto DNA accompanies DNA repair. This review will present evidence indicating that chromatin disassembly and reassembly occur during DNA repair and that these are critical processes for cell survival after DNA repair. Concomitantly, candidate proteins utilized for these processes will be highlighted. PMID:17303193

  19. Functional and mechanistic studies of XPC DNA-repair complex as transcriptional coactivator in embryonic stem cells

    PubMed Central

    Cattoglio, Claudia; Zhang, Elisa T.; Grubisic, Ivan; Chiba, Kunitoshi; Fong, Yick W.; Tjian, Robert

    2015-01-01

    The embryonic stem cell (ESC) state is transcriptionally controlled by OCT4, SOX2, and NANOG with cofactors, chromatin regulators, noncoding RNAs, and other effectors of signaling pathways. Uncovering components of these regulatory circuits and their interplay provides the knowledge base to deploy ESCs and induced pluripotent stem cells. We recently identified the DNA-repair complex xeroderma pigmentosum C (XPC)-RAD23B-CETN2 as a stem cell coactivator (SCC) required for OCT4/SOX2 transcriptional activation. Here we investigate the role of SCC genome-wide in murine ESCs by mapping regions bound by RAD23B and analyzing transcriptional profiles of SCC-depleted ESCs. We establish OCT4 and SOX2 as the primary transcription factors recruiting SCC to regulatory regions of pluripotency genes and identify the XPC subunit as essential for interaction with the two proteins. The present study reveals new mechanistic and functional aspects of SCC transcriptional activity, and thus underscores the diversified functions of this regulatory complex. PMID:25901318

  20. Ovarian carcinoma CDK12 mutations misregulate expression of DNA repair genes via deficient formation and function of the Cdk12/CycK complex

    PubMed Central

    Ekumi, Kingsley M.; Paculova, Hana; Lenasi, Tina; Pospichalova, Vendula; Bösken, Christian A.; Rybarikova, Jana; Bryja, Vitezslav; Geyer, Matthias; Blazek, Dalibor; Barboric, Matjaz

    2015-01-01

    The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer. PMID:25712099

  1. Catechols and 3-hydroxypyridones as inhibitors of the DNA repair complex ERCC1-XPF.

    PubMed

    Chapman, Timothy M; Gillen, Kevin J; Wallace, Claire; Lee, Maximillian T; Bakrania, Preeti; Khurana, Puneet; Coombs, Peter J; Stennett, Laura; Fox, Simon; Bureau, Emilie A; Brownlees, Janet; Melton, David W; Saxty, Barbara

    2015-10-01

    Catechol-based inhibitors of ERCC1-XPF endonuclease activity were identified from a high-throughput screen. Exploration of the structure-activity relationships within this series yielded compound 13, which displayed an ERCC1-XPF IC50 of 0.6 ?M, high selectivity against FEN-1 and DNase I and activity in nucleotide excision repair, cisplatin enhancement and ?H2AX assays in A375 melanoma cells. Screening of fragments as potential alternatives to the catechol group revealed that 3-hydroxypyridones are able to inhibit ERCC1-XPF with high ligand efficiency, and elaboration of the hit gave compounds 36 and 37 which showed promising ERCC1-XPF IC50 values of <10 ?M. PMID:26318993

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

    PubMed Central

    Schipler, Agnes; Iliakis, George

    2013-01-01

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

  3. Mutant huntingtin impairs Ku70-mediated DNA repair

    PubMed Central

    Enokido, Yasushi; Tamura, Takuya; Ito, Hikaru; Arumughan, Anup; Komuro, Akihiko; Shiwaku, Hiroki; Sone, Masaki; Foulle, Raphaele; Sawada, Hirohide; Ishiguro, Hiroshi; Ono, Tetsuya; Murata, Miho; Kanazawa, Ichiro; Tomilin, Nikolai; Tagawa, Kazuhiko; Wanker, Erich E.

    2010-01-01

    DNA repair defends against naturally occurring or disease-associated DNA damage during the long lifespan of neurons and is implicated in polyglutamine disease pathology. In this study, we report that mutant huntingtin (Htt) expression in neurons causes double-strand breaks (DSBs) of genomic DNA, and Htt further promotes DSBs by impairing DNA repair. We identify Ku70, a component of the DNA damage repair complex, as a mediator of the DNA repair dysfunction in mutant Htt–expressing neurons. Mutant Htt interacts with Ku70, impairs DNA-dependent protein kinase function in nonhomologous end joining, and consequently increases DSB accumulation. Expression of exogenous Ku70 rescues abnormal behavior and pathological phenotypes in the R6/2 mouse model of Huntington’s disease (HD). These results collectively suggest that Ku70 is a critical regulator of DNA damage in HD pathology. PMID:20439996

  4. The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex.

    PubMed

    Pebernard, Stephanie; Wohlschlegel, James; McDonald, W Hayes; Yates, John R; Boddy, Michael N

    2006-03-01

    Stabilization and processing of stalled replication forks is critical for cell survival and genomic integrity. We characterize a novel DNA repair heterodimer of Nse5 and Nse6, which are nonessential nuclear proteins critical for chromosome segregation in fission yeast. The Nse5/6 dimer facilitates DNA repair as part of the Smc5-Smc6 holocomplex (Smc5/6), the basic architecture of which we define. Nse5-Nse6 [corrected] (Nse5 and Nse6) [corrected] mutants display a high level of spontaneous DNA damage and mitotic catastrophe in the absence of the master checkpoint regulator Rad3 (hATR). Nse5/6 mutants are required for the response to genotoxic agents that block the progression of replication forks, acting in a pathway that allows the tolerance of irreparable UV lesions. Interestingly, the UV sensitivity of Nse5/6 [corrected] is suppressed by concomitant deletion of the homologous recombination repair factor, Rhp51 (Rad51). Further, the viability of Nse5/6 mutants depends on Mus81 and Rqh1, factors that resolve or prevent the formation of Holliday junctions. Consistently, the UV sensitivity of cells lacking Nse5/6 can be partially suppressed by overexpressing the bacterial resolvase RusA. We propose a role for Nse5/6 mutants in suppressing recombination that results in Holliday junction formation or in Holliday junction resolution. PMID:16478984

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  6. 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

  7. 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 the DNA repair-defective cell lines were smaller than those of normal cells, with the DNA-PK-deficient cells having RBEs near unity. To further investigate the sensitivity differences that were observed in ATM and NBS deficient cells, chromosomal aberrations were analyzed in normal lung fibroblast cells treated with KU-55933 (a specific ATM kinase inhibitor) or Mirin (an Mre11- Rad50-Nbs1 complex inhibitor involved in activation of ATM). We also performed siRNA knockdown of these proteins. Preliminary data indicate that chromosome exchanges increase in cells treated with the specific ATM inhibitor. Possible cytogenetic signatures of acute and low dose-rate gamma irradiation in ATM or nibrin deficient and suppressed cells will be discussed.

  8. DNA repair helicases as targets for anti-cancer therapy.

    PubMed

    Gupta, Rigu; Brosh, Robert M

    2007-01-01

    The genetic complexity of cancer has posed a formidable challenge to devising successful therapeutic treatments. Tumor resistance to cytotoxic chemotherapy drugs and radiation which induce DNA damage has limited their effectiveness. Targeting the DNA damage response is a strategy for combating cancer. The prospect for success of chemotherapy treatment may be improved by the selective inactivation of a DNA repair pathway. A key class of proteins involved in various DNA repair pathways is comprised of energy-driven nucleic acid unwinding enzymes known as helicases. DNA helicases have been either implicated or have proposed roles in nucleotide excision repair, mismatch repair, base excision repair, double strand break repair, and most recently cross-link repair. In addition to DNA repair, helicases have been implicated in the cellular processes of replication, recombination, transcription, and RNA stability/processing. The emerging evidence indicates that helicases have vital roles in pathways necessary for the maintenance of genomic stability. In support of this, a growing number of human genetic disorders are attributed to mutations in helicase genes. Because of their essential roles in nucleic acid metabolism, and more specifically the DNA damage response, helicases may be a suitable target of chemotherapy. In this review, we have explored this hypothesis and provided a conceptual framework for combinatorial treatments that might be used for combating cancer by inhibiting helicase function in tumor cells that already have compromised DNA repair and/or DNA damage signaling. This review is focused on helicase pathways, with a special emphasis on DNA cross-link repair and double strand break repair, that impact cancer biology and how cancer cells may be chemosensitized through the impairment of helicase function. PMID:17346143

  9. Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription.

    PubMed

    Liu, Zhihui; Lam, Norris; Thiele, Carol J

    2015-09-29

    The zinc finger transcription factor CASZ1 has been found to control neural fate-determination in flies, regulate murine and frog cardiac development, control murine retinal cell progenitor expansion and function as a tumor suppressor gene in humans. However, the molecular mechanism by which CASZ1 regulates gene transcription to exert these diverse biological functions has not been described. Here we identify co-factors that are recruited by CASZ1b to regulate gene transcription using co-immunoprecipitation (co-IP) and mass spectrometry assays. We find that CASZ1b binds to the nucleosome remodeling and histone deacetylase (NuRD) complex, histones and DNA repair proteins. Mutagenesis of the CASZ1b protein assay demonstrates that the N-terminus of CASZ1b is required for NuRD binding, and a poly(ADP-ribose) binding motif in the CASZ1b protein is required for histone H3 and DNA repair proteins binding. The N-terminus of CASZ1b fused to an artificial DNA-binding domain (GAL4DBD) causes a significant repression of transcription (5xUAS-luciferase assay), which could be blocked by treatment with an HDAC inhibitor. Realtime PCR results show that the transcriptional activity of CASZ1b mutants that abrogate NuRD or histone H3/DNA binding is significantly decreased. This indicates a model in which CASZ1b binds to chromatin and recruits NuRD complexes to orchestrate epigenetic-mediated transcriptional programs. PMID:26296975

  10. FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway.

    PubMed

    Ali, Abdullah Mahmood; Pradhan, Arun; Singh, Thiyam Ramsingh; Du, Changhu; Li, Jie; Wahengbam, Kebola; Grassman, Elke; Auerbach, Arleen D; Pang, Qishen; Meetei, Amom Ruhikanta

    2012-04-01

    Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20). We show that FAAP20 is an integral component of the FA nuclear core complex. We identify a region on FANCA that physically interacts with FAAP20, and show that FANCA regulates stability of this protein. FAAP20 contains a conserved ubiquitin-binding zinc-finger domain (UBZ), and binds K-63-linked ubiquitin chains in vitro. The FAAP20-UBZ domain is not required for interaction with FANCA, but is required for DNA-damage-induced chromatin loading of FANCA and the functional integrity of the FA pathway. These findings reveal critical roles for FAAP20 in the FA-BRCA pathway of DNA damage repair and genome maintenance. PMID:22343915

  11. XPD polymorphisms: effects on DNA repair proficiency.

    PubMed

    Lunn, R M; Helzlsouer, K J; Parshad, R; Umbach, D M; Harris, E L; Sanford, K K; Bell, D A

    2000-04-01

    XPD codes for a DNA helicase involved in transcription and nucleotide excision repair. Rare XPD mutations diminish nucleotide excision repair resulting in hypersensitivity to UV light and increased risk of skin cancer. Several polymorphisms in this gene have been identified but their impact on DNA repair is not known. We compared XPD genotypes at codons 312 and 751 with DNA repair proficiency in 31 women. XPD genotypes were measured by PCR-RFLP. DNA repair proficiency was assessed using a cytogenetic assay that detects X-ray induced chromatid aberrations (breaks and gaps). Chromatid aberrations were scored per 100 metaphase cells following incubation at 37 degrees C (1.5 h after irradiation) to allow for repair of DNA damage. Individuals with the Lys/Lys codon 751 XPD genotype had a higher number of chromatid aberrations (132/100 metaphase cells) than those having a 751Gln allele (34/100 metaphase cells). Individuals having greater than 60 chromatid breaks plus gaps were categorized as having sub-optimal repair. Possessing a Lys/Lys751 genotype increased the risk of sub-optimal DNA repair (odds ratio = 7.2, 95% confidence interval = 1.01-87.7). The Asp312Asn XPD polymorphism did not appear to affect DNA repair proficiency. These results suggest that the Lys751 (common) allele may alter the XPD protein product resulting in sub-optimal repair of X-ray-induced DNA damage. PMID:10753184

  12. The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD.

    PubMed

    Hegde, Pavana M; Dutta, Arijit; Sengupta, Shiladitya; Mitra, Joy; Adhikari, Sanjay; Tomkinson, Alan E; Li, Guo-Min; Boldogh, Istvan; Hazra, Tapas K; Mitra, Sankar; Hegde, Muralidhar L

    2015-08-21

    The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase ?, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ?8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells. PMID:26134572

  13. Complex Abdominal Wall Repair

    MedlinePLUS

    ... bowel and underwent placement of Veritas in an inlay. And as we talked about during the case, ... repair either utilizing an underlay technique or an inlay technique. I would not recommend placing this in ...

  14. Chromatin structure and DNA damage repair

    PubMed Central

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

    2008-01-01

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

  15. The efficiency of photolyase and indole complexes to repair DNA containing dimers of pyrimidine: A theoretical analysis of the electron transfer reactions

    NASA Astrophysics Data System (ADS)

    Volcov, Flávia; Goldman, Carla

    2004-02-01

    We analyze the effects of competing reactions to the efficiency of enzymatic splitting of pyrimidine dimers formed in DNA by the incidence of ultraviolet radiation. This is accomplished with the aid of a formula that expresses the efficiency of the repair in terms of parameters that regulate the reaction rates for primary and for back long-range electron transfers taking place in the process. Comparison of experimental data with estimations on account of this formula supports early conjectures in the literature that attribute the relative high performance of the enzymatic complexes of photolyase to its ability to suppress the back reaction.

  16. 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

  17. New insights into the mechanism of DNA mismatch repair.

    PubMed

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

    2015-12-01

    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 up-to-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

  18. 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.

  19. 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

  20. 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.

  1. Role of DNA repair protein ERCC1 in skin cancer 

    E-print Network

    Song, Liang

    2009-01-01

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

  2. 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)

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. DNA Repair 5 (2006) 145152 Mini review

    E-print Network

    Santos, Janine H.

    2006-01-01

    phosphorylation; the biosynthesis of heme, pyrimidines and steroids; calcium and iron homeostasis and programmed peroxide (H2O2) that in conjunction with its large iron stores can lead to a witch's brew of reactive. Published by Elsevier B.V. Keywords: DNA repair; Hydrogen peroxide; Mitochondrial DNA; Oxidative stress

  10. The awakening of DNA repair at Yale.

    PubMed

    Hanawalt, Philip C

    2013-12-01

    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

  11. 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

  12. 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

  13. Error-Prone Repair of DNA Double-Strand Breaks.

    PubMed

    Rodgers, Kasey; McVey, Mitch

    2016-01-01

    Preserving the integrity of the DNA double helix is crucial for the maintenance of genomic stability. Therefore, DNA double-strand breaks represent a serious threat to cells. In this review, we describe the two major strategies used to repair double strand breaks: non-homologous end joining and homologous recombination, emphasizing the mutagenic aspects of each. We focus on emerging evidence that homologous recombination, long thought to be an error-free repair process, can in fact be highly mutagenic, particularly in contexts requiring large amounts of DNA synthesis. Recent investigations have begun to illuminate the molecular mechanisms by which error-prone double-strand break repair can create major genomic changes, such as translocations and complex chromosome rearrangements. We highlight these studies and discuss proposed models that may explain some of the more extreme genetic changes observed in human cancers and congenital disorders. J. Cell. Physiol. 230: 15-24, 2016. © 2015 Wiley Periodicals, Inc. PMID:26033759

  14. 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.

  15. 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.

  16. Recombination and DNA Repair in Helicobacter pylori

    PubMed Central

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

    2013-01-01

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

  17. The RASSF1A Tumor Suppressor Regulates XPA-Mediated DNA Repair

    PubMed Central

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

    2014-01-01

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

  18. 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.

  19. RNase T in DNA repair DNA repair relies on various enzymes, including exonucleases that bind and trim

    E-print Network

    Chen, Sheng-Wei

    RNase T in DNA repair DNA repair relies on various enzymes, including exonucleases that bind and trim DNA at broken ends. However, we know little about how an exonuclease precisely selects and trims a DNA broken end in specific repair pathways. In this study, the enzyme RNase T, previously known

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

    PubMed Central

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

    2014-01-01

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

  1. Mutagenesis and DNA repair in mammalian cells

    SciTech Connect

    Glazer, P.M.

    1987-01-01

    Two aspects of DNA damage and repair in mammalian cells were investigated. Using a lambda phage shuttle vector, a system was developed to study mutations arising in the DNA of mammalian cells. This system was used to determine the spectrum of mutations induced in cellular DNA by ultraviolet light. Also, the repair of base pair mismatches in DNA was studied by the development of a method to detect a DNA mismatch repair activity in extracts made from cultured human cells. In order to study mutations arising in mammalian cells, stable mouse L cell lines were established with multiple copies of lambda phage vector which contains the supF gene of E. coli as a target for mutagenesis. Rescue of viable phage from high molecular weight mouse cell DNA using lambda in vitro packaging extracts was efficient and yielded a negligible background of phage with mutations in the supF gene. From mouse cells exposed to 12 J/m/sup 2/ of 254 nm ultraviolet (UV) light, 78,510 phage were rescued of which eight were found to have mutant supF genes. DNA sequence analysis of the mutants suggests that the primary site of UV mutagenesis in mammalian cells is at pyrimidine-cytosine (Py-C) sequences, and that the most frequent mutation at this site is a C to T transition.

  2. Intermediates of DNA double strand break repair in escherichia coli 

    E-print Network

    Mawer, Julia Sofia Pamela

    2012-06-22

    A DNA double-strand break (DSB) is a severe form of DNA damage. In fastgrowing cells, DSBs are commonly repaired by homologous recombination (HR) and in E. coli they are exclusively repaired by this mechanism. Failure ...

  3. DNA repair in hyperthermophilic and hyperradioresistant microorganisms.

    PubMed

    Ishino, Yoshizumi; Narumi, Issay

    2015-06-01

    The genome of a living cell is continuously under attack by exogenous and endogenous genotoxins. Especially, life at high temperature inflicts additional stress on genomic DNA, and very high rates of potentially mutagenic DNA lesions, including deamination, depurination, and oxidation, are expected. However, the spontaneous mutation rates in hyperthermophiles are similar to that in Escherichia coli, and it is interesting to determine how the hyperthermophiles preserve their genomes under such grueling environmental conditions. In addition, organisms with extremely radioresistant phenotypes are targets for investigating special DNA repair mechanisms in extreme environments. Multiple DNA repair mechanisms have evolved in all organisms to ensure genomic stability, by preventing impediments that result in genome destabilizing lesions. PMID:26056771

  4. Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes

    PubMed Central

    Wossidlo, Mark; Arand, Julia; Sebastiano, Vittorio; Lepikhov, Konstantin; Boiani, Michele; Reinhardt, Richard; Schöler, Hans; Walter, Jörn

    2010-01-01

    In mammalian zygotes, the 5-methyl-cytosine (5mC) content of paternal chromosomes is rapidly changed by a yet unknown but presumably active enzymatic mechanism. Here, we describe the developmental dynamics and parental asymmetries of DNA methylation in relation to the presence of DNA strand breaks, DNA repair markers and a precise timing of zygotic DNA replication. The analysis shows that distinct pre-replicative (active) and replicative (active and passive) phases of DNA demethylation can be observed. These phases of DNA demethylation are concomitant with the appearance of DNA strand breaks and DNA repair markers such as ?H2A.X and PARP-1, respectively. The same correlations are found in cloned embryos obtained after somatic cell nuclear transfer. Together, the data suggest that (1) DNA-methylation reprogramming is more complex and extended as anticipated earlier and (2) the DNA demethylation, particularly the rapid loss of 5mC in paternal DNA, is likely to be linked to DNA repair mechanisms. PMID:20442707

  5. Kiwifruit as a modulator of DNA damage and DNA repair.

    PubMed

    Collins, Andrew R

    2013-01-01

    Kiwifruit are a rich source of vitamin C and other antioxidants. We have demonstrated the capacity of kiwifruit to protect cellular DNA against oxidative damage in single-dose human experiments and in longer term supplementation trials using the comet assay to measure both DNA-strand breaks and oxidized bases. Enhanced antioxidant status following a single large dose of kiwifruit is shown by an increased resistance of lymphocyte DNA to oxidation by H(2)O(2)in vitro. After 3 weeks (or more) of supplementation, endogenous base oxidation is significantly decreased. In addition to its antioxidant potential, kiwifruit stimulates base excision repair as measured in an in vitro assay with DNA containing 8-oxoguanine as substrate. The relevance of DNA damage protection and modulation of DNA repair to cancer risk is discussed. PMID:23394994

  6. Early Days of DNA Repair: Discovery of Nucleotide Excision Repair and Homology-Dependent Recombinational Repair

    PubMed Central

    Rupp, W. Dean

    2013-01-01

    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. PMID:24348214

  7. Electrically monitoring DNA repair by photolyase

    NASA Astrophysics Data System (ADS)

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

    2005-08-01

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

  8. Introduction Telomere biology and DNA repair: Enemies with benefits

    E-print Network

    de Lange, Titia

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

  9. A lncRNA to repair DNA.

    PubMed

    Lukas, Jiri; Altmeyer, Matthias

    2015-11-01

    Long non-coding RNAs (lncRNAs) have emerged as regulators of various biological processes, but to which extent lncRNAs play a role in genome integrity maintenance is not well understood. In this issue of EMBO Reports, Sharma et al [1] identify the DNA damage-induced lncRNA DDSR1 as an integral player of the DNA damage response (DDR). DDSR1 has both an early role by modulating repair pathway choices, and a later function when it regulates gene expression. Sharma et al [1] thus uncover a dual role for a hitherto uncharacterized lncRNA during the cellular response to DNA damage. PMID:26420434

  10. DNA damage, DNA repair, and alcohol toxicity--a review.

    PubMed

    Brooks, P J

    1997-09-01

    Alcohol (ethanol) is clearly a toxic substance when consumed in excess. Chronic alcohol abuse results in a variety of pathological effects, including damage to the liver and brain, as well as other organs, and is associated with an increased risk of certain types of cancers. Alcohol consumption by pregnant women can result in fetal alcohol effects and fetal alcohol syndrome. All of these toxic effects are well documented. What is needed at present is a complete understanding of the molecular mechanisms by which alcohol causes these toxic effects. Such an understanding may lead to better treatments of some of these toxic effects. This review, focuses on the possibility that toxic effects of ethanol are mediated, at least in part, by damage to DNA. In particular, I emphasize data on the production of endogenous DNA-damaging molecules as a result of alcohol consumption and metabolism. Specific examples of DNA-damaging molecules to be considered are reactive oxygen species, including oxygen radicals, lipid peroxidation products, and acetaldehyde. The relevant DNA repair pathways that protect cells against DNA damage produced by these molecules will also be reviewed. The goal of this review is to integrate recent results from the fields of mutagenesis and DNA repair with the alcohol toxicity literature, with the aim of stimulating research into the role of DNA damage in different types of alcohol toxicity and the role of DNA repair in protecting cells from alcohol-related damage. PMID:9309320

  11. 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 design will not only assist in identifying novel functional moieties to replace the metabolically labile morpholino group but will also facilitate the design of molecules to target the DNA-PKcs/Ku80 interface or one of the autophosphorylation sites. PMID:23386830

  12. 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.

  13. The interplay between DNA repair and autophagy in cancer therapy.

    PubMed

    Zhang, Dan; Tang, Bo; Xie, Xia; Xiao, Yu-Feng; Yang, Shi-Ming; Zhang, Jian-Wei

    2015-01-01

    DNA is the prime target of anticancer treatments. DNA damage triggers a series of signaling cascades promoting cellular survival, including DNA repair, cell cycle arrest, and autophagy. The elevated basal and/or stressful levels of both DNA repair and autophagy observed in tumor cells, in contrast to normal cells, have been identified as the most important drug-responsive programs that impact the outcome of anticancer therapy. The exact relationship between DNA repair and autophagy in cancer cells remains unclear. On one hand, autophagy has been shown to regulate some of the DNA repair proteins after DNA damage by maintaining the balance between their synthesis, stabilization, and degradation. One the other hand, some evidence has demonstrated that some DNA repair molecular have a crucial role in the initiation of autophagy. In this review, we mainly discuss the interplay between DNA repair and autophagy in anticancer therapy and expect to enlighten some effective strategies for cancer treatment. PMID:25985143

  14. ISWI chromatin remodeling complexes in the DNA damage response

    PubMed Central

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

    2014-01-01

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

  15. Epigenetic changes of DNA repair genes in cancer

    PubMed Central

    Lahtz, Christoph; Pfeifer, Gerd P.

    2011-01-01

    ‘Every Hour Hurts, The Last One Kills'. That is an old saying about getting old. Every day, thousands of DNA damaging events take place in each cell of our body, but efficient DNA repair systems have evolved to prevent that. However, our DNA repair system and that of most other organisms are not as perfect as that of Deinococcus radiodurans, for example, which is able to repair massive amounts of DNA damage at one time. In many instances, accumulation of DNA damage has been linked to cancer, and genetic deficiencies in specific DNA repair genes are associated with tumor-prone phenotypes. In addition to mutations, which can be either inherited or somatically acquired, epigenetic silencing of DNA repair genes may promote tumorigenesis. This review will summarize current knowledge of the epigenetic inactivation of different DNA repair components in human cancer. PMID:21278452

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

    PubMed Central

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

    2014-01-01

    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. PMID:24947262

  17. DNA excision repair: where do all the dimers go?

    PubMed

    Kemp, Michael G; Sancar, Aziz

    2012-08-15

    Exposure of cells to UV light from the sun causes the formation of pyrimidine dimers in DNA that have the potential to lead to mutation and cancer. In humans, pyrimidine dimers are removed from the genome in the form of ~30 nt-long oligomers by concerted dual incisions. Though nearly 50 y of excision repair research has uncovered many details of UV photoproduct damage recognition and removal, the fate of the excised oligonucleotides and, in particular, the ultimate fate of the chemically very stable pyrimidine dimers remain unknown. Physiologically relevant UV doses introduce hundreds of thousands of pyrimidine dimers in diploid human cells, which are excised from the genome within ~24 h. Once removed from the genome, "where do all the dimers go?" In a recent study we addressed this question. Although our study did not determine the fate of the dimer itself, it revealed that the excised ~30-mer is released from the duplex in a tight complex with the transcription/repair factor TFIIH. This finding combined with recent reports that base and oligonucleotide products of the base and double-strand break repair pathways also make stable complexes with the cognate repair enzymes, and that these complexes activate the MAP kinase and checkpoint signaling pathways, respectively, raises the possibility that TFIIH-30-mer excision complexes may play a role in signaling reactions in response to UV damage. PMID:22825251

  18. Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA

    SciTech Connect

    Yang, Cai-Guang; Yi, Chengqi; Duguid, Erica M.; Sullivan, Christopher T.; Jian, Xing; Rice, Phoebe A.; He, Chuan

    2008-09-26

    Escherichia coli AlkB and its human homologues ABH2 and ABH3 repair DNA/RNA base lesions by using a direct oxidative dealkylation mechanism. ABH2 has the primary role of guarding mammalian genomes against 1-meA damage by repairing this lesion in double-stranded DNA (dsDNA), whereas AlkB and ABH3 preferentially repair single-stranded DNA (ssDNA) lesions and can repair damaged bases in RNA. Here we show the first crystal structures of AlkB-dsDNA and ABH2-dsDNA complexes, stabilized by a chemical cross-linking strategy. This study reveals that AlkB uses an unprecedented base-flipping mechanism to access the damaged base: it squeezes together the two bases flanking the flipped-out one to maintain the base stack, explaining the preference of AlkB for repairing ssDNA lesions over dsDNA ones. In addition, the first crystal structure of ABH2, presented here, provides a structural basis for designing inhibitors of this human DNA repair protein.

  19. 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. PMID:21600859

  20. DNA Computing Complexity Analysis Using DNA/DNA Hybridization Kinetics

    E-print Network

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

  1. RNA-directed repair of DNA double-strand breaks.

    PubMed

    Yang, Yun-Gui; Qi, Yijun

    2015-08-01

    DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, which if unrepaired or repaired incorrectly can cause cell death or genome instability that may lead to cancer. To counteract these adverse consequences, eukaryotes have evolved a highly orchestrated mechanism to repair DSBs, namely DNA-damage-response (DDR). DDR, as defined specifically in relation to DSBs, consists of multi-layered regulatory modes including DNA damage sensors, transducers and effectors, through which DSBs are sensed and then repaired via DNAprotein interactions. Unexpectedly, recent studies have revealed a direct role of RNA in the repair of DSBs, including DSB-induced small RNA (diRNA)-directed and RNA-templated DNA repair. Here, we summarize the recent discoveries of RNA-mediated regulation of DSB repair and discuss the potential impact of these novel RNA components of the DSB repair pathway on genomic stability and plasticity. PMID:25960340

  2. Structural dynamics in DNA damage signaling and repair

    PubMed Central

    Perry, J. Jefferson P.; Cotner-Gohara, Elizabeth; Ellenberger, Tom; Tainer, John A.

    2010-01-01

    Changing macromolecular conformations and complexes are critical features of cellular networks, typified by DNA damage response pathways that are essential to life. These fluctuations enhance specificity of macromolecular recognition and catalysis, and enable an integrated functioning of pathway components, ensuring efficiency while reducing off pathway reactions. Such dynamic complexes challenge classical detailed structural analyses, so there characterizations demand combining methods that provide detail with those that inform dynamics in solution. Small angle x-ray scattering, electron microscopy, hydrogen-deuterium-exchange and computation are complementing detailed structures from crystallography and NMR to provide comprehensive models for DNA damage searching, specificity, signaling and repair. Here, we review new approaches and results on DNA damage responses that advance structural biology in the fourth dimension, connecting proteins to pathways. PMID:20439160

  3. Comet assay to measure DNA repair: approach and applications

    PubMed Central

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

    2014-01-01

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

  4. Repair response of Escherichia coli to hydrogen peroxide DNA damage

    SciTech Connect

    Hagensee, M.E.; Moses, R.E.

    1986-12-01

    The repair response of Escherichia coli to hydrogen peroxide-induced DNA damage was investigated in intact and toluene-treated cells. Cellular DNA was cleaved after treatment by hydrogen peroxide as analyzed by alkaline sucrose sedimentation. The incision step did not require ATP or magnesium and was not inhibited by N-ethylmaleimide (NEM). An ATP-independent, magnesium-dependent incorporation of nucleotides was seen after the exposure of cells to hydrogen peroxide. This DNA repair synthesis was not inhibited by the addition of NEM or dithiothreitol. In dnaB (Ts) strain CRT266, which is thermolabile for DNA replication, normal levels of DNA synthesis were found at the restrictive temperature (43/sup 0/C), showing that DNA replication was not necessary for this DNA synthesis. Density gradient analysis also indicated that hydrogen peroxide inhibited DNA replication and stimulated repair synthesis. The subsequent reformation step required magnesium, did not require ATP, and was not inhibited by NEM, in agreement with the synthesis requirements. This suggests that DNA polymerase I was involved in the repair step. Furthermore, a strain defective in DNA polymerase I was unable to reform its DNA after peroxide treatment. Chemical cleavage of the DNA was shown by incision of supercoiled DNA with hydrogen peroxide in the presence of low concentration of ferric chloride. These findings suggest that hydrogen peroxide directly incises DNA, causing damage which is repaired by an incision repair pathway that requires DNA polymerase I.

  5. Transcriptional regulation of human DNA repair genes following genotoxic stress: trigger mechanisms, inducible responses and genotoxic adaptation

    PubMed Central

    Kaina, Bernd

    2013-01-01

    DNA repair is the first barrier in the defense against genotoxic stress. In recent years, mechanisms that recognize DNA damage and activate DNA repair functions through transcriptional upregulation and post-translational modification were the focus of intensive research. Most DNA repair pathways are complex, involving many proteins working in discrete consecutive steps. Therefore, their balanced expression is important for avoiding erroneous repair that might result from excessive base removal and DNA cleavage. Amelioration of DNA repair requires both a fine-tuned system of lesion recognition and transcription factors that regulate repair genes in a balanced way. Transcriptional upregulation of DNA repair genes by genotoxic stress is counteracted by DNA damage that blocks transcription. Therefore, induction of DNA repair resulting in an adaptive response is only visible through a narrow window of dose. Here, we review transcriptional regulation of DNA repair genes in normal and cancer cells and describe mechanisms of promoter activation following genotoxic exposures through environmental carcinogens and anticancer drugs. The data available to date indicate that 25 DNA repair genes are subject to regulation following genotoxic stress in rodent and human cells, but for only a few of them, the data are solid as to the mechanism, homeostatic regulation and involvement in an adaptive response to genotoxic stress. PMID:23892398

  6. 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.

  7. 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.

  8. Hierarchies of DNA repair in mammalian cells: biological consequences.

    PubMed

    Mullenders, L H; Vrieling, H; Venema, J; van Zeeland, A A

    1991-01-01

    Mammalian cells exposed to genotoxic agents exhibit heterogeneous levels of repair of certain types of DNA damage in various genomic regions. For UV-induced cyclobutane pyrimidine dimers we propose that at least three levels of repair exist: (1) slow repair of inactive (X-chromosomal) genes, (2) fast repair of active housekeeping genes, and (3) accelerated repair of the transcribed strand of active genes. These hierarchies of repair may be related to chromosomal banding patterns as obtained by Giemsa staining. The possible consequences of defective DNA repair in one or more of these levels may be manifested in different clinical features associated with UV-sensitive human syndromes. Moreover, molecular analysis of hprt mutations reveals that mutations are primarily generated by DNA damage in the poorly repaired non-transcribed strand of the gene. PMID:1944339

  9. DNA Computing Complexity Analysis Using DNA/DNA Hybridization Kinetics

    E-print Network

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

  10. Human DNA polymerase ? 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 ? protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Breast, lung and oral cancers over-express polymerase ?, 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 ?, 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 ? 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 ? grasps the primer to bypass DNA lesions, or extend poorly annealed DNA termini to mediate end-joining. PMID:25775267

  11. Structure of the DNA repair helicase XPD.

    PubMed

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

    2008-05-30

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

  12. Gemcitabine Functions Epigenetically by Inhibiting Repair Mediated DNA Demethylation

    PubMed Central

    Schäfer, Andrea; Schomacher, Lars; Barreto, Guillermo; Döderlein, Gabi; Niehrs, Christof

    2010-01-01

    Gemcitabine is a cytotoxic cytidine analog, which is widely used in anti-cancer therapy. One mechanism by which gemcitabine acts is by inhibiting nucleotide excision repair (NER). Recently NER was implicated in Gadd45 mediated DNA demethylation and epigenetic gene activation. Here we analyzed the effect of gemcitabine on DNA demethylation. We find that gemcitabine inhibits specifically Gadd45a mediated reporter gene activation and DNA demethylation, similar to the topoisomerase I inhibitor camptothecin, which also inhibits NER. In contrast, base excision repair inhibitors had no effect on DNA demethylation. In Xenopus oocytes, gemcitabine inhibits DNA repair synthesis accompanying demethylation of oct4. In mammalian cells, gemcitabine induces DNA hypermethylation and silencing of MLH1. The results indicate that gemcitabine induces epigenetic gene silencing by inhibiting repair mediated DNA demethylation. Thus, gemcitabine can function epigenetically and provides a tool to manipulate DNA methylation. PMID:21124914

  13. Lack of CAK complex accumulation at DNA damage sites in XP-B and XP-B/CS fibroblasts reveals differential regulation of CAK anchoring to core TFIIH by XPB and XPD helicases during nucleotide excision repair

    PubMed Central

    Zhu, Qianzheng; Wani, Gulzar; Sharma, Nidhi; Wani, Altaf

    2012-01-01

    Transcription factor II H (TFIIH) is composed of core TFIIH and Cdk-activating kinase (CAK) complexes. Besides transcription, TFIIH also participates in nucleotide excision repair (NER), verifying DNA lesions through its helicase components XPB and XPD. The assembly state of TFIIH is known to be affected by truncation mutations in Xeroderma pigmentosum group G/Cockayne syndrome (XP-G/CS). Here, we showed that CAK component MAT1 was rapidly recruited to UV-induced DNA damage sites, co-localizing with core TFIIH component p62, and dispersed from the damage sites upon completion of DNA repair. While the core TFIIH-CAK association remained intact, MAT1 failed to accumulate at DNA damage sites in fibroblasts harboring XP-B or XP-B/CS mutations. Nevertheless, MAT1, XPD and XPC as well as XPG were able to accumulate at damage sites in XP-D fibroblasts, in which the core TFIIH-CAK association also remained intact. Interestingly, XPG recruitment was impaired in XP-B/CS fibroblasts derived from patients with mild phenotype, but persisted in XP-B/CS fibroblasts from severely affected patients resulting in a nonfunctional preincision complex. An examination of steady-state levels of RNA polymerase II (RNAPII) indicated that UV-induced RNAPII phosphorylation was dramatically reduced in XP-B/CS fibroblasts. These results demonstrated that the CAK rapidly disassociates from the core TFIIH upon assembly of nonfunctional preincision complex in XP-B and XP-B/CS cells. The persistency of nonfunctional preincision complex correlates with the severity exhibited by XP-B patients. The results suggest that XPB and XPD helicases differentially regulate the anchoring of CAK to core TFIIH during damage verification step of NER. PMID:23083890

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

    PubMed

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

    2015-12-15

    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 the cell-cycle kinase CDK1 and nuclear DNA repair. The basal and radiation-induced mitochondrial ATP generation is reduced significantly in cells harboring CDK1 phosphorylation-deficient mutant complex I subunits. Similarly, mitochondrial ATP generation and nuclear DNA repair are also compromised severely in cells harboring mitochondrially targeted, kinase-deficient CDK1. These results demonstrate a mechanism governing the communication between mitochondria and the nucleus by which CDK1 boosts mitochondrial bioenergetics to meet the increased cellular fuel demand for DNA repair and cell survival under genotoxic stress conditions. PMID:26670043

  15. Supramolecular Complexes of DNA

    NASA Astrophysics Data System (ADS)

    Zuber, G.; Scherman, D.

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

  16. UV sensitivity and impaired nucleotide excision repair in DNA-dependent protein kinase mutant cells.

    PubMed Central

    Muller, C; Calsou, P; Frit, P; Cayrol, C; Carter, T; Salles, B

    1998-01-01

    DNA-dependent protein kinase (DNA-PK), a member of the phosphatidyl-inositol (PI)3-kinase family, is involved in the repair of DNA double-strand breaks. Its regulatory subunit, Ku, binds to DNA and recruits the kinase catalytic subunit (DNA-PKcs). We show here a new role of DNA-PK in the modulation of the process of nucleotide excision repair (NER) in vivo since, as compared with their respective parental cell lines, DNA-PK mutants (scid , V-3 and xrs 6 cells) exhibit sensitivity to UV-C irradiation (2.0- to 2.5-fold) and cisplatin ( approximately 3- to 4-fold) associated with a decreased activity (40-55%) of unscheduled DNA synthesis after UV-C irradiation. Moreover, we observed that wortmannin sensitized parental cells in vivo when combined with either cisplatin or UV-C light, but had no effect on the DNA-PKcs deficient scid cells. Despite a lower repair synthesis activity (approximately 2-fold) measured in vitro with nuclear cell extracts from DNA-PK mutants, a direct involvement of DNA-PK in the NER reaction in vitro has not been observed. This study establishes a regulatory function of DNA-PK in the NER process in vivo but rules out a physical role of the complex in the repair machinery at the site of the DNA lesion. PMID:9490781

  17. 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.

  18. Damage and repair of nerve cell DNA in toxic stress.

    PubMed

    Kisby, G E; Kabel, H; Hugon, J; Spencer, P

    1999-08-01

    It is generally agreed that ALS/PDC is triggered by a disappearing environmental factor peculiar to the lifestyle of people of the western Pacific (i.e., Guam, Irian Jaya, Indonesia, and the Kii Peninsula of Japan). A strong candidate is the cycad plant genotoxin cycasin, the beta-D-glucoside of methylazoxymethanol (MAM). We propose that prenatal or postnatal exposure to low levels of cycasin/MAM may damage neuronal DNA, compromise DNA repair, perturb neuronal gene expression, and irreversibly alter cell function to precipitate a slowly evolving disease ("slow-toxin" hypothesis). In support of our hypothesis, we have demonstrated the following: 1. DNA from postmitotic rodent central nervous system neurons is particularly sensitive to damage by MAM. 2. MAM reduces DNA repair in human and rodent neurons, whereas DNA-repair inhibitors potentiate MAM-induced DNA damage and toxicity in mature rodent nervous tissue. 3. Human neurons (SY5Y neuroblastoma) that are deficient in DNA repair are susceptible to MAM-induced cytotoxicity and DNA damage, whereas overexpression of DNA repair in similar cells is protective. 4. MAM alters gene expression in SY5Y human neuroblastoma cells and, in the presence of DNA damage and reduced DNA repair, enhances glutamate-modulated expression of tau mRNA in rat primary neurons; the corresponding protein (TAU) is elevated in ALS/PDC and Alzheimer's disease. These findings support a direct relationship between MAM-induced DNA damage and neurotoxicity and suggest the genotoxin may operate in a similar manner in vivo. More broadly, a combination of genotoxin-induced DNA damage (via exogenous and/or endogenous agents) and disturbed DNA repair may be important contributing factors in the slow and progressive degeneration of neurons that is characteristic of sporadic neurodegenerative disease. Preliminary studies demonstrate that DNA repair is reduced in the brain of subjects with western Pacific ALS/PDC, ALS, and Alzheimer's disease, which would increase the susceptibility of brain tissue to DNA damage by endogenous/exogenous genotoxins. Interindividual differences in the extent of prior exposure to DNA-damaging agents and/or the efficiency of its repair might produce population variety in the rate of damage accumulation and explain the susceptibility of certain individuals to sporadic neurodegenerative disease. Studies are underway using DNA-repair proficient and deficient neuronal cell cultures and mutant mice to explore gene-environment interplay with respect to MAM treatment, DNA damage, and DNA repair, and the age-related appearance of neurobehavioral and neuropathological compromise. PMID:10461542

  19. Strategies for DNA interstrand crosslink repair: insights from worms, flies, frogs, and slime molds.

    PubMed

    McVey, Mitch

    2010-07-01

    DNA interstrand crosslinks (ICLs) are complex lesions that covalently link both strands of the DNA double helix and impede essential cellular processes such as DNA replication and transcription. Recent studies suggest that multiple repair pathways are involved in their removal. Elegant genetic analysis has demonstrated that at least three distinct sets of pathways cooperate in the repair and/or bypass of ICLs in budding yeast. Although the mechanisms of ICL repair in mammals appear similar to those in yeast, important differences have been documented. In addition, mammalian crosslink repair requires other repair factors, such as the Fanconi anemia proteins, whose functions are poorly understood. Because many of these proteins are conserved in simpler metazoans, nonmammalian models have become attractive systems for studying the function(s) of key crosslink repair factors. This review discusses the contributions that various model organisms have made to the field of ICL repair. Specifically, it highlights how studies performed with C. elegans, Drosophila, Xenopus, and the social amoeba Dictyostelium serve to complement those from bacteria, yeast, and mammals. Together, these investigations have revealed that although the underlying themes of ICL repair are largely conserved, the complement of DNA repair proteins utilized and the ways in which each of the proteins is used can vary substantially between different organisms. PMID:20143343

  20. DNA Damage Repair in the Context of Plant Chromatin.

    PubMed

    Donà, Mattia; Mittelsten Scheid, Ortrun

    2015-08-01

    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

  1. Mutation Research 509 (2002) 127151 Mitochondrial DNA repair and aging

    E-print Network

    Santos, Janine H.

    2002-01-01

    Mutation Research 509 (2002) 127­151 Mitochondrial DNA repair and aging Bhaskar S. Mandavilli species that damage DNA, RNA and proteins in the cell. Oxidative damage to the mitochondrial DNA, Drosophila and yeast. Research in the last several years shows that mitochondrial DNA is more susceptible

  2. The BLM dissolvasome in DNA replication and repair

    PubMed Central

    Manthei, Kelly A.; Keck, James L.

    2013-01-01

    RecQ DNA helicases are critical for proper maintenance of genomic stability and mutations in multiple human RecQ genes are linked with genetic disorders characterized by a predisposition to cancer. RecQ proteins are conserved from prokaryotes to humans and in all cases form higher-order complexes with other proteins to efficiently execute their cellular functions. The focus of this review is a conserved complex that is formed between RecQ helicases and type-I topoisomerases. In humans, this complex is referred to as the BLM dissolvasome or BTR complex, and is comprised of the RecQ helicase BLM, topoisomerase III?, and the RMI proteins. The BLM dissolvasome functions to resolve linked DNA intermediates without exchange of genetic material, which is critical in somatic cells. We will review the history of this complex and highlight its roles in DNA replication, recombination, and repair. Additionally, we will review recently established interactions between BLM dissolvasome and a second set of genome maintenance factors (the Fanconi anemia proteins) that appear to allow coordinated genome maintenance efforts between the two systems. PMID:23543275

  3. The Role of the COP9 Signalosome and Neddylation in DNA Damage Signaling and Repair

    PubMed Central

    Chung, Dudley; Dellaire, Graham

    2015-01-01

    The maintenance of genomic integrity is an important process in organisms as failure to sense and repair damaged DNA can result in a variety of diseases. Eukaryotic cells have developed complex DNA repair response (DDR) mechanisms to accurately sense and repair damaged DNA. Post-translational modifications by ubiquitin and ubiquitin-like proteins, such as SUMO and NEDD8, have roles in coordinating the progression of DDR. Proteins in the neddylation pathway have also been linked to regulating DDR. Of interest is the COP9 signalosome (CSN), a multi-subunit metalloprotease present in eukaryotes that removes NEDD8 from cullins and regulates the activity of cullin-RING ubiquitin ligases (CRLs). This in turn regulates the stability and turnover of a host of CRL-targeted proteins, some of which have established roles in DDR. This review will summarize the current knowledge on the role of the CSN and neddylation in DNA repair. PMID:26437438

  4. The Role of the COP9 Signalosome and Neddylation in DNA Damage Signaling and Repair.

    PubMed

    Chung, Dudley; Dellaire, Graham

    2015-01-01

    The maintenance of genomic integrity is an important process in organisms as failure to sense and repair damaged DNA can result in a variety of diseases. Eukaryotic cells have developed complex DNA repair response (DDR) mechanisms to accurately sense and repair damaged DNA. Post-translational modifications by ubiquitin and ubiquitin-like proteins, such as SUMO and NEDD8, have roles in coordinating the progression of DDR. Proteins in the neddylation pathway have also been linked to regulating DDR. Of interest is the COP9 signalosome (CSN), a multi-subunit metalloprotease present in eukaryotes that removes NEDD8 from cullins and regulates the activity of cullin-RING ubiquitin ligases (CRLs). This in turn regulates the stability and turnover of a host of CRL-targeted proteins, some of which have established roles in DDR. This review will summarize the current knowledge on the role of the CSN and neddylation in DNA repair. PMID:26437438

  5. DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

    PubMed Central

    Boiteux, Serge; Jinks-Robertson, Sue

    2013-01-01

    DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage. PMID:23547164

  6. 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) component of sunlight. NER can be divided into two classes based on where the repair occurs. NER occurring in DNA that is not undergoing transcription (i.e., most of the genome) is called global genome repair (GGR or GGNER), while NER taking place in the transcribed strand of active genes is called transcription-coupled repair (TCR or TC-NER). We will explore NER in more detail below. Mismatch repair (MMR) is another type of excision repair that specifically removes mispaired bases resulting from replication errors. DNA damage can also result in breaks in the DNA backbone, in one or both strands. Single-strand breaks (SSBs) are efficiently repaired by a mechanism that shares common features with the later steps in BER. Double-strand breaks (DSBs) are especially devastating since by definition there is no intact complementary strand to serve as a template for repair, and even one unrepaired DSB can be lethal [3]. In cells that have replicated their DNA prior to cell division, the missing information can be supplied by the duplicate copy, or sister chromatid, and DSBs in these cells are faithfully repaired by homologous recombination involving the exchange of strands of DNA between the two copies. However, most cells in the body are non-dividing, and in these cells the major mechanism for repairing DSBs is by non-homologous end joining (NHEJ), which as the name implies involves joining two broken DNA ends together without a requirement for homologous sequence and which therefore has a high potential for loss of genetic information.

  7. Distinct structural alterations in PCNA block DNA mismatch repair

    PubMed Central

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

    2013-01-01

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

  8. Nuclear GIT2 Is an ATM Substrate and Promotes DNA Repair

    PubMed Central

    Lu, Daoyuan; Cai, Huan; Park, Sung-Soo; Siddiqui, Sana; Premont, Richard T.; Schmalzigaug, Robert; Paramasivam, Manikandan; Seidman, Michael; Bodogai, Ionoa; Biragyn, Arya; Daimon, Caitlin M.; Martin, Bronwen

    2015-01-01

    Insults to nuclear DNA induce multiple response pathways to mitigate the deleterious effects of damage and mediate effective DNA repair. G-protein-coupled receptor kinase-interacting protein 2 (GIT2) regulates receptor internalization, focal adhesion dynamics, cell migration, and responses to oxidative stress. Here we demonstrate that GIT2 coordinates the levels of proteins in the DNA damage response (DDR). Cellular sensitivity to irradiation-induced DNA damage was highly associated with GIT2 expression levels. GIT2 is phosphorylated by ATM kinase and forms complexes with multiple DDR-associated factors in response to DNA damage. The targeting of GIT2 to DNA double-strand breaks was rapid and, in part, dependent upon the presence of H2AX, ATM, and MRE11 but was independent of MDC1 and RNF8. GIT2 likely promotes DNA repair through multiple mechanisms, including stabilization of BRCA1 in repair complexes; upregulation of repair proteins, including HMGN1 and RFC1; and regulation of poly(ADP-ribose) polymerase activity. Furthermore, GIT2-knockout mice demonstrated a greater susceptibility to DNA damage than their wild-type littermates. These results suggest that GIT2 plays an important role in MRE11/ATM/H2AX-mediated DNA damage responses. PMID:25605334

  9. Repair of a Complex Congenital Cardiac Defect

    MedlinePLUS Videos and Cool Tools

    ... 00:00:14 ANNOUNCER: Over the next hour, live from Children's Hospital Boston's cardiac OR, see cardiac surgeons repair a complex congenital cardiac defect. Each year the pediatric cardiac surgery program provides surgical care to approximately 1,100 patients, including more than 700 cases of open ...

  10. Structural and Functional Interaction Between the Human DNA Repair Proteins DNA ligase IV and XRCC4

    SciTech Connect

    Wu, P.; Meesala, S; Dauvillier, S; Modesti, M; Andres, S; Huang, Y; Sekiguchi, J; Calsou, P; Salles, B; Junop, M

    2009-01-01

    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.

  11. 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.

  12. DNA end recognition by the Mre11 nuclease dimer: insights into resection and repair of damaged DNA

    PubMed Central

    Sung, Sihyun; Li, Fuyang; Park, Young Bong; Kim, Jin Seok; Kim, Ae-Kyoung; Song, Ok-kyu; Kim, Jiae; Che, Jun; Lee, Sang Eun; Cho, Yunje

    2014-01-01

    The Mre11–Rad50–Nbs1 (MRN) complex plays important roles in sensing DNA damage, as well as in resecting and tethering DNA ends, and thus participates in double-strand break repair. An earlier structure of Mre11 bound to a short duplex DNA molecule suggested that each Mre11 in a dimer recognizes one DNA duplex to bridge two DNA ends at a short distance. Here, we provide an alternative DNA recognition model based on the structures of Methanococcus jannaschii Mre11 (MjMre11) bound to longer DNA molecules, which may more accurately reflect a broken chromosome. An extended stretch of B-form DNA asymmetrically runs across the whole dimer, with each end of this DNA molecule being recognized by an individual Mre11 monomer. DNA binding induces rigid-body rotation of the Mre11 dimer, which could facilitate melting of the DNA end and its juxtaposition to an active site of Mre11. The identified Mre11 interface binding DNA duplex ends is structurally conserved and shown to functionally contribute to efficient resection, non-homologous end joining, and tolerance to DNA-damaging agents when other resection enzymes are absent. Together, the structural, biochemical, and genetic findings presented here offer new insights into how Mre11 recognizes damaged DNA and facilitates DNA repair. PMID:25107472

  13. Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage ?

    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 ? 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

  14. 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.

  15. 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.

  16. 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.

  17. Proteins that participate in nucleotide excision repair of DNA in mammalian cells.

    PubMed

    Wood, R D

    1995-01-30

    The most versatile strategy for repair of damage to DNA, and the main process for repair of UV-induced damage, is nucleotide excision repair. In mammalian cells, the complete mechanism involves more than 20 polypeptides, and defects in many of these are associated with various forms of inherited disorders in humans. The syndrome xeroderma pigmentosum (XP) is associated with mutagen hypersensitivity and increased cancer frequency, and studies of the nucleotide excision repair defect in this disease have been particularly informative. Many of the XP proteins are now being characterized. XPA binds to DNA, with a preference for damaged base pairs. XPC activity is part of a protein complex with single-stranded DNA binding activity. The XPG protein is a nuclease. PMID:7746857

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

    E-print Network

    Kowalczykowski, Stephen C.

    Intelligent Design | Overwhelming Evidence Human DNA repair process video - by chance? Uncommon Descent - Sat, 2009-01-31 00:42 More details of DNA repair have been revealed. See: Human DNA repair process recorded in action (Video) (PhysOrg.com) -- A key phase in the repair process of damaged human DNA

  19. Single cell visualization of DNA repair in vivo.

    NASA Astrophysics Data System (ADS)

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

    2008-03-01

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

  20. Molecular regulation of UV-induced DNA repair.

    PubMed

    Shah, Palak; He, Yu-Ying

    2015-01-01

    Ultraviolet (UV) radiation from sunlight is a major etiologic factor for skin cancer, the most prevalent cancer in the United States, as well as premature skin aging. In particular, UVB radiation causes formation of specific DNA damage photoproducts between pyrimidine bases. These DNA damage photoproducts are repaired by a process called nucleotide excision repair, also known as UV-induced DNA repair. When left unrepaired, UVB-induced DNA damage leads to accumulation of mutations, predisposing people to carcinogenesis as well as to premature aging. Genetic loss of nucleotide excision repair leads to severe disorders, namely, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS), which are associated with predisposition to skin carcinogenesis at a young age as well as developmental and neurological conditions. Regulation of nucleotide excision repair is an attractive avenue to preventing or reversing these detrimental consequences of impaired nucleotide excision repair. Here, we review recent studies on molecular mechanisms regulating nucleotide excision repair by extracellular cues and intracellular signaling pathways, with a special focus on the molecular regulation of individual repair factors. PMID:25534312

  1. A Fluorescent Probe to Measure DNA Damage and Repair

    PubMed Central

    Condie, Allison G.; Yan, Yan; Gerson, Stanton L.; Wang, Yanming

    2015-01-01

    DNA damage and repair is a fundamental process that plays an important role in cancer treatment. Base excision repair (BER) is a major repair pathway that often leads to drug resistance in DNA-targeted cancer chemotherapy. In order to measure BER, we have developed a near infrared (NIR) fluorescent probe. This probe binds to a key intermediate, termed apurinic/apyrimidinic (AP) site, in the BER pathway where DNA damage and repair occurs. We have developed an assay to show the efficacy of the probe binding to AP sites and have shown that it can distinguish AP sites in DNA extract from chemotherapy treated cells. This probe has potential application in monitoring patient response to chemotherapy and evaluating new drugs in development. PMID:26309022

  2. Redox signaling between DNA repair proteins for efficient lesion detection

    E-print Network

    Newman, Dianne K.

    Base excision repair (BER) enzymes maintain the integrity of the genome, and in humans, BER mutations are associated with cancer. Given the remarkable sensitivity of DNA-mediated charge transport (CT) to mismatched and ...

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

    SciTech Connect

    Not Available

    1994-07-01

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

  4. 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.

  5. Modeling Damage Complexity-Dependent Non-Homologous End-Joining Repair Pathway

    PubMed Central

    Li, Yongfeng; Reynolds, Pamela; O'Neill, Peter; Cucinotta, Francis A.

    2014-01-01

    Non-homologous end joining (NHEJ) is the dominant DNA double strand break (DSB) repair pathway and involves several repair proteins such as Ku, DNA-PKcs, and XRCC4. It has been experimentally shown that the choice of NHEJ proteins is determined by the complexity of DSB. In this paper, we built a mathematical model, based on published data, to study how NHEJ depends on the damage complexity. Under an appropriate set of parameters obtained by minimization technique, we can simulate the kinetics of foci track formation in fluorescently tagged mammalian cells, Ku80-EGFP and DNA-PKcs-YFP for simple and complex DSB repair, respectively, in good agreement with the published experimental data, supporting the notion that simple DSB undergo fast repair in a Ku-dependent, DNA-PKcs-independent manner, while complex DSB repair requires additional DNA-PKcs for end processing, resulting in its slow repair, additionally resulting in slower release rate of Ku and the joining rate of complex DNA ends. Based on the numerous experimental descriptions, we investigated several models to describe the kinetics for complex DSB repair. An important prediction of our model is that the rejoining of complex DSBs is through a process of synapsis formation, similar to a second order reaction between ends, rather than first order break filling/joining. The synapsis formation (SF) model allows for diffusion of ends before the synapsis formation, which is precluded in the first order model by the rapid coupling of ends. Therefore, the SF model also predicts the higher number of chromosomal aberrations observed with high linear energy transfer (LET) radiation due to the higher proportion of complex DSBs compared to low LET radiation, and an increased probability of misrejoin following diffusion before the synapsis is formed, while the first order model does not provide a mechanism for the increased effectiveness in chromosomal aberrations observed. PMID:24520318

  6. 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 act as molecular level transformers that typically reshape the DNA and sometimes themselves to achieve extraordinary specificity and efficiency. PMID:24754999

  7. Defective DNA repair and increased chromatin binding of DNA repair factors in Down syndrome fibroblasts.

    PubMed

    Necchi, Daniela; Pinto, Antonella; Tillhon, Micol; Dutto, Ilaria; Serafini, Melania Maria; Lanni, Cristina; Govoni, Stefano; Racchi, Marco; Prosperi, Ennio

    2015-10-01

    Down syndrome (DS) is characterized by genetic instability, neurodegeneration, and premature aging. However, the molecular mechanisms leading to this phenotype are not yet well understood. Here, we report that DS fibroblasts from both fetal and adult donors show the presence of oxidative DNA base damage, such as dihydro-8-oxoguanine (8-oxodG), and activation of a DNA damage response (DDR), already during unperturbed growth conditions. DDR with checkpoint activation was indicated by histone H2AX and Chk2 protein phosphorylation, and by increased p53 protein levels. In addition, both fetal and adult DS fibroblasts were more sensitive to oxidative DNA damage induced by potassium bromate, and were defective in the removal of 8-oxodG, as compared with age-matched cells from control healthy donors. The analysis of core proteins participating in base excision repair (BER), such as XRCC1 and DNA polymerase ?, showed that higher amounts of these factors were bound to chromatin in DS than in control cells, even in the absence of DNA damage. These findings occurred in concomitance with increased levels of phosphorylated XRCC1 detected in DS cells. These results indicate that DS cells exhibit a BER deficiency, which is associated with prolonged chromatin association of core BER factors. PMID:26258283

  8. 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.

  9. 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.

  10. 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. PMID:23684800

  11. Repair of Alkylation Damage in Eukaryotic Chromatin Depends on Searching Ability of Alkyladenine DNA Glycosylase.

    PubMed

    Zhang, Yaru; O'Brien, Patrick J

    2015-11-20

    Human alkyladenine DNA glycosylase (AAG) initiates the base excision repair pathway by excising alkylated and deaminated purine lesions. In vitro biochemical experiments demonstrate that AAG uses facilitated diffusion to efficiently search DNA to find rare sites of damage and suggest that electrostatic interactions are critical to the searching process. However, it remains an open question whether DNA searching limits the rate of DNA repair in vivo. We constructed AAG mutants with altered searching ability and measured their ability to protect yeast from alkylation damage in order to address this question. Each of the conserved arginine and lysine residues that are near the DNA binding interface were mutated, and the functional impacts were evaluated using kinetic and thermodynamic analysis. These mutations do not perturb catalysis of N-glycosidic bond cleavage, but they decrease the ability to capture rare lesion sites. Nonspecific and specific DNA binding properties are closely correlated, suggesting that the electrostatic interactions observed in the specific recognition complex are similarly important for DNA searching complexes. The ability of the mutant proteins to complement repair-deficient yeast cells is positively correlated with the ability of the proteins to search DNA in vitro, suggesting that cellular resistance to DNA alkylation is governed by the ability to find and efficiently capture cytotoxic lesions. It appears that chromosomal access is not restricted and toxic sites of alkylation damage are readily accessible to a searching protein. PMID:26317160

  12. DNA repair in premature aging disorders and neurodegeneration.

    PubMed

    Coppedè, Fabio; Migliore, Lucia

    2010-02-01

    The accumulation of DNA damage has been widely implicated in premature aging and neurodegeneration. Progeroid syndromes with defects in the cellular response to DNA damage suggest that progressive genome instability represents an important aspect of the aging process. Moreover, most of the major neurodegenerative diseases are characterized by the accumulation of neuronal DNA damage, suggesting that impaired DNA repair mechanisms might be relevant to both premature aging and neurodegeneration. Two progeroid syndromes, Hutchinson-Gilford progeria syndrome and Werner's syndrome, are characterized by clinical features mimicking physiological aging at an early age and molecular studies have implicated decreased cell proliferation and altered DNA-damage responses as common causal mechanisms in the pathogenesis of both diseases. Defects in nucleotide excision repair cause three distinct human diseases: xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy; each of them is characterized by premature onset of pathologies that overlap with those associated with old age in humans. Increasing evidence also suggests that an impaired DNA repair, particularly the base excision repair pathway, might play a fundamental role in the development of age-related neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and Huntington' s disease. Here, we review the current knowledge on the role of DNA repair in premature aging and neurodegenerative diseases. PMID:20298165

  13. 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.

  14. 53BP1: pro choice in DNA repair.

    PubMed

    Zimmermann, Michal; de Lange, Titia

    2014-02-01

    The DNA damage response factor 53BP1 functions at the intersection of two major double strand break (DSB) repair pathways--promoting nonhomologous end-joining (NHEJ) and inhibiting homology-directed repair (HDR)--and integrates cellular inputs to ensure their timely execution in the proper cellular contexts. Recent work has revealed that 53BP1 controls 5' end resection at DNA ends, mediates synapsis of DNA ends, promotes the mobility of damaged chromatin, improves DSB repair in heterochromatic regions, and contributes to lethal mis-repair of DSBs in BRCA1-deficient cells. Here we review these aspects of 53BP1 and discuss new data revealing how 53BP1 is loaded onto chromatin and uses its interacting factors Rif1 and PTIP to promote NHEJ and inhibit HDR. PMID:24094932

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

    PubMed Central

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

    2014-01-01

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

  16. 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 remaining four genes (XPC, ERCC2, XRCC1, and MLH1) in either the experimental or control animals. PMID:21080036

  17. Interactions of DNA repair gene variants modulate chromosomal aberrations in healthy subjects.

    PubMed

    Vodicka, Pavel; Musak, Ludovit; Frank, Christoph; Kazimirova, Alena; Vymetalkova, Veronika; Barancokova, Magdalena; Smolkova, Bozena; Dzupinkova, Zuzana; Jiraskova, Katerina; Vodenkova, Sona; Kroupa, Michal; Osina, Oto; Naccarati, Alessio; Palitti, Fabrizio; Försti, Asta; Dusinska, Maria; Vodickova, Ludmila; Hemminki, Kari

    2015-11-01

    Human cancers are often associated with numerical and structural chromosomal instability. Structural chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBL) arise as consequences of direct DNA damage or due to replication on a damaged DNA template. In both cases, DNA repair is critical and inter-individual differences in its capacity are probably due to corresponding genetic variations. We investigated functional variants in DNA repair genes (base and nucleotide excision repair, double-strand break repair) in relation to CAs, chromatid-type aberrations (CTAs) and chromosome-type aberrations (CSAs) in healthy individuals. Chromosomal damage was determined by conventional cytogenetic analysis. The genotyping was performed by both restriction fragment length polymorphism and TaqMan allelic discrimination assays. Multivariate logistic regression was applied for testing individual factors on CAs, CTAs and CSAs. Pair-wise genotype interactions of 11 genes were constructed for all possible pairs of single-nucleotide polymorphisms. Analysed individually, we observed significantly lower CTA frequencies in association with XPD Lys751Gln homozygous variant genotype [odds ratio (OR) 0.64, 95% confidence interval (CI) 0.48-0.85, P = 0.004; n = 1777]. A significant association of heterozygous variant genotype in RAD54L with increased CSA frequency (OR 1.96, 95% CI 1.01-4.02, P = 0.03) was determined in 282 subjects with available genotype. By addressing gene-gene interactions, we discovered 14 interactions significantly modulating CAs, 9 CTAs and 12 CSAs frequencies. Highly significant interactions included always pairs from two different pathways. Although individual variants in genes encoding DNA repair proteins modulate CAs only modestly, several gene-gene interactions in DNA repair genes evinced either enhanced or decreased CA frequencies suggesting that CAs accumulation requires complex interplay between different DNA repair pathways. PMID:26354780

  18. Physical and functional interactions between Drosophila homologue of Swc6/p18Hamlet subunit of the SWR1/SRCAP chromatin-remodeling complex with the DNA repair/transcription factor TFIIH.

    PubMed

    Herrera-Cruz, Mariana; Cruz, Grisel; Valadez-Graham, Viviana; Fregoso-Lomas, Mariana; Villicaña, Claudia; Vázquez, Martha; Reynaud, Enrique; Zurita, Mario

    2012-09-28

    The multisubunit DNA repair and transcription factor TFIIH maintains an intricate cross-talk with different factors to achieve its functions. The p8 subunit of TFIIH maintains the basal levels of the complex by interacting with the p52 subunit. Here, we report that in Drosophila, the homolog of the p8 subunit (Dmp8) is encoded in a bicistronic transcript with the homolog of the Swc6/p18(Hamlet) subunit (Dmp18) of the SWR1/SRCAP chromatin remodeling complex. The SWR1 and SRCAP complexes catalyze the exchange of the canonical histone H2A with the H2AZ histone variant. In eukaryotic cells, bicistronic transcripts are not common, and in some cases, the two encoded proteins are functionally related. We found that Dmp18 physically interacts with the Dmp52 subunit of TFIIH and co-localizes with TFIIH in the chromatin. We also demonstrated that Dmp18 genetically interacts with Dmp8, suggesting that a cross-talk might exist between TFIIH and a component of a chromatin remodeler complex involved in histone exchange. Interestingly, our results also show that when the level of one of the two proteins is decreased and the other maintained, a specific defect in the fly is observed, suggesting that the organization of these two genes in a bicistronic locus has been selected during evolution to allow co-regulation of both genes. PMID:22865882

  19. DNA repair pathway gene expression score correlates with repair proficiency and tumor sensitivity to chemotherapy.

    PubMed

    Pitroda, Sean P; Pashtan, Itai M; Logan, Hillary L; Budke, Brian; Darga, Thomas E; Weichselbaum, Ralph R; Connell, Philip P

    2014-03-26

    Mutagenesis is a hallmark of malignancy, and many oncologic treatments function by generating additional DNA damage. Therefore, DNA damage repair is centrally important in both carcinogenesis and cancer treatment. Homologous recombination (HR) and nonhomologous end joining are alternative pathways of double-strand DNA break repair. We developed a method to quantify the efficiency of DNA repair pathways in the context of cancer therapy. The recombination proficiency score (RPS) is based on the expression levels for four genes involved in DNA repair pathway preference (Rif1, PARI, RAD51, and Ku80), such that high expression of these genes yields a low RPS. Carcinoma cells with low RPS exhibit HR suppression and frequent DNA copy number alterations, which are characteristic of error-prone repair processes that arise in HR-deficient backgrounds. The RPS system was clinically validated in patients with breast or non-small cell lung carcinomas (NSCLCs). Tumors with low RPS were associated with greater mutagenesis, adverse clinical features, and inferior patient survival rates, suggesting that HR suppression contributes to the genomic instability that fuels malignant progression. This adverse prognosis associated with low RPS was diminished if NSCLC patients received adjuvant chemotherapy, suggesting that HR suppression and associated sensitivity to platinum-based drugs counteract the adverse prognosis associated with low RPS. Therefore, RPS may help oncologists select which therapies will be effective for individual patients, thereby enabling more personalized care. PMID:24670686

  20. 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.

  1. Dynamics and mechanisms of DNA repair by photolyase

    PubMed Central

    Liu, Zheyun; Wang, Lijuan; Zhong, Dongping

    2015-01-01

    Photolyase, a class of flavoproteins, uses blue light to repair two types of ultraviolet-induced DNA damage, cyclobutane pyrimidine dimer (CPD) and pyrimidine-pyrimidone (6–4) photoproduct (6–4PP). In this perspective, we review the recent progress on the repair dynamics and mechanisms of both types of DNA restoration by photolyases. We first report the spectroscopic characterization of flavin in various redox states and the active-site solvation dynamics in photolyases. We then systematically summarize the detailed repair dynamics of damaged DNA by photolyases and a biomimetic system through resolving all elementary steps on the ultrafast timescales, including multiple intermolecular electron- and proton-transfer reactions and bond-breaking and -making processes. We determined the unique electron tunneling pathways, identified the key functional residues and revealed the molecular origin of high repair efficiency, and thus elucidate the molecular mechanisms and repair photocycles at the most fundamental level. We finally conclude that the active sites of photolyases, unlike aqueous solution for the biomimetic system, provide a unique electrostatic environment and local flexibility and thus a dedicated synergy for all elementary dynamics to maximize the repair efficiency. This repair photomachine is the first enzyme that the entire functional evolution is completely mapped out in real time. PMID:25870862

  2. Dynamic regulation of cerebral DNA repair genes by psychological stress.

    PubMed

    Forsberg, Kristin; Aalling, Nadia; Wörtwein, Gitta; Loft, Steffen; Møller, Peter; Hau, Jann; Hageman, Ida; Jørgensen, Martin B; Jørgensen, Anders

    2015-01-15

    Neuronal genotoxic insults from oxidative stress constitute a putative molecular link between stress and depression on the one hand, and cognitive dysfunction and dementia risk on the other. Oxidative modifications to DNA are repaired by specific enzymes; a process that plays a critical role for maintaining genomic integrity. The aim of the present study was to characterize the pattern of cerebral DNA repair enzyme regulation after stress through the quantification of a targeted range of gene products involved in different types of DNA repair. 72 male Sprague-Dawley rats were subjected to either restraint stress (6 h/day) or daily handling (controls), and sacrificed after 1, 7 or 21 stress sessions. The mRNA expression of seven genes (Ogg1, Ape1, Ung1, Neil1, Xrcc1, Ercc1, Nudt1) involved in the repair of oxidatively damaged DNA was determined by quantitative real time polymerase chain reaction in the prefrontal cortex (PFC) and hippocampus (HC). DNA repair gene expression in PFC exhibited a general trend towards an induction after acute stress and a decrease after subchronic exposure compared to control animals. After chronic stress, a normalization towards control levels was observed. A similar pattern was seen in HC, but with overall smaller effects and without the induction after acute stress. Nuclear DNA damage from oxidation as measured by the comet assay was unaffected by stress in both regions. We conclude that psychological stress have a dynamic influence on brain DNA repair gene expression; however, since we were unable to identify concurrent changes in DNA damage from oxidation, the down-stream consequences of this regulation, if any, remains unclear. PMID:25726146

  3. Human Rad50 is physically associated with human Mre11: identification of a conserved multiprotein complex implicated in recombinational DNA repair.

    PubMed Central

    Dolganov, G M; Maser, R S; Novikov, A; Tosto, L; Chong, S; Bressan, D A; Petrini, J H

    1996-01-01

    In this report, we describe the identification and molecular characterization of a human RAD50 homolog, hRAD50. hRAD50 was included in a collection of cDNAs which were isolated by a direct cDNA selection strategy focused on the chromosomal interval spanning 5q23 to 5q31. Alterations of the 5q23-q31 interval are frequently observed in myelodysplasia and myeloid leukemia. This strategy was thus undertaken to create a detailed genetic map of that region. Saccharomyces cerevisiae RAD50 (ScRAD50) is one of three yeast RAD52 epistasis group members (ScRAD50, ScMRE11, and ScXRS2) in which mutations eliminate meiotic recombination but confer a hyperrecombinational phenotype in mitotic cells. The yeast Rad50, Mre11, and Xrs2 proteins appear to act in a multiprotein complex, consistent with the observation that the corresponding mutants confer essentially identical phenotypes. In this report, we demonstrate that the human Rad50 and Mre11 proteins are stably associated in a protein complex which may include three other proteins. hRAD50 is expressed in all tissues examined, but mRNA levels are significantly higher in the testis. Other human RAD52 epistasis group homologs exhibit this expression pattern, suggesting the involvement of human RAD52 epistasis group proteins in meiotic recombination. Human RAD52 epistasis group proteins are highly conserved and act in protein complexes that are analogous to those of their yeast counterparts. These findings indicate that the function of the RAD52 epistasis group is conserved in human cells. PMID:8756642

  4. 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.

  5. Electron transfer mechanisms of DNA repair by photolyase.

    PubMed

    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. PMID:25830375

  6. DNA damage and repair in children with Down's syndrome.

    PubMed

    Morawiec, Zbigniew; Janik, Katarzyna; Kowalski, Michal; Stetkiewicz, Tomasz; Szaflik, Jerzy; Morawiec-Bajda, Alina; Sobczuk, Anna; Blasiak, Janusz

    2008-01-01

    Down's syndrome (DS) is associated with the presence of a third 21 chromosome and is generally considered as a non-cancer-prone genetic disease. However, leukaemias occur more frequently in children with the syndrome than in general population and there is an open question, whether the presence of an additional chromosome may contribute to genomic instability, which, in turn, may play a role in a higher susceptibility to cancer and leukaemias in particular. In order to assess genomic instability associated with the presence of a third 21 chromosome, we determined the level of endogenous DNA damage and susceptibility to a genotoxic stress-inducing factor, hydrogen peroxide and N-methyl-N'-nitro-N-nitrosoguanidyne (MNNG) as well as the ability to remove DNA damage in the peripheral blood lymphocytes of children with DS and healthy kids. The level of DNA damage and the kinetics of DNA repair were evaluated by alkaline comet assay. Oxidative DNA damage was assayed with DNA repair enzymes: endonuclease III-like NTH1 and formamidopyrimidine-DNA glycosylase. The cells taken from children with DS did not display an effective DNA repair after treatment with 10 mM hydrogen peroxide. No difference in the sensitivity to DNA-damaging agents and the efficacy of DNA repair due to age and gender in DS children was observed. These results suggest that children with DS may be characterized by the increased sensitivity to the DNA-damaging agents impaired cellular reaction to DNA damage, which, in turn, may increase the probability of cancers in these children. Therefore, a special care to avoid exposure to potential mutagenic factor my be considered in these children. PMID:17765270

  7. Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution.

    PubMed

    Tagua, Victor G; Pausch, Marcell; Eckel, Maike; Gutiérrez, Gabriel; Miralles-Durán, Alejandro; Sanz, Catalina; Eslava, Arturo P; Pokorny, Richard; Corrochano, Luis M; Batschauer, Alfred

    2015-12-01

    DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryptochromes (cry-DASH) belong to a family of flavoproteins acting as repair enzymes for UV-B-induced DNA lesions (photolyases) or as UV-A/blue light photoreceptors (cryptochromes). They are present in plants, bacteria, various vertebrates, and fungi and were originally considered as sensory photoreceptors because of their incapability to repair cyclobutane pyrimidine dimer (CPD) lesions in duplex DNA. However, cry-DASH can repair CPDs in single-stranded DNA, but their role in DNA repair in vivo remains to be clarified. The genome of the fungus Phycomyces blakesleeanus contains a single gene for a protein of the cryptochrome/photolyase family (CPF) encoding a cry-DASH, cryA, despite its ability to photoreactivate. Here, we show that cryA expression is induced by blue light in a Mad complex-dependent manner. Moreover, we demonstrate that CryA is capable of binding flavin (FAD) and methenyltetrahydrofolate (MTHF), fully complements the Escherichia coli photolyase mutant and repairs in vitro CPD lesions in single-stranded and double-stranded DNA with the same efficiency. These results support a role for Phycomyces cry-DASH as a photolyase and suggest a similar role for cry-DASH in mucoromycotina fungi. PMID:26578805

  8. Effect of donor age on DNA repair by articular chondrocytes

    SciTech Connect

    Lipman, J.M.

    1986-05-01

    The hypothesis that aging of articular chondrocytes at a cellular level results from loss of DNA repair capability was studied by two different measures: unscheduled DNA synthesis (UDS) and O/sup 6/-methylguanine acceptor protein (MGAP) activity. UDS following damage by 254 nm ultraviolet irradiation (20J/m/sup 2/) was examined in intact articular cartilage from rabbits of different ages. Semiconservative DNA synthesis was suppressed with hydroxurea and repair followed by the incorporation of (/sup 3/H)-thymidine ((/sup 3/H)-dThd). After repair the cartilage was digested in proteinase K (0.5mg/ml) with dodecyl sodium sulfate (0.2%) and DNA determined with Hoechst 33258 dye. UDS (dpm (/sup 3/H)-dThd/..mu..g DNA) was greater in articular cartilage from 3- than 39-month-old rabbits. MGAP was studied in cell extracts of cultured human and rabbit chondrocytes by transfer of (/sup 3/H) O/sup 6/-methyl groups from exogenous DNA to protein. It was significantly less in rabbit than in human cells on a per protein or DNA basis. There was no decline in this activity in human chondrocytes from newborn to 60 years of age; and rabbits from 3- to 36-months-old. The data indicate that in the two different repair mechanisms, age differences are found with resting but not dividing chondrocytes.

  9. Higher-order chromatin structure-dependent repair of DNA double-strand breaks: involvement of the V(D)J recombination double-strand break repair pathway.

    PubMed

    Johnston, P J; MacPhail, S H; Stamato, T D; Kirchgessner, C U; Olive, P L

    1998-05-01

    Repair of DNA double-strand breaks (DSBs) is linked to the V(D)J recombination pathway through investigations of radiation-sensitive mutants. Here we report a possible association between the distribution of DSBs within higher-order chromatin structures and this pathway. Both murine severe combined immunodeficient (SCID) and Chinese hamster XR-1 cells exhibit defective DNA DSB repair and defective V(D)J recombination. The DSB repair defect is not complete, with only a subset of slowly repairing lesions affected by the mutations in these cell lines. We used a modified neutral filter elution procedure which retained elements of higher-order chromatin structures, namely nuclear matrix-DNA interactions. X-ray-induced DSBs that occurred as multiples within looped DNA structures were nonrepairable in SCID and XR-1 cells. In contrast, these lesions were repaired in radioresistant wild-type cells. Cell lines complemented with human DNA containing the respective complementing genes (XRCC7 and XRCC4) showed an increased rate of DSB repair. These results agree with previous findings with xrs5 cells (a member of the XRCC5 group). Xrs5 cells are defective for the Ku p80 subunit of the V(D)J recombination complex and show repair and V(D)J recombination defects similar to those of SCID and XR-1 cells. PMID:9588356

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

    PubMed Central

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

    2014-01-01

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

  11. The current state of eukaryotic DNA base damage and repair.

    PubMed

    Bauer, Nicholas C; Corbett, Anita H; Doetsch, Paul W

    2015-12-01

    DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-strand break damage resulting from oxidation, alkylation, deamination, and spontaneous hydrolysis. If left unrepaired, such lesions can become fixed in the genome as permanent mutations. Thus, evolution has led to the creation of several highly conserved, partially redundant pathways to repair or mitigate the effects of DNA base damage. The biochemical mechanisms of these pathways have been well characterized and the impact of this work was recently highlighted by the selection of Tomas Lindahl, Aziz Sancar and Paul Modrich as the recipients of the 2015 Nobel Prize in Chemistry for their seminal work in defining DNA repair pathways. However, how these repair pathways are regulated and interconnected is still being elucidated. This review focuses on the classical base excision repair and strand incision pathways in eukaryotes, considering both Saccharomyces cerevisiae and humans, and extends to some important questions and challenges facing the field of DNA base damage repair. PMID:26519467

  12. The current state of eukaryotic DNA base damage and repair

    PubMed Central

    Bauer, Nicholas C.; Corbett, Anita H.; Doetsch, Paul W.

    2015-01-01

    DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-strand break damage resulting from oxidation, alkylation, deamination, and spontaneous hydrolysis. If left unrepaired, such lesions can become fixed in the genome as permanent mutations. Thus, evolution has led to the creation of several highly conserved, partially redundant pathways to repair or mitigate the effects of DNA base damage. The biochemical mechanisms of these pathways have been well characterized and the impact of this work was recently highlighted by the selection of Tomas Lindahl, Aziz Sancar and Paul Modrich as the recipients of the 2015 Nobel Prize in Chemistry for their seminal work in defining DNA repair pathways. However, how these repair pathways are regulated and interconnected is still being elucidated. This review focuses on the classical base excision repair and strand incision pathways in eukaryotes, considering both Saccharomyces cerevisiae and humans, and extends to some important questions and challenges facing the field of DNA base damage repair. PMID:26519467

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

    PubMed Central

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

    1997-01-01

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

  14. Heterochromatin formation via recruitment of DNA repair proteins

    PubMed Central

    Kirkland, Jacob G.; Peterson, Misty R.; Still, Christopher D.; Brueggeman, Leo; Dhillon, Namrita; Kamakaka, Rohinton T.

    2015-01-01

    Heterochromatin formation and nuclear organization are important in gene regulation and genome fidelity. Proteins involved in gene silencing localize to sites of damage and some DNA repair proteins localize to heterochromatin, but the biological importance of these correlations remains unclear. In this study, we examined the role of double-strand-break repair proteins in gene silencing and nuclear organization. We find that the ATM kinase Tel1 and the proteins Mre11 and Esc2 can silence a reporter gene dependent on the Sir, as well as on other repair proteins. Furthermore, these proteins aid in the localization of silenced domains to specific compartments in the nucleus. We identify two distinct mechanisms for repair protein–mediated silencing—via direct and indirect interactions with Sir proteins, as well as by tethering loci to the nuclear periphery. This study reveals previously unknown interactions between repair proteins and silencing proteins and suggests insights into the mechanism underlying genome integrity. PMID:25631822

  15. Curcumin Triggers DNA Damage and Inhibits Expression of DNA Repair Proteins in Human Lung Cancer Cells.

    PubMed

    Ting, Chien-Yi; Wang, Hsin-Ell; Yu, Chien-Chih; Liu, Hsin-Chung; Liu, Yu-Chang; Chiang, I-Tsang

    2015-07-01

    The study goal was to evaluate the effects of curcumin on DNA damage and expression of DNA-repair proteins in human lung cancer. Thus, NCI-H460 cells were used to study the effects of curcumin on DNA damage and repair in vitro. We investigated curcumin induces DNA damage by comet the assay and 4',6-diamidino-2-phenylindole (DAPI) staining. The DNA damage/repair-related protein levels were examined and monitored by western blotting and confocal microscopy. Curcumin significantly increased the length of comet tails and DNA condensation in NCI-H460 cells. Curcumin reduced expression of DNA-repair proteins such as 14-3-3 protein sigma (14-3-3?), O6-methylguanine-DNA methyltransferase (MGMT), breast cancer susceptibility gene 1 (BRCA1), and mediator of DNA damage checkpoint 1 (MDC1). Curcumin also increased phosphorylation of p53 and Histone H2A.X (S140) in the nuclei of NCI-H460 cells. Taken together, our findings indicated that curcumin triggered DNA damage and inhibited expression of DNA-repair-associated proteins in NCI-H460 cells. PMID:26124332

  16. Charge transport-mediated recruitment of DNA repair enzymes

    E-print Network

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

    2008-11-18

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

  17. 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 DH5? E. coli strain. Currently, alternative vectors are being designed for more efficient genetic manipulations in S. solfataricus.

  18. Functional Aspects of PARP1 in DNA Repair and Transcription

    PubMed Central

    Ko, Hui Ling; Ren, Ee Chee

    2012-01-01

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

  19. Stress and DNA repair biology of the Fanconi anemia pathway

    PubMed Central

    Longerich, Simonne; Li, Jian; Xiong, Yong; Sung, Patrick

    2014-01-01

    Fanconi anemia (FA) represents a paradigm of rare genetic diseases, where the quest for cause and cure has led to seminal discoveries in cancer biology. Although a total of 16 FA genes have been identified thus far, the biochemical function of many of the FA proteins remains to be elucidated. FA is rare, yet the fact that 5 FA genes are in fact familial breast cancer genes and FA gene mutations are found frequently in sporadic cancers suggest wider applicability in hematopoiesis and oncology. Establishing the interaction network involving the FA proteins and their associated partners has revealed an intersection of FA with several DNA repair pathways, including homologous recombination, DNA mismatch repair, nucleotide excision repair, and translesion DNA synthesis. Importantly, recent studies have shown a major involvement of the FA pathway in the tolerance of reactive aldehydes. Moreover, despite improved outcomes in stem cell transplantation in the treatment of FA, many challenges remain in patient care. PMID:25237197

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

    SciTech Connect

    Cornetta, Tommaso; Festa, Fabiola; Testa, Antonella; Cozzi, Renata Prof. . E-mail: cozzi@uniroma3.it

    2006-10-01

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

  1. Silibinin Preferentially Radiosensitizes Prostate Cancer by Inhibiting DNA Repair Signaling.

    PubMed

    Nambiar, Dhanya K; Rajamani, Paulraj; Deep, Gagan; Jain, Anil K; Agarwal, Rajesh; Singh, Rana P

    2015-12-01

    Radiotherapy, a frequent mode of cancer treatment, is often restricted by dose-related toxicity and development of therapeutic resistance. To develop a novel and selective radiosensitizer, we studied the radiosensitizing effects and associated mechanisms of silibinin in prostate cancer. The radiosensitizing effect of silibinin with ionizing radiation (IR) was assessed on radioresistant prostate cancer cell lines by clonogenic, cell cycle, cell death, and DNA repair assays. Tumor xenograft growth, immunohistochemical (IHC) analysis of tumor tissues, and toxicity-related parameters were measured in vivo. Silibinin (25 ?mol/L) enhanced IR (2.5-10 Gy)-caused inhibition (up to 96%, P < 0.001) of colony formation selectively in prostate cancer cells, and prolonged and enhanced IR-caused G2-M arrest, apoptosis, and ROS production. Mechanistically, silibinin inhibited IR-induced DNA repair (ATM and Chk1/2) and EGFR signaling and attenuated the levels of antiapoptotic proteins. Specifically, silibinin suppressed IR-induced nuclear translocation of EGFR and DNA-PK, an important mediator of DSB repair, leading to an increased number of ?-H2AX (ser139) foci suggesting lesser DNA repair. In vivo, silibinin strongly radiosensitized DU145 tumor xenograft inhibition (84%, P < 0.01) with higher apoptotic response (10-fold, P < 0.01) and reduced repair of DNA damage, and rescued the mice from IR-induced toxicity and hematopoietic injury. Overall, silibinin enhanced the radiotherapeutic response via suppressing IR-induced prosurvival signaling and DSB repair by inhibiting nuclear translocation of EGFR and DNA-PK. Because silibinin is already in phase II clinical trial for prostate cancer patients, the present finding has translational relevance for radioresistant prostate cancer. Mol Cancer Ther; 14(12); 2722-34. ©2015 AACR. PMID:26516160

  2. DNA damage and repair in plants – from models to crops

    PubMed Central

    Manova, Vasilissa; Gruszka, Damian

    2015-01-01

    The genomic integrity of every organism is constantly challenged by endogenous and exogenous DNA-damaging factors. Mutagenic agents cause reduced stability of plant genome and have a deleterious effect on development, and in the case of crop species lead to yield reduction. It is crucial for all organisms, including plants, to develop efficient mechanisms for maintenance of the genome integrity. DNA repair processes have been characterized in bacterial, fungal, and mammalian model systems. The description of these processes in plants, in contrast, was initiated relatively recently and has been focused largely on the model plant Arabidopsis thaliana. Consequently, our knowledge about DNA repair in plant genomes - particularly in the genomes of crop plants - is by far more limited. However, the relatively small size of the Arabidopsis genome, its rapid life cycle and availability of various transformation methods make this species an attractive model for the study of eukaryotic DNA repair mechanisms and mutagenesis. Moreover, abnormalities in DNA repair which proved to be lethal for animal models are tolerated in plant genomes, although sensitivity to DNA damaging agents is retained. Due to the high conservation of DNA repair processes and factors mediating them among eukaryotes, genes and proteins that have been identified in model species may serve to identify homologous sequences in other species, including crop plants, in which these mechanisms are poorly understood. Crop breeding programs have provided remarkable advances in food quality and yield over the last century. Although the human population is predicted to “peak” by 2050, further advances in yield will be required to feed this population. Breeding requires genetic diversity. The biological impact of any mutagenic agent used for the creation of genetic diversity depends on the chemical nature of the induced lesions and on the efficiency and accuracy of their repair. More recent targeted mutagenesis procedures also depend on host repair processes, with different pathways yielding different products. Enhanced understanding of DNA repair processes in plants will inform and accelerate the engineering of crop genomes via both traditional and targeted approaches. PMID:26557130

  3. 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 XPG as well as the C-terminal region of WRN. The physical interaction between XPG and WRN links NER, (made evident by the disease XP) with DSBR, which imparts additional knowledge of the overlapping nature of these two proteins and the previously distinct DNA repair pathways they are associated with. Since genomic integrity is constantly threatened by both endogenous and exogenous (internal and external) damage, understanding the roles of these proteins in coordinating DNA repair processes with replication will signifi cantly further understanding how defects instigate physiological consequences in response to various DNA damaging sources. This ultimately contributes to our understanding of cancer and premature aging.

  4. Finite element analysis to model complex mitral valve repair.

    PubMed

    Labrosse, Michel; Mesana, Thierry; Baxter, Ian; Chan, Vincent

    2016-01-01

    Although finite element analysis has been used to model simple mitral repair, it has not been used to model complex repair. A virtual mitral valve model was successful in simulating normal and abnormal valve function. Models were then developed to simulate an edge-to-edge repair and repair employing quadrangular resection. Stress contour plots demonstrated increased stresses along the mitral annulus, corresponding to the annuloplasty. The role of finite element analysis in guiding clinical practice remains undetermined. PMID:24904177

  5. Circadian Modulation of 8-Oxoguanine DNA Damage Repair.

    PubMed

    Manzella, Nicola; Bracci, Massimo; Strafella, Elisabetta; Staffolani, Sara; Ciarapica, Veronica; Copertaro, Alfredo; Rapisarda, Venerando; Ledda, Caterina; Amati, Monica; Valentino, Matteo; Tomasetti, Marco; Stevens, Richard G; Santarelli, Lory

    2015-01-01

    The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours. PMID:26337123

  6. Circadian Modulation of 8-Oxoguanine DNA Damage Repair

    PubMed Central

    Manzella, Nicola; Bracci, Massimo; Strafella, Elisabetta; Staffolani, Sara; Ciarapica, Veronica; Copertaro, Alfredo; Rapisarda, Venerando; Ledda, Caterina; Amati, Monica; Valentino, Matteo; Tomasetti, Marco; Stevens, Richard G.; Santarelli, Lory

    2015-01-01

    The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours. PMID:26337123

  7. A New Epistasis Group for the Repair of DNA Damage in Bacteriophage T4: Replication Repair

    PubMed Central

    Wachsman, Joseph T.; Drake, John W.

    1987-01-01

    The gene 32 mutation amA453 sensitizes bacteriophage T4 to the lethal effects of ultraviolet (UV) irradiation, methyl methanesulfonate and angelicin-mediated photodynamic irradiation when treated particles are plated on amber-suppressing host cells. The increased UV sensitivity caused by amA453 is additive to that caused by mutations in both the T4 excision repair (denV) and recombination repair (uvsWXY) systems, suggesting the operation of a third kind of repair system. The mutation uvs79, with many similarities to amA453 but mapping in gene 41, is largely epistatic to amA453. The mutation mms1, also with many similarities to amA453, maps close to amA453 within gene 32 and is largely epistatic to uvs79. Neither amA453 nor uvs79 affect the ratio of UV-induced mutational to lethal hits, nor does amA453 affect spontaneous or UV-enhanced recombination frequencies. Gene 32 encodes the major T4 ssDNA-binding protein (the scaffolding of DNA replication) and gene 41 encodes a DNA helicase, both being required for T4 DNA replication. We conclude that a third repair process operates in phage T4 and suggest that it acts during rather than before or after DNA replication. PMID:3552872

  8. Breaking bad: The mutagenic effect of DNA repair.

    PubMed

    Chen, Jia; Furano, Anthony V

    2015-08-01

    Species survival depends on the faithful replication of genetic information, which is continually monitored and maintained by DNA repair pathways that correct 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 CpGs than at non-CpG positions. Interestingly, the neutral mutation rate at non-CpG sites is positively correlated with CpG content. Although the basis of this correlation was not immediately apparent, some bioinformatic results were consistent with the induction of non-CpG mutations by DNA repair at 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 fact induce 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-preferring single-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 error-prone processes during DNA repair would have important implications for disease, aging and evolution. This perspective briefly reviews both the bioinformatic and biochemical literature relevant to repair-induced mutagenesis and discusses future directions required to understand the mechanistic basis of this process. PMID:26073774

  9. Productive and Nonproductive Complexes of Ku and DNA-Dependent Protein Kinase at DNA Termini

    PubMed Central

    West, Robert B.; Yaneva, Mariana; Lieber, Michael R.

    1998-01-01

    DNA-dependent protein kinase (DNA-PK) is the only eukaryotic protein kinase known to be specifically activated by double-stranded DNA (dsDNA) termini, accounting for its importance in repair of dsDNA breaks and its role in physiologic processes involving dsDNA breaks, such as V(D)J recombination. In this study we conducted kinase and binding analyses using DNA-PK on DNA termini of various lengths in the presence and absence of Ku. We confirmed our previous observations that DNA-PK can bind DNA termini in the absence of Ku, and we determined rate constants for binding. However, in the presence of Ku, DNA-PK can assume either a productive or a nonproductive configuration, depending on the length of the DNA terminus. For dsDNA greater than 26 bp, the productive mode is achieved and Ku increases the affinity of the DNA-PK for the Ku:DNA complex. The change in affinity is achieved by increases in both the kinetic association rate and reduction in the kinetic dissociation rate. For dsDNA smaller than 26 bp, the nonproductive mode, in which DNA-PK is bound to Ku:DNA but is inactive as a kinase, is assumed. Both the productive and nonproductive configurations are likely to be of physiologic importance, depending on the distance of the dsDNA break site to other protein complexes, such as nucleosomes. PMID:9742108

  10. Transcription-coupled repair of oxidative DNA damage in human cells: Mechanisms and consequences

    E-print Network

    Tsutakawa, Susan; Cooper, Priscilla K.

    2001-01-01

    1993. Molecular mechanism of transcription-repair coupling.transcription elongation on non-damaged DNA by the same mechanismTranscription-Coupled Repair of Oxidative DNA Damage in Human Cells: Mechanisms

  11. Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis

    E-print Network

    Nagel, Zachary D.

    The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair ...

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

    SciTech Connect

    Ammermann, D.

    1988-05-01

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

  13. DNA Repair at Telomeres: Keeping the Ends Intact

    PubMed Central

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

    2013-01-01

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

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

    SciTech Connect

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

    2002-03-01

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

  15. DNA repair of pyrimidine dimers and 6-4 photoproducts in the ribosomal DNA.

    PubMed

    Balajee, A S; May, A; Bohr, V A

    1999-06-15

    The nucleolus is a unique structural component of interphase nuclei where the ribosomal genes, trans-cribed by RNA polymerase I (RNA pol I), are organized. In the present study, the repair of UV-induced photolesions was investigated in the ribosomal DNA (rDNA) in relation to RNA pol I transcription. We used hamster cells because their repair phenotype permits the separate analysis of the major photo-products induced by UV light. Immunofluorescent labeling of UV-induced DNA repair and transcription sites showed that the nucleolar regions were defic-ient in DNA repair despite the presence of abundant RNA pol I transcription foci. Immunological staining indicated that various NER proteins, including TFIIH (subunits p62 and p89), p53, Gadd 45 and prolifer-ating cell nuclear antigen are all enriched in the nuclei but distinctly absent in nucleoli. This lack of enrichment of NER factors in the nucleolus may be responsible for the inefficient repair of photo-products in the rDNA. UV irradiation generates two major photoproducts, the cyclobutane pyrimidine dimers (CPDs) and the 6-4 photoproducts (6-4 PPs). The repair kinetics of these two lesions were assessed simultaneously by the immunological isolation of bromodeoxyuridine (BudR) containing excision repair patches using an antibody to BudR. We found that the repair of the photolesions was less efficient in the rDNA compared to that of the endo-genous housekeeping gene, dihydrofolate reductase (DHFR). Gene specific repair of each of these two photoproducts was then measured separately in the rDNA and in the DHFR gene, which is transcribed by RNA pol II. The removal of CPDs was deficient in the rDNA as compared to the DHFR gene. On the contrary, 6-4 PPs were removed efficiently from the rDNA although somewhat slower than from the DHFR gene. The relatively efficient repair of 6-4 PPs in the rDNA is consistent with the notion that the 6-4 PPs are repaired efficiently in different genomic regions by the global genome repair pathway. PMID:10352180

  16. 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

    2016-01-01

    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. PMID:26559971

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

    NASA Technical Reports Server (NTRS)

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

    2007-01-01

    When cells are exposed to radiation, cellular lesions are induced in the DNA including double strand breaks (DSBs), single strand breaks and clustered DNA damage, which if not repaired with high fidelity may lead to detrimental biological consequences. Complex DSBs are induced by ionizing radiation and characterized by the presence of base lesions close to the break termini. They are believed to be one of the major causes of the biological effects of IR. The complexity of DSBs increases with the ionization density of the radiation and these complex DSBs are distinct from the damage induced by sparsely ionizing gamma-radiation. It has been hypothesized that complex DSBs produced by heavy ions in space pose problems to the DNA repair machinery. We have used imm uno-cyto-chemical staining of phosphorylated histone H2AX (gamma-H2AX) foci, as a marker of DSBs. We have investigated the formation and loss of gamma-H2AX foci and RAD51 foci (a protein involved in the homologous recombination pathway) in mammalian cells induced by low fluences of low-LET gamma-radiation and high-LET Fe-56 ions (1GeV/n, 151 keV/micron LET). M059J and M059K cells, which are deficient and proficient in DNA-PK(sub cs) activity respectively, were used to examine the role of DNA-PK(sub cs), a key protein in the non-homologous end joining (NHEJ) pathway of DSB repair, along with HF19 human fibroblasts. Followi ng irradiation with Fe-56 ions the rate of repair was slower in M059J cells compared with that in M059K, indicating a role for DNA-PK(sub cs) in the repair of DSB induced by Fe-56 ions. However a small percentage of DSBs induced are rejoined within 5 h although many DSBs still persist up to 24 h. When RAD51 was examined in M059J/K cells, RAD51 foci are visible 24 hours after irradiation in approximately 40% of M059J cells compared with <5% of M059K cells indicating that persistent DSBs or those formed at stalled replication forks recruit RAD51 in DNA-PK(sub cs) deficient cells. Following 1 Gy gamma-radiation the induction of gamma-H2AX foci is similar in M059J and M059K cells. However, the repair rate of DSBs is slower in M059J cells than in M059K as shown previously but faster than seen with DSB induced by 56Fe ions. Vanillin, an inhibitor of DNA-PK(sub cs), reduces significantly the rate of DSB repair in HF19 cells following 1 Gy gamma-radiation but at 0.25 Gy gamma-irradiation the rate of DSB repair is similar in the presence or absence vanillin, thus suggesting the repair of a sub-set of DSBs induced by low dose, low-LET radiation does not require DNA-PK(sub cs). This sub-set of DSBs is formed in lower yield with high LET radiation. T he complexity of DNA DSBs induced by HZE radiation will be discussed in terms of reduced repair efficiency and provide scope to model different sub-classes of DSBs as precursors that may lead to the detrimental health effects of HZE radiation.

  18. Ancient bacteria show evidence of DNA repair Sarah Stewart Johnson*

    E-print Network

    Nielsen, Rasmus

    Ancient bacteria show evidence of DNA repair Sarah Stewart Johnson* , Martin B. Hebsgaard , Torben for review June 14, 2007) Recent claims of cultivable ancient bacteria within sealed environ- ments highlight-term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence

  19. RIF1 in DNA break repair pathway choice.

    PubMed

    Daley, James M; Sung, Patrick

    2013-03-01

    New findings on the RIF1 protein by four research groups, including Chapman et al. (2013) and Escribano-Díaz et al. (2013) in this issue, provide insights into DNA double-strand break repair pathway choice in mammalian cells. PMID:23473603

  20. 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…

  1. DNA repair variants, indoor tanning, and risk of melanoma.

    PubMed

    Torres, Salina M; Luo, Li; Lilyquist, Jenna; Stidley, Christine A; Flores, Kristina; White, Kirsten A M; Erdei, Esther; Gonzales, Melissa; Paine, Susan; Vogel, Rachel I; Lazovich, Deann; Berwick, Marianne

    2013-09-01

    Although ultraviolet radiation (UV) exposure from indoor tanning has been linked to an increased risk of melanoma, the role of DNA repair genes in this process is unknown. We evaluated the association of 92 single nucleotide polymorphisms (SNPs) in 20 DNA repair genes with the risk of melanoma and indoor tanning among 929 patients with melanoma and 817 controls from the Minnesota Skin Health Study. Significant associations with melanoma risk were identified for SNPs in ERCC4, ERCC6, RFC1, XPC, MGMT, and FBRSL1 genes; with a cutoff of P < 0.05. ERCC6 and FBRSL1 gene variants and haplotypes interacted with indoor tanning. However, none of the 92 SNPs tested met the correction criteria for multiple comparisons. This study, based on an a priori interest in investigating the role of DNA repair capacity using variants in base excision and nucleotide excision repair, identified several genes that may play a role in resolving UV-induced DNA damage. PMID:23659246

  2. Polymorphisms in DNA Repair Genes, Recreational Physical Activity and Breast Cancer Risk

    PubMed Central

    McCullough, Lauren E.; Santella, Regina M.; Cleveland, Rebecca J.; Millikan, Robert C.; Olshan, Andrew F.; North, Kari E.; Bradshaw, Patrick T.; Eng, Sybil M.; Terry, Mary Beth; Shen, Jing; Crew, Katherine D.; Rossner, Pavel; Teitelbaum, Susan L.; Neugut, Alfred I.; Gammon, Marilie D.

    2013-01-01

    The mechanisms driving the inverse association between recreational physical activity (RPA) and breast cancer risk are complex. While exercise is associated with increased reactive oxygen species production it may also improve damage repair systems, particularly those that operate on single-strand breaks including base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR). Of these repair pathways, the role of MMR in breast carcinogenesis is least investigated. Polymorphisms in MMR or other DNA repair gene variants may modify the association between RPA and breast cancer incidence. We investigated the individual and joint effects of variants in three MMR pathway genes (MSH3, MLH1 and MSH2) on breast cancer occurrence using resources from the Long Island Breast Cancer Study Project. We additionally characterized interactions between RPA and genetic polymorphisms in MMR, BER and NER pathways. We found statistically significant multiplicative interactions (p<0.05) between MSH2 and MLH1, as well as between postmenopausal RPA and four variants in DNA repair (XPC-Ala499Val, XPF-Arg415Gln, XPG-Asp1104His and MLH1-lle219Val). Significant risk reductions were observed among highly active women with the common genotype for XPC (OR=0.54; 95% CI, 0.36–0.81) and XPF (OR=0.62; 95% CI, 0.44–0.87), as well as among active women who carried at least one variant allele in XPG (OR=0.46; 95% CI, 0.29–0.77) and MLH1 (OR=0.46; 95% CI, 0.30–0.71). Our data show that women with minor alleles in both MSH2 and MLH1 could be at increased breast cancer risk. RPA may be modified by genes in the DNA repair pathway, and merit further investigation. PMID:23852586

  3. Investigating the Role of Vitamin D and DNA Repair in Influencing Cancer Presentation and Outcomes 

    E-print Network

    Syed, Moinuddin Mohammed

    2014-09-26

    in (http://www.1000genomes.org/) with known genotypes of human repair genes will be quantified for DNA repair capacity using comet assay, cell cycle analysis, and gene expression of key DNA repair genes (for both ethnic groups) after exposure to DNA...

  4. Investigating the role of vitamin D and DNA repair in influencing cancer presentation and outcome 

    E-print Network

    Pidaparti, Divya

    2015-04-24

    in (http://www.1000genomes.org/) with known genotypes of human repair genes will be quantified for DNA repair capacity using comet assay, cell cycle analysis, and gene expression of key DNA repair genes (for both ethnic groups) after exposure to DNA...

  5. Investigating the role of vitamin D and DNA repair in influencing cancer presentation and outcomes 

    E-print Network

    Varrier, Shilpa

    2015-04-23

    in (http://www.1000genomes.org/) with known genotypes of human repair genes will be quantified for DNA repair capacity using comet assay, cell cycle analysis, and gene expression of key DNA repair genes (for both ethnic groups) after exposure to DNA...

  6. 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

  7. 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.

  8. DNA Double Strand Break Repair Pathway Choice Is Directed by Distinct MRE11 Nuclease Activities

    PubMed Central

    Shibata, Atsushi; Moiani, Davide; Arvai, Andrew S.; Perry, J. Jefferson P.; Harding, Shane M.; Genois, Marie-Michelle; Maity, Ranjan; van Rossum-Fikkert, Sari; Kertokalio, Aryandi; Romoli, Filippo; Ismail, Amani; Ismalaj, Ermal; Petricci, Elena; Matthew, J Neale; Bristow, Robert G; Masson, Jean-Yves; Wyman, Claire; Jeggo, Penny; Tainer, John A.

    2014-01-01

    SUMMARY MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection and signaling; yet, how its endo- and exonuclease activities regulate DSB repair by non-homologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors we examined repair pathway choice at DSBs generated in G2 following radiation exposure. Whilst endo- or exonuclease inhibition impairs radiation-induced RPA chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, whilst exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exo and EXO1/BLM bidirectional resection towards and away from the DNA end, which commits to HR. PMID:24316220

  9. A role for nuclear envelope–bridging complexes in homology-directed repair

    PubMed Central

    Swartz, Rebecca K.; Rodriguez, Elisa C.; King, Megan C.

    2014-01-01

    Unless efficiently and faithfully repaired, DNA double-strand breaks (DSBs) cause genome instability. We implicate a Schizosaccharomyces pombe nuclear envelope–spanning linker of nucleoskeleton and cytoskeleton (LINC) complex, composed of the Sad1/Unc84 protein Sad1 and Klarsicht/Anc1/SYNE1 homology protein Kms1, in the repair of DSBs. An induced DSB associates with Sad1 and Kms1 in S/G2 phases of the cell cycle, connecting the DSB to cytoplasmic microtubules. DSB resection to generate single-stranded DNA and the ATR kinase drive the formation of Sad1 foci in response to DNA damage. Depolymerization of microtubules or loss of Kms1 leads to an increase in the number and size of DSB-induced Sad1 foci. Further, Kms1 and the cytoplasmic microtubule regulator Mto1 promote the repair of an induced DSB by gene conversion, a type of homology-directed repair. kms1 genetically interacts with a number of genes involved in homology-directed repair; these same gene products appear to attenuate the formation or promote resolution of DSB-induced Sad1 foci. We suggest that the connection of DSBs with the cytoskeleton through the LINC complex may serve as an input to repair mechanism choice and efficiency. PMID:24943839

  10. 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.

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

    PubMed Central

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

    2014-01-01

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

  12. Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma

    PubMed Central

    Rivera, M; Wu, Q; Hamerlik, P; Hjelmeland, A B; Bao, S; Rich, J N

    2015-01-01

    Glioblastoma (GBM), the most prevalent type of primary intrinsic brain cancer in adults, remains universally fatal despite maximal therapy, including radiotherapy and chemotherapy. Cytotoxic therapy generates double-stranded DNA breaks (DSBs), most commonly repaired by homologous recombination (HR). We hypothesized that cancer cells coopt meiotic repair machinery as DSBs are generated during meiosis and repaired by molecular complexes distinct from genotoxic responses in somatic tissues. Indeed, we found that gliomas express meiotic repair genes and their expression informed poor prognosis. We interrogated the function of disrupted meiotic cDNA1 (DMC1), a homolog of RAD51, the primary recombinase used in mitotic cells to search and recombine with the homologous DNA template. DMC1, whose only known function is as an HR recombinase, was expressed by GBM cells and induced by radiation. Although targeting DMC1 in non-neoplastic cells minimally altered cell growth, DMC1 depletion in GBM cells decreased proliferation, induced activation of CHK1 and expression of p21CIP1/WAF1, and increased RPA foci, suggesting increased replication stress. Combining loss of DMC1 with ionizing radiation inhibited activation of DNA damage responses and increased radiosensitivity. Furthermore, loss of DMC1 reduced tumor growth and prolonged survival in vivo. Our results suggest that cancers coopt meiotic genes to augment survival under genotoxic stress, offering molecular targets with high therapeutic indices. PMID:25906155

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

    PubMed Central

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

    2015-01-01

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

  14. Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

    SciTech Connect

    Iwanaga, Kentaro; Sueoka, Naoko; Sato, Akemi; Hayashi, Shinichiro; Sueoka, Eisaburo . E-mail: sueokae@post.saga-med.ac.jp

    2005-08-05

    Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression.

  15. Crystal Structure of the Human Hsmar1-Derived Transposase Domain in the DNA Repair Enzyme Metnase

    SciTech Connect

    Goodwin, Kristie D.; He, Hongzhen; Imasaki, Tsuyoshi; Lee, Suk-Hee; Georgiadis, Millie M.

    2010-08-12

    Although the human genome is littered with sequences derived from the Hsmar1 transposon, the only intact Hsmar1 transposase gene exists within a chimeric SET-transposase fusion protein referred to as Metnase or SETMAR. Metnase retains many of the transposase activities including terminal inverted repeat (TIR) specific DNA-binding activity, DNA cleavage activity, albeit uncoupled from TIR-specific binding, and the ability to form a synaptic complex. However, Metnase has evolved as a DNA repair protein that is specifically involved in nonhomologous end joining. Here, we present two crystal structures of the transposase catalytic domain of Metnase revealing a dimeric enzyme with unusual active site plasticity that may be involved in modulating metal binding. We show through characterization of a dimerization mutant, F460K, that the dimeric form of the enzyme is required for its DNA cleavage, DNA-binding, and nonhomologous end joining activities. Of significance is the conservation of F460 along with residues that we propose may be involved in the modulation of metal binding in both the predicted ancestral Hsmar1 transposase sequence as well as in the modern enzyme. The Metnase transposase has been remarkably conserved through evolution; however, there is a clustering of substitutions located in alpha helices 4 and 5 within the putative DNA-binding site, consistent with loss of transposition specific DNA cleavage activity and acquisition of DNA repair specific cleavage activity.

  16. 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 ?M) formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair. Conclusions/Significance A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks. PMID:24722772

  17. DNA repair activity in fish and interest in ecotoxicology: a review.

    PubMed

    Kienzler, Aude; Bony, Sylvie; Devaux, Alain

    2013-06-15

    The knowledge of DNA repair in a target species is of first importance as it is the primary line of defense against genotoxicants, and a better knowledge of DNA repair capacity in fish could help to interpret genotoxicity data and/or assist in the choice of target species, developmental stage and tissues to focus on, both for environmental biomonitoring studies and DNA repair testing. This review focuses in a first part on what is presently known on a mechanistic basis, about the various DNA repair systems in fish, in vivo and in established cell lines. Data on base excision repair (BER), direct reversal with O?-alkylguanine transferase and double strand breaks repair, although rather scarce, are being reviewed, as well as nucleotide excision repair (NER) and photoreactivation repair (PER), which are by far the most studied repair mechanisms in fish. Most of these repair mechanisms seem to be strongly species and tissue dependent; they also depend on the developmental stage of the organisms. BER is efficient in vivo, although no data has been found on in vitro models. NER activity is quite low or even inexistent depending on the studies; however this lack is partly compensated by a strong PER activity, especially in early developmental stage. In a second part, a survey of the ecotoxicological studies integrating DNA repair as a parameter responding to single or mixture of contaminant is realized. Three main approaches are being used: the measurement of DNA repair gene expression after exposure, although it has not yet been clearly established whether gene expression is indicative of repair capacity; the monitoring of DNA damage removal by following DNA repair kinetics; and the modulation of DNA repair activity following exposure in situ, in order to assess the impact of exposure history on DNA repair capacity. Since all DNA repair processes are possible targets for environmental pollutants, we can also wonder at which extent such a modulation of repair capacities in fish could be the base for the development of new biomarkers of genotoxicity. Knowing the importance of the germ cell DNA integrity in the reproductive success of aquatic organisms, the DNA repair capacity of such cells deserve to be more studied, as well as DNA repair capacities of established fish cell lines. The limited amount of available data, which shows low/slow DNA repair capacities of fish cell lines compared with mammalian cell lines, concerned mainly the NER system; thus this point merits to be explored more deeply. Additionally, since some of the DNA repair systems appear more efficient in embryo larval stages, it would be of interest to consider embryonic cell lineages more closely. PMID:23571068

  18. Unraveling the Base Excision Repair Mechanism of Human DNA Glycosylase.

    PubMed

    Sadeghian, Keyarash; Ochsenfeld, Christian

    2015-08-12

    Human DNA glycosylase, hOGG1, is known to perform DNA repair by cleaving oxidized guanine (8OG) from the DNA. Despite numerous experimental and theoretical investigations, the underlying selective molecular mechanism has remained a mystery. Here we present a mechanism that explains how hOGG1's catalytic pocket is able to host an undamaged guanine base, but is not able to cleave it from the DNA. Using linear-scaling quantum mechanics/molecular mechanics (QM/MM) techniques with more than 500 atoms in the QM-region, we have investigated previously proposed mechanisms that all rely on protonating the 8OG nucleobase. We have found that the repair mechanisms propagated in the literature to this date are not capable of differentiating between the G and 8OG nucleobase. Besides this nonselectivity, they also involve reaction barriers that are too high, hence rendering the corresponding reaction intermediates inaccessible. Instead, we present a completely different reaction mechanism, where hOGG1 initially targets the ribose moiety of the substrate and cleaves the glycosidic bond at the very last stage. Our ribose-protonated repair mechanism is not only energetically more preferable, but also explains the selectivity utilized by hOGG1 to block processing a guanine base. PMID:26226322

  19. Diversity of Endonuclease V: From DNA Repair to RNA Editing

    PubMed Central

    Kuraoka, Isao

    2015-01-01

    Deamination of adenine occurs in DNA, RNA, and their precursors via a hydrolytic reaction and a nitrosative reaction. The generated deaminated products are potentially mutagenic because of their structural similarity to natural bases, which in turn leads to erroneous nucleotide pairing and subsequent disruption of cellular metabolism. Incorporation of deaminated precursors into the nucleic acid strand occurs during nucleotide synthesis by DNA and RNA polymerases or base modification by DNA- and/or RNA-editing enzymes during cellular functions. In such cases, removal of deaminated products from DNA and RNA by a nuclease might be required depending on the cellular function. One such enzyme, endonuclease V, recognizes deoxyinosine and cleaves 3' end of the damaged base in double-stranded DNA through an alternative excision repair mechanism in Escherichia coli, whereas in Homo sapiens, it recognizes and cleaves inosine in single-stranded RNA. However, to explore the role of endonuclease V in vivo, a detailed analysis of cell biology is required. Based on recent reports and developments on endonuclease V, we discuss the potential functions of endonuclease V in DNA repair and RNA metabolism. PMID:26404388

  20. Detection of the early stage of recombinational DNA repair by silicon nanowire transistors.

    PubMed

    Chiesa, Marco; Cardenas, Paula P; Otón, Francisco; Martinez, Javier; Mas-Torrent, Marta; Garcia, Fernando; Alonso, Juan C; Rovira, Concepció; Garcia, Ricardo

    2012-03-14

    A silicon nanowire-based biosensor has been designed and applied for label-free and ultrasensitive detection of the early stage of recombinational DNA repair by RecA protein. Silicon nanowires transistors were fabricated by atomic force microscopy nanolithography and integrated into a microfluidic environment. The sensor operates by measuring the changes in the resistance of the nanowire as the biomolecular reactions proceed. We show that the nanoelectronic sensor can detect and differentiate several steps in the binding of RecA to a single-stranded DNA filament taking place on the nanowire-aqueous interface. We report relative changes in the resistance of 3.5% which are related to the interaction of 250 RecA·single-stranded DNA complexes. Spectroscopy data confirm the presence of the protein-DNA complexes on the functionalized silicon surfaces. PMID:22364265

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

    PubMed Central

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

    2012-01-01

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

  2. 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.

  3. Inter-individual variation in DNA repair capacity: A need for multi-pathway functional assays to promote translational DNA repair research

    E-print Network

    Nagel, Zachary D.

    Why does a constant barrage of DNA damage lead to disease in some individuals, while others remain healthy? This article surveys current work addressing the implications of inter-individual variation in DNA repair capacity ...

  4. Visualization of a DNA-PK/PARP1 complex

    PubMed Central

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

    2012-01-01

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

  5. DNA Repair and the Accumulation of Oxidatively Damaged DNA Are Affected by Fruit Intake in Mice

    PubMed Central

    Croteau, Deborah L.; de Souza-Pinto, Nadja C.; Harboe, Charlotte; Keijzers, Guido; Zhang, Yongqing; Becker, Kevin; Sheng, Shan

    2010-01-01

    AGING is associated with elevated oxidative stress and DNA damage. To achieve healthy aging, we must begin to understand how diet affects cellular processes. We postulated that fruit-enriched diets might initiate a program of enhanced DNA repair and thereby improve genome integrity. C57Bl/6 J mice were fed for 14 weeks a control diet or a diet with 8% peach or nectarine extract. The activities of DNA repair enzymes, the level of DNA damage, and gene expression changes were measured. Our study showed that repair of various oxidative DNA lesions was more efficient in liver extracts derived from mice fed fruit-enriched diets. In support of these findings, gas chromatography–mass spectrometry analysis revealed that there was a decrease in the levels of formamidopyrimidines in peach-fed mice compared with the controls. Additionally, microarray analysis revealed that NTH1 was upregulated in peach-fed mice. Taken together, these results suggest that an increased intake of fruits might modulate the efficiency of DNA repair, resulting in altered levels of DNA damage. PMID:20847039

  6. 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.

  7. 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.

  8. ASSOCIATIONS BETWEEN POLYMORPHISMS IN DNA REPAIR GENES AND GLIOBLASTOMA

    PubMed Central

    McKean-Cowdin, Roberta; Barnholtz-Sloan, Jill; Inskip, Peter; Ruder, Avima; Butler, MaryAnn; Rajaraman, Preetha; Razavi, Pedram; Patoka, Joe; Wiencke, John; Bondy, Melissa; Wrensch, Margaret

    2009-01-01

    A pooled analysis was conducted to examine the association between select variants in DNA repair genes and glioblastoma multiforme (GBM), the most common and deadliest form of adult brain tumors. Genetic data for approximately 1,000 GBM cases and 2,000 controls were combined from four centers in the United States that have conducted case-control studies of adult GBM including the National Cancer Institute, the National Institute for Occupational Safety and Health, the University of Texas M.D. Anderson Cancer Center, and the University of California at San Francisco. Twelve DNA repair SNPs were selected for investigation in the pilot collaborative project. The C allele of the PARP1 rs1136410 variant was associated with a 20% reduction in risk of GBM (ORCT or CC =0.80; 95%CI 0.67–0.95). A 44% increase in risk of GBM was found for individuals homozygous for the G allele of the PRKDC rs7003908 variant (ORGG 1.44; 95%CI 1.13–1.84); there was a statistically significant trend (p=0.009) with increasing number of G alleles. A significant, protective effect was found when 3 SNPs (ERCC2 rs13181, ERCC1 rs3212986, and GLTSCR1 rs1035938) located near each other on chromosome 19 were modeled as a haplotype. The most common haplotype (AGC) was associated with a 23% reduction in risk (p=0.03) compared to all other haplotypes combined. Few studies have reported on the associations between variants in DNA repair genes and brain tumors, and few specifically have examined their impact on GBMs. Our results suggest that common variation in DNA repair genes may be associated with risk of GBMs. PMID:19318434

  9. Genome-wide Transcriptome Profiling of Homologous Recombination DNA Repair

    PubMed Central

    Peng, Guang; Lin, Curtis Chun-Jen; Mo, Wei; Dai, Hui; Park, Yun-Yong; Kim, Soo-Mi; Peng, Yang; Mo, Qianxing; Siwko, Stefan; Hu, Ruozhen; Lee, Ju-Seog; Hennessy, Bryan; Hanash, Samir; Mills, Gordon B.; Lin, Shiaw-Yih

    2014-01-01

    Homologous recombination (HR) repair deficiency predisposes to cancer development, but also sensitizes cancer cells to DNA-damage-inducing therapeutics. Here we identify an HR-defect (HRD) gene signature, which can be used to functionally assess HR repair status without interrogating individual genetic alterations in cells. By using this HRD gene signature as a functional network analysis tool, we discover that simultaneous loss of two major tumor suppressors BRCA1 and PTEN extensively rewire the HR repair-deficient phenotype, which is found in cells with defects in either BRCA1 or PTEN alone. Moreover, the HRD gene signature serves as an effective drug discovery platform to identify agents targeting HR repair as potential chemo/radio-sensitizers. More importantly, this HRD gene signature is able to predict clinical outcomes across multiple cancer lineages. Our findings, therefore, provide a molecular profile of HR repair to assess its status at a functional network level, which can provide both biological insights and have clinical implications in cancer. PMID:24553445

  10. A novel cell permeable DNA replication and repair marker

    PubMed Central

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

    2014-01-01

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

  11. Repairing of N-mustard derivative BO-1055 induced DNA damage requires NER, HR, and MGMT-dependent DNA repair mechanisms.

    PubMed

    Kuo, Ching-Ying; Chou, Wen-Cheng; Wu, Chin-Chung; Wong, Teng-Song; Kakadiya, Rajesh; Lee, Te-Chang; Su, Tsann-Long; Wang, Hui-Chun

    2015-09-22

    Alkylating agents are frequently used as first-line chemotherapeutics for various newly diagnosed cancers. Disruption of genome integrity by such agents can lead to cell lethality if DNA lesions are not removed. Several DNA repair mechanisms participate in the recovery of mono- or bi-functional DNA alkylation. Thus, DNA repair capacity is correlated with the therapeutic response. Here, we assessed the function of novel water-soluble N-mustard BO-1055 (ureidomustin) in DNA damage response and repair mechanisms. As expected, BO-1055 induces ATM and ATR-mediated DNA damage response cascades, including downstream Chk1/Chk2 phosphorylation, S/G2 cell-cycle arrest, and cell death. Further investigation revealed that cell survival sensitivity to BO-1055 is comparable to that of mitomycin C. Both compounds require nucleotide excision repair and homologous recombination, but not non-homologous end-joining, to repair conventional cross-linking DNA damage. Interestingly and unlike mitomycin C and melphalan, MGMT activity was also observed in BO-1055 damage repair systems, which reflects the occurrence of O-alkyl DNA lesions. Combined treatment with ATM/ATR kinase inhibitors significantly increases BO-1055 sensitivity. Our study pinpoints that BO-1055 can be used for treating tumors that with deficient NER, HR, and MGMT DNA repair genes, or for synergistic therapy in tumors that DNA damage response have been suppressed. PMID:26208482

  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. The COP9 signalosome is vital for timely repair of DNA double-strand breaks

    PubMed Central

    Meir, Michal; Galanty, Yaron; Kashani, Lior; Blank, Michael; Khosravi, Rami; Fernández-Ávila, María Jesús; Cruz-García, Andrés; Star, Ayelet; Shochot, Lea; Thomas, Yann; Garrett, Lisa J.; Chamovitz, Daniel A.; Bodine, David M.; Kurz, Thimo; Huertas, Pablo; Ziv, Yael; Shiloh, Yosef

    2015-01-01

    The DNA damage response is vigorously activated by DNA double-strand breaks (DSBs). The chief mobilizer of the DSB response is the ATM protein kinase. We discovered that the COP9 signalosome (CSN) is a crucial player in the DSB response and an ATM target. CSN is a protein complex that regulates the activity of cullin ring ubiquitin ligase (CRL) complexes by removing the ubiquitin-like protein, NEDD8, from their cullin scaffold. We find that the CSN is physically recruited to DSB sites in a neddylation-dependent manner, and is required for timely repair of DSBs, affecting the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair (HRR). The CSN is essential for the processivity of deep end-resection—the initial step in HRR. Cullin 4a (CUL4A) is recruited to DSB sites in a CSN- and neddylation-dependent manner, suggesting that CSN partners with CRL4 in this pathway. Furthermore, we found that ATM-mediated phosphorylation of CSN subunit 3 on S410 is critical for proper DSB repair, and that loss of this phosphorylation site alone is sufficient to cause a DDR deficiency phenotype in the mouse. This novel branch of the DSB response thus significantly affects genome stability. PMID:25855810

  14. DNA Repair Gene Polymorphisms and Their Relation With DNA Damage, DNA Repair, and Total Antioxidant Capacity in Childhood Acute Lymphoblastic Leukemia Survivors.

    PubMed

    Dincer, Yildiz; Yüksel, Selin; Batar, Bahadir; Güven, Mehmet; Onaran, Ilhan; Celkan, Tiraje

    2015-07-01

    Oxidative stress and defective DNA repair are major contributory factors in the initiation and progression of carcinogenesis. Chemotherapy and radiotherapy cause oxidative DNA damage, consume antioxidant capacity, and impair DNA repair activity. These effects of chemotherapy and radiotherapy may be contributory factors in the development of secondary malignancy in cancer survivors. Basal, H2O2-induced, and postrepair DNA damage; urinary 8-hydroxydeoxyguanosine level as a marker of oxidatively damaged DNA; and serum total antioxidant capacity were measured; XPD Lys751Gln, XRCC1 Arg399Gln, and XRCC1 Arg194Trp polymorphisms were analyzed in childhood acute lymphoblastic leukemia (ALL) survivors. Basal and H2O2-induced DNA damage were found to be higher in the ALL survivor group versus the control group, however, there was no significant difference between the other parameters. No association was found between the examined parameters and polymorphisms of XPD 751 and XRCC1 399 and both the groups. XRCC1 194Trp allele was found to be associated with a low level of postrepair DNA damage in the ALL survivors. In conclusion, basal DNA damage and susceptibility to oxidation are high in childhood ALL survivors. This situation which may easily lead to occurrence of a secondary cancer does not seem to be a result of deficient DNA repair. PMID:24577548

  15. Electron trap for DNA-bound repair enzymes: a strategy for DNA-mediated signaling.

    PubMed

    Yavin, Eylon; Stemp, Eric D A; O'shea, Valerie L; David, Sheila S; Barton, Jacqueline K

    2006-03-01

    Despite a low copy number within the cell, base excision repair (BER) enzymes readily detect DNA base lesions and mismatches. These enzymes also contain [Fe4S4] clusters, yet a redox role for these iron cofactors had been unclear. Here, we provide evidence that BER proteins may use DNA-mediated redox chemistry as part of a signaling mechanism to detect base lesions. By using chemically modified bases, we show electron trapping on DNA in solution with bound BER enzymes by electron paramagnetic resonance (EPR) spectroscopy. We demonstrate electron transfer from two BER proteins, Endonuclease III (EndoIII) and MutY, to modified bases in DNA containing oxidized nitroxyl radical EPR probes. Electron trapping requires that the modified base is coupled to the DNA pi-stack, and trapping efficiency is increased when a noncleavable MutY substrate analogue is located distally to the trap. These results are consistent with DNA binding leading to the activation of the repair proteins toward oxidation. Significantly, these results support a mechanism for DNA repair that involves DNA-mediated charge transport. PMID:16505354

  16. 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.

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

    PubMed

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

    2014-01-01

    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

  18. A moonlighting metabolic protein influences repair at DNA double-stranded breaks.

    PubMed

    Torres-Machorro, Ana Lilia; Aris, John P; Pillus, Lorraine

    2015-02-18

    Catalytically active proteins with divergent dual functions are often described as 'moonlighting'. In this work we characterize a new, chromatin-based function of Lys20, a moonlighting protein that is well known for its role in metabolism. Lys20 was initially described as homocitrate synthase (HCS), the first enzyme in the lysine biosynthetic pathway in yeast. Its nuclear localization led to the discovery of a key role for Lys20 in DNA damage repair through its interaction with the MYST family histone acetyltransferase Esa1. Overexpression of Lys20 promotes suppression of DNA damage sensitivity of esa1 mutants. In this work, by taking advantage of LYS20 mutants that are active in repair but not in lysine biosynthesis, the mechanism of suppression of esa1 was characterized. First we analyzed the chromatin landscape of esa1 cells, finding impaired histone acetylation and eviction. Lys20 was recruited to sites of DNA damage, and its overexpression promoted enhanced recruitment of the INO80 remodeling complex to restore normal histone eviction at the damage sites. This study improves understanding of the evolutionary, structural and biological relevance of independent activities in a moonlighting protein and links metabolism to DNA damage repair. PMID:25628362

  19. Ultraviolet radiation-induced non-melanoma skin cancer: Regulation of DNA damage repair and inflammation

    PubMed Central

    Kim, Young; He, Yu-Ying

    2014-01-01

    Exposure to ultraviolet (UV) radiation is associated with approximately 65% of melanoma cases, and 90% of non-melanoma skin cancers (NMSC), including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). While the incidence of most other malignancies has either stabilized or declined, that of NMSC has increased and is developing even in younger age groups. NMSCs account for nearly 15,000 deaths, 3.5 million new cases, and more than 3 billion dollars a year in medical costs in the United States alone, representing a major public health concern. As sun protection efforts have not been proven effective, targeted chemoprevention strategies are much needed. Skin carcinogenesis by DNA damage is considered a predominant paradigm for UV toxicity. Exposure to UV radiation can activate various oncogenes while inactivating tumor suppressor genes, resulting in inappropriate survival and proliferation of keratinocytes that harbor these damages. Moreover, increasing evidence demonstrate that inflammatory responses by the immune cells within the tumor microenvironment also contribute significantly to skin tumorigenesis. Initiation and progression of skin carcinogenesis mediated by UV radiation involve complex pathways, including those of apoptosis, proliferation, autophagy, DNA repair, checkpoint signaling, metabolism, and inflammation. In this review, we highlight the recent advances in two of these key molecular processes that result in UV-mediated skin carcinogenesis. In particular, we discuss 1) pathways that regulate DNA damage repair and 2) the regulation of the inflammatory process its crosstalk with DNA repair potentially leading to non-melanoma skin carcinogenesis. PMID:25642450

  20. Regulation of nucleotide excision repair by UV-DDB: prioritization of damage recognition to internucleosomal DNA.

    PubMed

    Fei, Jia; Kaczmarek, Nina; Luch, Andreas; Glas, Andreas; Carell, Thomas; Naegeli, Hanspeter

    2011-10-01

    How tightly packed chromatin is thoroughly inspected for DNA damage is one of the fundamental unanswered questions in biology. In particular, the effective excision of carcinogenic lesions caused by the ultraviolet (UV) radiation of sunlight depends on UV-damaged DNA-binding protein (UV-DDB), but the mechanism by which this DDB1-DDB2 heterodimer stimulates DNA repair remained enigmatic. We hypothesized that a distinctive function of this unique sensor is to coordinate damage recognition in the nucleosome repeat landscape of chromatin. Therefore, the nucleosomes of human cells have been dissected by micrococcal nuclease, thus revealing, to our knowledge for the first time, that UV-DDB associates preferentially with lesions in hypersensitive, hence, highly accessible internucleosomal sites joining the core particles. Surprisingly, the accompanying CUL4A ubiquitin ligase activity is necessary to retain the xeroderma pigmentosum group C (XPC) partner at such internucleosomal repair hotspots that undergo very fast excision kinetics. This CUL4A complex thereby counteracts an unexpected affinity of XPC for core particles that are less permissive than hypersensitive sites to downstream repair subunits. That UV-DDB also adopts a ubiquitin-independent function is evidenced by domain mapping and in situ protein dynamics studies, revealing direct but transient interactions that promote a thermodynamically unfavorable ?-hairpin insertion of XPC into substrate DNA. We conclude that the evolutionary advent of UV-DDB correlates with the need for a spatiotemporal organizer of XPC positioning in higher eukaryotic chromatin. PMID:22039351

  1. From Breaking Bad to Worse: Exploiting Homologous DNA Repair Deficiency in Cancer

    E-print Network

    Hemann, Michael

    DNA repair deficiencies are common among cancer cells and represent a potential vulnerability that might be exploited by targeting compensatory repair pathways. However, the identification of synthetically lethal combinations ...

  2. Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment.

    PubMed

    Eyboulet, Fanny; Cibot, Camille; Eychenne, Thomas; Neil, Helen; Alibert, Olivier; Werner, Michel; Soutourina, Julie

    2013-12-01

    Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3' endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes. PMID:24298055

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

    E-print Network

    Braithwaite, Elena K.

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

  4. Nucleosome Disruption by DNA Ligase III-XRCC1 Promotes Efficient Base Excision Repair ? ‡

    PubMed Central

    Odell, Ian D.; Barbour, Joy-El; Murphy, Drew L.; Della-Maria, Julie A.; Sweasy, Joann B.; Tomkinson, Alan E.; Wallace, Susan S.; Pederson, David S.

    2011-01-01

    Each day, approximately 20,000 oxidative lesions form in the DNA of every nucleated human cell. The base excision repair (BER) enzymes that repair these lesions must function in a chromatin milieu. We have determined that the DNA glycosylase hNTH1, apurinic endonuclease (APE), and DNA polymerase ? (Pol ?), which catalyze the first three steps in BER, are able to process their substrates in both 601- and 5S ribosomal DNA (rDNA)-based nucleosomes. hNTH1 formed a discrete ternary complex that was displaced by the addition of APE, suggesting an orderly handoff of substrates from one enzyme to the next. In contrast, DNA ligase III?-XRCC1, which completes BER, was appreciably active only at concentrations that led to nucleosome disruption. Ligase III?-XRCC1 was also able to bind and disrupt nucleosomes containing a single base gap and, because of this property, enhanced both its own activity and that of Pol ? on nucleosome substrates. Collectively, these findings provide insights into rate-limiting steps that govern BER in chromatin and reveal a unique role for ligase III?-XRCC1 in enhancing the efficiency of the final two steps in the BER of lesions in nucleosomes. PMID:21930793

  5. 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...

  6. Whole transcriptome analysis reveals a role for OGG1-initiated DNA repair signaling in airway remodeling.

    PubMed

    Aguilera-Aguirre, Leopoldo; Hosoki, Koa; Bacsi, Attila; Radák, Zsolt; Sur, Sanjiv; Hegde, Muralidhar L; Tian, Bing; Saavedra-Molina, Alfredo; Brasier, Allan R; Ba, Xueqing; Boldogh, Istvan

    2015-12-01

    Reactive oxygen species (ROS) generated by environmental exposures, and endogenously as by-products of respiration, oxidatively modify biomolecules including DNA. Accumulation of ROS-induced DNA damage has been implicated in various diseases that involve inflammatory processes, and efficient DNA repair is considered critical in preventing such diseases. One of the most abundant DNA base lesions is 7,8-dihydro-8-oxoguanine (8-oxoG), which is repaired by the 8-oxoguanine DNA glycosylase 1 (OGG1)-initiated base-excision repair (OGG1-BER) pathway. Recent studies have shown that the OGG1-BER by-product 8-oxoG base forms a complex with cytosolic OGG1, activating small GTPases and downstream cell signaling in cultured cells and lungs. This implies that persistent OGG1-BER could result in signaling leading to histological changes in airways. To test this, we mimicked OGG1-BER by repeatedly challenging airways with its repair product 8-oxoG base. Gene expression was analyzed by RNA sequencing (RNA-Seq) and qRT-PCR, and datasets were evaluated by gene ontology and statistical tools. RNA-Seq analysis identified 3252 differentially expressed transcripts (2435 up- and 817 downregulated, ?3-fold change). Among the upregulated transcripts, 2080 mRNAs were identified whose encoded protein products were involved in modulation of the actin family cytoskeleton, extracellular matrix, cell adhesion, cadherin, and cell junctions, affecting biological processes such as tissue development, cell-to-cell adhesion, cell communication, and the immune system. These data are supported by histological observations showing epithelial alterations, subepithelial fibrosis, and collagen deposits in the lungs. These data imply that continuous challenge by the environment and consequent OGG1-BER-driven signaling trigger gene expression consistent with airway remodeling. PMID:26187872

  7. Biochemical studies of DNA strand break repair and molecular characterization of mei-41, a gene involved in DNA break repair

    SciTech Connect

    Oliveri, D.R.

    1989-01-01

    The ability to repair X-irradiation induced single-strand DNA breaks was examined in mutagen-sensitive mutants of Drosophila melanogaster. This analysis demonstrated that examined stocks possess a normal capacity to repair X-ray induced single-strand breaks. One of the mutants in this study, mei-41, has been shown to be involved in a number of DNA metabolizing functions. A molecular characterization of this mutant is presented. A cDNA hybridizing to genomic DNA both proximal and distal to a P element inducing a mei-41 mutation was isolated from both embryonic and adult female recombinant lambda phage libraries. A 2.2 kilobase embryonic cDNA clone was sequenced; the sequence of an open reading frame was identified which would predict a protein of 384 amino acids with a molecular weight of 43,132 daltons. An examination of homologies to sequences in protein and nucleic acid data bases revealed no sequences with significant homology to mei-41, however, two potential Zinc-finger domains were identified. Analysis of RNA hybridizing to the embryonic cDNA demonstrated the existence of a major 2.2 kilobase transcript expressed primarily in embryos and adult flies. An examination of the transcription of this gene in mei-41 mutants revealed significant variation from wild-type, an indication that the embryonic cDNA does represent a mei-41 transcript. Expression in tissues from adult animals demonstrated that the 2.2 kilobase RNA is expressed primarily in reproductive tissues. A 3.8kb transcript is the major species of RNA in the adult head and thorax. Evidence is presented which implies that expression of the mei-41 gene is strongly induced by exposure of certain cells to mutagens.

  8. Two-ventricle repair for complex congenital heart defects palliated towards single-ventricle repair

    PubMed Central

    Kottayil, Brijesh P.; Sunil, Gopalraj S.; Kappanayil, Mahesh; Mohanty, Sweta Harish; Francis, Edwin; Vaidyanathan, Balu; Balachandran, Rakhi; Nair, Suresh G.; Kumar, Raman Krishna

    2014-01-01

    OBJECTIVES Complex congenital heart defects that present earlier in life are sometimes channelled towards single-ventricle repair, because of anatomical or logistic challenges involved in two-ventricle correction. Given the long-term functional and survival advantage, we have been consciously exploring the feasibility of a biventricular repair in these patients when they present later for Fontan completion. METHODS Since June 2009, 71 patients were referred for staged completion of the Fontan procedure. Following detailed evaluation that included three-dimensional echocardiography and magnetic resonance imaging, 10 patients (Group 1—median age 6 years) were identified and later underwent complex biventricular repair with takedown of Glenn shunt, while completion of extracardiac Fontan repair was done in 61 patients (Group 2—median age 7 years). RESULTS Two-ventricle repair was accomplished in all the 10 Group 1 patients. One patient developed complete heart block requiring permanent pacemaker insertion. Late patch dehiscence occurred in another (awaiting repair). At a median follow-up of 15 months, there was no mortality among the Group 1 patients and all except for 1 patient were symptom free. There were 2 early deaths (3.3%) in the Group 2 patients. CONCLUSIONS Two-ventricular repair, although surgically challenging, should be considered in all patients with two functional ventricles who come for Fontan completion. Comprehensive preoperative imaging and meticulous planning helps in identifying suitable candidates. PMID:24310735

  9. PTIP associates with Artemis to dictate DNA repair pathway choice

    PubMed Central

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

    2014-01-01

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

  10. PTIP associates with Artemis to dictate DNA repair pathway choice.

    PubMed

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

    2014-12-15

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

  11. Cellular Pathways for DNA Repair and Damage Tolerance of Formaldehyde-Induced DNA-Protein Crosslinks

    PubMed Central

    de Graaf, Bendert; Clore, Adam; McCullough, Amanda K.

    2009-01-01

    Although it is well established that DNA-protein crosslinks are formed as a consequence of cellular exposure to agents such as formaldehyde, transplatin, ionizing and ultraviolet radiation, the biochemical pathways that promote cellular survival via repair or tolerance of these lesions are poorly understood. To investigate the mechanisms that function to limit DNA-protein crosslink-induced cytotoxicity, the Saccharomyces cerevisiae non-essential gene deletion library was screened for increased sensitivity to formaldehyde exposure. Following low-dose, chronic exposure, strains containing deletions in genes mediating homologous recombination showed the greatest sensitivity, while under the same exposure conditions, deletions in genes associated with nucleotide excision repair conferred only low to moderate sensitivities. However, when the exposure regime was changed to a high-dose acute (short-term) formaldehyde treatment, the genes that conferred maximal survival switched to the nucleotide excision repair pathway, with little contribution of the homologous recombination genes. Data are presented which suggest that following acute formaldehyde exposure, repair and/or tolerance of DNA-protein crosslinks proceeds via formation of nucleotide excision repair-dependent single-strand break intermediates and without a detectable accumulation of double-strand breaks. These data clearly demonstrate a differential pathway response to chronic versus acute formaldehyde exposures and may have significance and implications for risk extrapolation in human exposure studies. PMID:19625222

  12. SUMOylation of xeroderma pigmentosum group C protein regulates DNA damage recognition during nucleotide excision repair

    PubMed Central

    Akita, Masaki; Tak, Yon-Soo; Shimura, Tsutomu; Matsumoto, Syota; Okuda-Shimizu, Yuki; Shimizu, Yuichiro; Nishi, Ryotaro; Saitoh, Hisato; Iwai, Shigenori; Mori, Toshio; Ikura, Tsuyoshi; Sakai, Wataru; Hanaoka, Fumio; Sugasawa, Kaoru

    2015-01-01

    The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions. In the absence of SUMOylation, XPC is normally recruited to the sites with photolesions, but then immobilized profoundly by the UV-damaged DNA-binding protein (UV-DDB) complex. Since the absence of UV-DDB alleviates the NER defect caused by impaired SUMOylation of XPC, we propose that this modification is critical for functional interactions of XPC with UV-DDB, which facilitate the efficient damage handover between the two damage recognition factors and subsequent initiation of NER. PMID:26042670

  13. 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.

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

    PubMed Central

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

    2014-01-01

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

  15. Structural complexity of DNA sequence.

    PubMed

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

    2013-01-01

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

  16. Structural Complexity of DNA Sequence

    PubMed Central

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

    2013-01-01

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

  17. 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

  18. A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis.

    PubMed

    Makarova, Kira S; Aravind, L; Grishin, Nick V; Rogozin, Igor B; Koonin, Eugene V

    2002-01-15

    During a systematic analysis of conserved gene context in prokaryotic genomes, a previously undetected, complex, partially conserved neighborhood consisting of more than 20 genes was discovered in most Archaea (with the exception of Thermoplasma acidophilum and Halobacterium NRC-1) and some bacteria, including the hyperthermophiles Thermotoga maritima and Aquifex aeolicus. The gene composition and gene order in this neighborhood vary greatly between species, but all versions have a stable, conserved core that consists of five genes. One of the core genes encodes a predicted DNA helicase, often fused to a predicted HD-superfamily hydrolase, and another encodes a RecB family exonuclease; three core genes remain uncharacterized, but one of these might encode a nuclease of a new family. Two more genes that belong to this neighborhood and are present in most of the genomes in which the neighborhood was detected encode, respectively, a predicted HD-superfamily hydrolase (possibly a nuclease) of a distinct family and a predicted, novel DNA polymerase. Another characteristic feature of this neighborhood is the expansion of a superfamily of paralogous, uncharacterized proteins, which are encoded by at least 20-30% of the genes in the neighborhood. The functional features of the proteins encoded in this neighborhood suggest that they comprise a previously undetected DNA repair system, which, to our knowledge, is the first repair system largely specific for thermophiles to be identified. This hypothetical repair system might be functionally analogous to the bacterial-eukaryotic system of translesion, mutagenic repair whose central components are DNA polymerases of the UmuC-DinB-Rad30-Rev1 superfamily, which typically are missing in thermophiles. PMID:11788711

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

    SciTech Connect

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

    2006-07-03

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

  20. 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. The result indicates that the negative charge transfer may proceed by hopping. [1] J. P. Pouget, T. Douki, M. J. Richard, J. Cadet, Chem. Res. Toxicol. 2000, 13, 541. [2] E. C. Friedberg, DNA repair and mutagenesis, ASM Press, Washington, D.C., 1995. [3] R. E. Holmlin, P. J. Dandlicker, J. K. Barton, Angew. Chem. Int. Ed. 1997, 36, 2715. [4] B. Giese, Acc. Chem. Res. 2000, 33, 631. [5] F. D. Lewis, R. L. Letsinger, M. R. Wasielewski, Acc. Chem. Res. 2001, 34, 159. [6] G. B. Schuster, Acc. Chem. Res. 2000, 33, 253. [7] A. Schwögler, L. T. Burgdorf, T. Carell, Angew. Chem. Int. Ed. 2000, 39, 3918. [8] A. Schwögler, T. Carell, Org. Lett. 2000, 2, 1415. [9] M. K. Cichon, C. H. Haas, F. Grolle, A. Mees, T. Carell.J. Am. Chem. Soc. 2002, 124, 13984-13985.

  1. Tetrameric Ctp1 coordinates DNA binding and bridging in DNA double strand break repair

    PubMed Central

    Andres, Sara N.; Appel, C. Denise; Westmoreland, Jim; Williams, Jessica S.; Nguyen, Yvonne; Robertson, Patrick D.; Resnick, Michael A.; Williams, R. Scott

    2014-01-01

    Ctp1 (aka CtIP or Sae2) collaborates with Mre11–Rad50–Nbs1 to initiate repair of DNA double strand breaks (DSBs), but its function(s) remain enigmatic. We report that tetrameric Schizosaccharomyces pombe Ctp1 harbors multivalent DNA-binding and bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal “RHR” DNA interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA bridging activity in vitro and both the THDD and RHR are required for efficient DSB repair in S. pombe. Our results establish non-nucleolytic roles for Ctp1 in binding and coordination of DSB repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CTIP-linked Seckel and Jawad syndromes. PMID:25580577

  2. Investigating the role of vitamin D and DNA repair in influencing cancer presentation and outcome 

    E-print Network

    Stonecipher, Ashley

    2015-04-23

    exposure to DNA damaging chemotherapeutic agents. Chi-square based population association approach will be used to associate genotypes of DNA repair genes to DRC capacity, thus providing the basis of population difference in the cancer phenotype....

  3. A novel bisindole-PBD conjugate causes DNA damage induced apoptosis via inhibition of DNA repair pathway.

    PubMed

    Sarma, Pranjal; Ramaiah, M Janaki; Kamal, Ahmed; Bhadra, Utpal; Bhadra, Manika Pal

    2014-10-01

    DNA damage response (DDR) that includes cell cycle check points, DNA repair, apoptosis, and senescence is intimately linked with cancer. It shields an organism against cancer development when genomic integrity fails. DNA repair pathways protect the cells from tumor progression caused as a result of DNA damage induced by irradiation or due to chemotherapeutic treatment. Many promising anticancer agents have been identified that target specific DNA repair pathways in response to DNA damage thereby leading to apoptosis. Here we identified a novel bisindole-PBD conjugate that possess potent anticancer activity in breast cancer cells. Further studies aimed at understanding the mechanism of action of the molecule showed its role in DNA damage induced apoptosis via inhibition of DNA repair pathway. Trypan blue and BrdU assay exhibited a dose-dependent effect. Single-stranded DNA damage was observed by COMET assay. In addition DNA damage induced ROS generation with simultaneous activation of ATM and ATR upon compound treatment was observed. Further downregulation of Bcl-XL and activation of Bax showed DNA damage induced apoptosis in MCF-7 and MDAMB-231 cells. In conclusion, it can be summarized that bisindole-PBD conjugate induces DNA damage in a dose dependent (2, 4, and 8 ?M) manner by inhibiting the DNA repair genes. PMID:25010292

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

    NASA Astrophysics Data System (ADS)

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

    2002-04-01

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

  5. Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

    SciTech Connect

    Chen, Xi; Ballin, Jeff D.; Della-Maria, Julie; Tsai, Miaw-Sheue; White, Elizabeth J.; Tomkinson, Alan E.; Wilson, Gerald M.

    2009-05-11

    The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIII{beta} and the hLigIII{alpha}/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.

  6. In TFIIH, XPD helicase is exclusively devoted to DNA repair.

    PubMed

    Kuper, Jochen; Braun, Cathy; Elias, Agnes; Michels, Gudrun; Sauer, Florian; Schmitt, Dominik R; Poterszman, Arnaud; Egly, Jean-Marc; Kisker, Caroline

    2014-09-01

    The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER). Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue) to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription. PMID:25268380

  7. New insights into tumor dormancy: Targeting DNA repair pathways

    PubMed Central

    Evans, Elizabeth B; Lin, Shiaw-Yih

    2015-01-01

    Over the past few decades, major strides have advanced the techniques for early detection and treatment of cancer. However, metastatic tumor growth still accounts for the majority of cancer-related deaths worldwide. In fact, breast cancers are notorious for relapsing years or decades after the initial clinical treatment, and this relapse can vary according to the type of breast cancer. In estrogen receptor-positive breast cancers, late tumor relapses frequently occur whereas relapses in estrogen receptor-negative cancers or triple negative tumors arise early resulting in a higher mortality risk. One of the main causes of metastasis is tumor dormancy in which cancer cells remain concealed, asymptomatic, and untraceable over a prolonged period of time. Under certain conditions, dormant cells can re-enter into the cell cycle and resume proliferation leading to recurrence. However, the molecular and cellular regulators underlying this transition remain poorly understood. To date, three mechanisms have been identified to trigger tumor dormancy including cellular, angiogenic, and immunologic dormancies. In addition, recent studies have suggested that DNA repair mechanisms may contribute to the survival of dormant cancer cells. In this article, we summarize the recent experimental and clinical evidence governing cancer dormancy. In addition, we will discuss the role of DNA repair mechanisms in promoting the survival of dormant cells. This information provides mechanistic insight to explain why recurrence occurs, and strategies that may enhance therapeutic approaches to prevent disease recurrence. PMID:26468441

  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. 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).

  10. DNA lesions, inducible DNA repair, and cell division: three key factors in mutagenesis and carcinogenesis.

    PubMed Central

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

    1993-01-01

    DNA lesions that escape repair have a certain probability of giving rise to mutations when the cell divides. Endogenous DNA damage is high: 10(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 mutation and therefore cancer. There is little cancer from nondividing cells. Endogenous cell division rates can be influenced by hormone levels, decreased by calorie restriction, or increased 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). PMID:8013423

  11. 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

  12. Smc5p Promotes Faithful Chromosome Transmission and DNA Repair in Saccharomyces cerevisiae

    PubMed Central

    Cost, Gregory J.; Cozzarelli, Nicholas R.

    2006-01-01

    Heterodimers of structural maintenance of chromosomes (SMC) proteins form the core of several protein complexes involved in the organization of DNA, including condensation and cohesion of the chromosomes at metaphase. The functions of the complexes with a heterodimer of Smc5p and Smc6p are less clear. To better understand them, we created two S. cerevisiae strains bearing temperature-sensitive alleles of SMC5. When shifted to the restrictive temperature, both mutants lose viability gradually, concomitant with the appearance of nuclear abnormalities and phosphorylation of the Rad53p DNA damage checkpoint protein. Removal of Rad52p or overexpression of the SUMO ligase Mms21p partially suppresses the temperature sensitivity of smc5 strains and increases their survival at the restrictive temperature. At the permissive temperature, smc5-31 but not smc5-33 cells exhibit hypersensitivity to several DNA-damaging agents despite induction of the DNA damage checkpoint. Similarly, smc5-31 but not smc5-33 cells are killed by overexpression of the SUMO ligase-defective Mms21-SAp but not by overexpression of wild-type Mms21p. Both smc5 alleles are synthetically lethal with mms21-SA and exhibit Rad52p-independent chromosome fragmentation and loss at semipermissive temperatures. Our data indicate a critical role for the S. cerevisiae Smc5/6-containing complexes in both DNA repair and chromosome segregation. PMID:16452135

  13. Disruption of PARP1 function inhibits base excision repair of a sub-set of DNA lesions

    PubMed Central

    Reynolds, Pamela; Cooper, Sarah; Lomax, Martine; O'Neill, Peter

    2015-01-01

    The repair of endogenously induced DNA damage is essential to maintain genomic integrity. It has been shown that XRCC1 and PARP1 are involved in the repair of base lesions and SSBs, although the exact mode of action has yet to be determined. Here we show that XRCC1 is involved in the repair of base lesions and SSBs independent of the cell cycle. However, the rate of repair of damage requiring XRCC1 does reflect the damage complexity. The repair of induced DNA damage occurs by PARP1-dependent and PARP1-independent sub-pathways of BER. It is suggested that the repair of SSBs and purine base damage is by a sub-pathway of BER that requires both XRCC1 and PARP1. Repair of pyrimidine base damage may require XRCC1 but does not require PARP1 activity. Therefore, although BER of simple lesions occurs rapidly, pathway choice and the involvement of PARP1 are highly dependent on the types of lesion induced. PMID:25813046

  14. Disruption of PARP1 function inhibits base excision repair of a sub-set of DNA lesions.

    PubMed

    Reynolds, Pamela; Cooper, Sarah; Lomax, Martine; O'Neill, Peter

    2015-04-30

    The repair of endogenously induced DNA damage is essential to maintain genomic integrity. It has been shown that XRCC1 and PARP1 are involved in the repair of base lesions and SSBs, although the exact mode of action has yet to be determined. Here we show that XRCC1 is involved in the repair of base lesions and SSBs independent of the cell cycle. However, the rate of repair of damage requiring XRCC1 does reflect the damage complexity. The repair of induced DNA damage occurs by PARP1-dependent and PARP1-independent sub-pathways of BER. It is suggested that the repair of SSBs and purine base damage is by a sub-pathway of BER that requires both XRCC1 and PARP1. Repair of pyrimidine base damage may require XRCC1 but does not require PARP1 activity. Therefore, although BER of simple lesions occurs rapidly, pathway choice and the involvement of PARP1 are highly dependent on the types of lesion induced. PMID:25813046

  15. Complex kinetics of DNA condensation revealed through DNA twist tracing

    NASA Astrophysics Data System (ADS)

    Li, Wei; Wong, Wei Juan; Lim, Ci Ji; Ju, Hai-Peng; Li, Ming; Yan, Jie; Wang, Peng-Ye

    2015-08-01

    Toroid formation is an important mechanism for DNA condensation in cells. The length change during DNA condensation was investigated in previous single-molecule experiments. However, DNA twist is key to understanding the topological kinetics of DNA condensation. In this study, DNA twist as well as DNA length was traced during the DNA condensation by the freely orbiting magnetic tweezers and the tilted magnetic tweezers combined with Brownian dynamics simulations. The experimental results disclose the complex relationship between DNA extension and backbone rotation. Brownian dynamics simulations show that the toroid formation follows a wiggling pathway which leads to the complex DNA backbone rotation as revealed in our experiments. These findings provide the complete description of multivalent cation-dependent DNA toroid formation under tension.

  16. Calculation of complex DNA damage induced by ions

    E-print Network

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

    2012-01-27

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

  17. Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences.

    PubMed

    Cilli, Piera; Minoprio, Anna; Bossa, Cecilia; Bignami, Margherita; Mazzei, Filomena

    2015-10-23

    The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase ? (POL ?) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL ? incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL ? was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences. PMID:26338705

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

    NASA Technical Reports Server (NTRS)

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

    2009-01-01

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

  19. 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.

  20. Accelerated Search Kinetics Mediated by Redox Reactions of DNA Repair Enzymes

    E-print Network

    Levine, Alex J.

    Accelerated Search Kinetics Mediated by Redox Reactions of DNA Repair Enzymes Pak-Wing Fok and Tom to explain the localization of base excision repair (BER) enzymes to lesions on DNA. The CT mechanism relies on redox reactions of iron-sulfur cofactors that modify the enzyme's binding affinity. These redox

  1. 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

  2. Medical Electronics High-throughput Arrayed Comets for DNA Damage and Repair ............................................................................................................. MED.1

    E-print Network

    Reif, Rafael

    Medical Electronics High-throughput Arrayed Comets for DNA Damage and RepairEctronics MEDical ElEctronics High-throughput arrayed comets for Dna Damage and repair D. K. Wood, D. M. Weingeist method for this work is the well-established comet assay, in which cells are embedded in agarose, exposed

  3. Temperature effects on survival and DNA repair in four freshwater cladoceran Daphnia species exposed to UV radiation.

    PubMed

    Connelly, Sandra J; Moeller, Robert E; Sanchez, Guillermo; Mitchell, David L

    2009-01-01

    The biological responses of four freshwater daphniid species, Daphnia middendorffiana, D. pulicaria, D. pulex and D. parvula, to a single acute dose of ultraviolet B radiation (UVB) were compared. In addition to survival, we compared the induction of DNA damage (i.e. cyclobutane pyrimidine dimers) between species as well as the ability to repair this damage in the presence or absence of photoreactivating light. All four species showed high levels of shielding against DNA damage when compared to damage induced in purified DNA dosimeters at the same time and dose. Significant variation in survival was observed between species depending on temperature and light conditions. Contrary to our expectations, all species showed significantly higher survival and light-dependent DNA damage removal rates at 10 degrees C compared to 20 degrees C, suggesting that the enhanced rate of photoenzymatic repair (PER) at the lower temperature contributed significantly to the recovery of these organisms from UVB. PER was highly effective in promoting survival of three of the four species at 10 degrees C, but at 20 degrees C it was only partially effective in two species, and ineffective in two others. None of the species showed significant dark repair at 20 degrees C and only D. pulicaria showed a significant capacity at 10 degrees C. Two species, D. middendorffiana and D. pulex, showed some short-term survival at 10 degrees C in absence of PER despite their inability to repair any appreciable amount of DNA damage in the dark. All species died rapidly at 20 degrees C in absence of PER, as predicted from complete or near-absence of nucleotide excision repair (NER). Overall, the protective effects of tissue structure and pigmentation were similar in all Daphnia species tested and greatly mitigated the absorption of UVB by DNA and its damaging effects. Surprisingly, the visibly melanotic D. middendorffiana was not better shielded from DNA damage than the three non-melanotic species, and in fact suffered the highest damage rates. Melanin content in this species was not temperature dependent under the experimental growth conditions, and so did not contribute to temperature-dependent responses. It is evident that different species within the same genus have developed diverse biological responses to UVB. Our data strongly suggest that DNA damage is lethal to Daphnia and that photoenzymatic repair is the primary mechanism for removing these lesions. In the absence of light, few species are capable of removing any DNA damage. Surprisingly, the single species in which significant excision repair was detected did so only at reduced temperature. This temperature-dependence of excision repair is striking and may reflect adaptations of certain organisms to stress in a complex and changing environment. PMID:18700864

  4. Chromatin Modifications during Repair of Environmental Exposure-Induced DNA Damage: A Potential Mechanism for Stable Epigenetic Alterations

    PubMed Central

    O’Hagan, Heather M.

    2014-01-01

    Exposures to environmental toxicants and toxins cause epigenetic changes that likely play a role in the development of diseases associated with exposure. The mechanism behind these exposure-induced epigenetic changes is currently unknown. One commonality between most environmental exposures is that they cause DNA damage either directly or through causing an increase in reactive oxygen species, which can damage DNA. Like transcription, DNA damage repair must occur in the context of chromatin requiring both histone modifications and ATP-dependent chromatin remodeling. These chromatin changes aid in DNA damage accessibility and signaling. Several proteins and complexes involved in epigenetic silencing during both development and cancer have been found to be localized to sites of DNA damage. The chromatin-based response to DNA damage is considered a transient event, with chromatin being restored to normal as DNA damage repair is completed. However, in individuals chronically exposed to environmental toxicants or with chronic inflammatory disease, repeated DNA damage-induced chromatin rearrangement may ultimately lead to permanent epigenetic alterations. Understanding the mechanism behind exposure-induced epigenetic changes will allow us to develop strategies to prevent or reverse these changes. This review focuses on epigenetic changes and DNA damage induced by environmental exposures, the chromatin changes that occur around sites of DNA damage, and how these transient chromatin changes may lead to heritable epigenetic alterations at sites of chronic exposure. PMID:24259318

  5. Synthetic-viability genomic screening defines Sae2 function in DNA repair

    E-print Network

    Puddu, Fabio; Oelschlaegel, Tobias; Guerini, Ilaria; Geisler, Nicola J.; Niu, Hengyao; Herzog, Mareike; Salguero, Israel; Ochoa-Montaño, Bernardo; Viré, Emmanuelle; Sung, Patrick; Adams, David J.; Keane, Thomas M.; Jackson, Stephen P.

    2015-04-21

    DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3' single-stranded DNA (ssDNA) generation by 5' DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB...

  6. DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from

    E-print Network

    Stuart, Jeffrey A.

    DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA J.A. Stuart, K. Hashiguchi, D.M. Wilson III, W.C. Copeland1 , N.C. Souza Received December 31, 2003; Revised and Accepted March 21, 2004 ABSTRACT Mitochondrial DNA (mtDNA) contains

  7. The impact of base excision DNA repair in age-related neurodegenerative diseases.

    PubMed

    Leandro, Giovana S; Sykora, Peter; Bohr, Vilhelm A

    2015-06-01

    The aging process and several age-related neurodegenerative disorders have been linked to elevated levels of DNA damage induced by ROS and deficiency in DNA repair mechanisms. DNA damage induced by ROS is a byproduct of cellular respiration and accumulation of damage over time, is a fundamental aspect of a main theory of aging. Mitochondria have a pivotal role in generating cellular oxidative stress, and mitochondrial dysfunction has been associated with several diseases. DNA base excision repair is considered the major pathway for repair of oxidized bases in DNA both in the nuclei and in mitochondria, and in neurons this mechanism is particularly important because non-diving cells have limited back-up DNA repair mechanisms. An association between elevated oxidative stress and a decrease in BER is strongly related to the aging process and has special relevance in age-related neurodegenerative diseases. Here, we review the role of DNA repair in aging, focusing on the implications of the DNA base excision repair pathways and how alterations in expression of these DNA repair proteins are related to the aging process and to age-related neurodegenerative diseases. PMID:26255938

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

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  10. 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

  11. DNA damage response factors from diverse pathways, including DNA crosslink repair, mediate alternative end joining.

    PubMed

    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

  12. 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

    2014-11-01

    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. PMID:25437539

  13. A novel role for the transcriptional modulator NusA in DNA repair/damage tolerance pathways in Escherichia coli

    E-print Network

    Cohen, Susan E., Ph. D. Massachusetts Institute of Technology

    2009-01-01

    All organisms must contend with the consequences of DNA damage, induced by a variety of both endogenous and exogenous sources. Mechanisms of DNA repair and DNA damage tolerance are crucial for cellular survival after DNA ...

  14. SMCHD1 accumulates at DNA damage sites and facilitates the repair of DNA double-strand breaks

    PubMed Central

    Coker, Heather; Brockdorff, Neil

    2014-01-01

    ABSTRACT SMCHD1 is a structural maintenance of chromosomes (SMC) family protein involved in epigenetic gene silencing and chromosome organisation on the female inactive X chromosome and at a limited number of autosomal loci. Here, we demonstrate that SMCHD1 also has a role in DNA repair of double-strand breaks; SMCHD1 is recruited to sites of laser micro-irradiated damage along with other DNA repair factors, including Ku80 (also known as XRCC5 in mammals) and RAD51. Cells deficient in SMCHD1 show evidence of decreased efficiency of repair and cell viability after DNA damage. We suggest that SMCHD1 responds to DNA double-strand breaks in a manner that is likely to involve its ability to alter chromatin states to facilitate DNA repair. PMID:24790221

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

    PubMed Central

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

    2014-01-01

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

  16. The Apollo 5' exonuclease functions together with TRF2 to protect telomeres from DNA repair.

    PubMed

    Lenain, Christelle; Bauwens, Serge; Amiard, Simon; Brunori, Michele; Giraud-Panis, Marie-Josèphe; Gilson, Eric

    2006-07-11

    A major issue in telomere research is to understand how the integrity of chromosome ends is preserved . The human telomeric protein TRF2 coordinates several pathways that prevent checkpoint activation and chromosome fusions. In this work, we identified hSNM1B, here named Apollo, as a novel TRF2-interacting factor. Interestingly, the N-terminal domain of Apollo is closely related to that of Artemis, a factor involved in V(D)J recombination and DNA repair. Both proteins belong to the beta-CASP metallo-beta-lactamase family of DNA caretaker proteins. Apollo appears preferentially localized at telomeres in a TRF2-dependent manner. Reduced levels of Apollo exacerbate the sensitivity of cells to TRF2 inhibition, resulting in severe growth defects and an increased number of telomere-induced DNA-damage foci and telomere fusions. Purified Apollo protein exhibits a 5'-to-3' DNA exonuclease activity. We conclude that Apollo is a novel component of the human telomeric complex and works together with TRF2 to protect chromosome termini from being recognized and processed as DNA damage. These findings unveil a previously undescribed telomere-protection mechanism involving a DNA 5'-to-3' exonuclease. PMID:16730175

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

    PubMed Central

    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. PMID:26146487

  18. 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

  19. 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.

  20. 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 ?-galactosidase A. In addition to accumulation of ?-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. PMID:26046974

  1. Epigenetic regulation of DNA repair machinery in Helicobacter pylori-induced gastric carcinogenesis

    PubMed Central

    Santos, Juliana Carvalho; Ribeiro, Marcelo Lima

    2015-01-01

    Although thousands of DNA damaging events occur in each cell every day, efficient DNA repair pathways have evolved to counteract them. The DNA repair machinery plays a key role in maintaining genomic stability by avoiding the maintenance of mutations. The DNA repair enzymes continuously monitor the chromosomes to correct any damage that is caused by exogenous and endogenous mutagens. If DNA damage in proliferating cells is not repaired because of an inadequate expression of DNA repair genes, it might increase the risk of cancer. In addition to mutations, which can be either inherited or somatically acquired, epigenetic silencing of DNA repair genes has been associated with carcinogenesis. Gastric cancer represents the second highest cause of cancer mortality worldwide. The disease develops from the accumulation of several genetic and epigenetic changes during the lifetime. Among the risk factors, Helicobacter pylori (H. pylori) infection is considered the main driving factor to gastric cancer development. Thus, in this review, we summarize the current knowledge of the role of H. pylori infection on the epigenetic regulation of DNA repair machinery in gastric carcinogenesis. PMID:26290630

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

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

  3. 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 ? (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

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

    E-print Network

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

  5. XRCC1 deficiency increased the DNA damage induced by ?-ray in HepG2 cell: Involvement of DSB repair and cell cycle arrest.

    PubMed

    Niu, Yujie; Zhang, Xing; Zheng, Yuxin; Zhang, Rong

    2013-09-01

    ?-ray irradiation can induce DNA damages which include base damages, single-strand breaks and double-strand breaks in various type cells. The DNA repair protein XRCC1, as a part of the BER pathway, forms complexes with DNA polymerase beta, DNA ligase III and poly-ADP-ribose polymerase (PARP) in the repair of DNA single strand breaks and also affects the repair of double strand breaks. However, it is still not known well whether XRCC1 contributes to affect the irradiation sensitivity and DNA damage in HepG2 cell and the potential mechanism. Hence, the purpose of this study was to explore whether abrogation of XRCC1 gene expression by shRNA could reduce DNA repair and thus sensitize HepG2 cells to ?-ray. Cell viability was measured by Trypan blue staining and cloning efficiency assay. The DNA damage was detected by Comet assay. Apoptosis and cell cycle were detected by flow cytometry. The DNA-PKcs and gadd153 mRNA expression were determined by Real-time PCR. Our results showed that abrogation of XRCC 1 could sensitize HepG2 cells to ?-ray. This enhanced sensitivity could be attributed to the increased DNA damage and increased cell cycle arrest, which might be related with the increasing of DNA-PKcs and gadd153 mRNA expression. Therefore, our results suggested that the ?-ray irradiation sensitivity could be increased by targeting inhibition of XRCC1 in HepG2 cell. PMID:23708312

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  7. ATPase-Dependent Control of the Mms21 SUMO Ligase during DNA Repair

    PubMed Central

    Sánchez, Humberto; Bueno, Andrés; Guasch, Clàudia; Almedawar, Seba; Bru-Virgili, Sergi; Garí, Eloi; Wyman, Claire; Reverter, David; Colomina, Neus; Torres-Rosell, Jordi

    2015-01-01

    Modification of proteins by SUMO is essential for the maintenance of genome integrity. During DNA replication, the Mms21-branch of the SUMO pathway counteracts recombination intermediates at damaged replication forks, thus facilitating sister chromatid disjunction. The Mms21 SUMO ligase docks to the arm region of the Smc5 protein in the Smc5/6 complex; together, they cooperate during recombinational DNA repair. Yet how the activity of the SUMO ligase is controlled remains unknown. Here we show that the SUMO ligase and the chromosome disjunction functions of Mms21 depend on its docking to an intact and active Smc5/6 complex, indicating that the Smc5/6-Mms21 complex operates as a large SUMO ligase in vivo. In spite of the physical distance separating the E3 and the nucleotide-binding domains in Smc5/6, Mms21-dependent sumoylation requires binding of ATP to Smc5, a step that is part of the ligase mechanism that assists Ubc9 function. The communication is enabled by the presence of a conserved disruption in the coiled coil domain of Smc5, pointing to potential conformational changes for SUMO ligase activation. In accordance, scanning force microscopy of the Smc5-Mms21 heterodimer shows that the molecule is physically remodeled in an ATP-dependent manner. Our results demonstrate that the ATP-binding activity of the Smc5/6 complex is coordinated with its SUMO ligase, through the coiled coil domain of Smc5 and the physical remodeling of the molecule, to promote sumoylation and chromosome disjunction during DNA repair. PMID:25764370

  8. ATPase-dependent control of the Mms21 SUMO ligase during DNA repair.

    PubMed

    Bermúdez-López, Marcelino; Pociño-Merino, Irene; Sánchez, Humberto; Bueno, Andrés; Guasch, Clàudia; Almedawar, Seba; Bru-Virgili, Sergi; Garí, Eloi; Wyman, Claire; Reverter, David; Colomina, Neus; Torres-Rosell, Jordi

    2015-03-01

    Modification of proteins by SUMO is essential for the maintenance of genome integrity. During DNA replication, the Mms21-branch of the SUMO pathway counteracts recombination intermediates at damaged replication forks, thus facilitating sister chromatid disjunction. The Mms21 SUMO ligase docks to the arm region of the Smc5 protein in the Smc5/6 complex; together, they cooperate during recombinational DNA repair. Yet how the activity of the SUMO ligase is controlled remains unknown. Here we show that the SUMO ligase and the chromosome disjunction functions of Mms21 depend on its docking to an intact and active Smc5/6 complex, indicating that the Smc5/6-Mms21 complex operates as a large SUMO ligase in vivo. In spite of the physical distance separating the E3 and the nucleotide-binding domains in Smc5/6, Mms21-dependent sumoylation requires binding of ATP to Smc5, a step that is part of the ligase mechanism that assists Ubc9 function. The communication is enabled by the presence of a conserved disruption in the coiled coil domain of Smc5, pointing to potential conformational changes for SUMO ligase activation. In accordance, scanning force microscopy of the Smc5-Mms21 heterodimer shows that the molecule is physically remodeled in an ATP-dependent manner. Our results demonstrate that the ATP-binding activity of the Smc5/6 complex is coordinated with its SUMO ligase, through the coiled coil domain of Smc5 and the physical remodeling of the molecule, to promote sumoylation and chromosome disjunction during DNA repair. PMID:25764370

  9. 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. PMID:23000446

  10. Impact of Age-Associated Cyclopurine Lesions on DNA Repair Helicases

    PubMed Central

    Banerjee, Taraswi; Sommers, Joshua A.; Kaplan, Daniel L.; Kuper, Jochen; Kisker, Caroline; Brosh, Robert M.

    2014-01-01

    8,5? cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ helicases (RECQ1, BLM, WRN, RecQ) were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD) and SF4 (DnaB) tolerated cPu in either strand. SF2 Fe-S helicases (FANCJ, DDX11 (ChlR1), DinG, XPD) displayed marked differences in their ability to unwind the cPu DNA substrates. Archaeal Thermoplasma acidophilum XPD (taXPD), homologue to the human XPD helicase involved in NER DNA damage verification, was impeded by cPu in the non-translocating strand, while FANCJ was uniquely inhibited by the cPu in the translocating strand. Sequestration experiments demonstrated that FANCJ became trapped by the translocating strand cPu whereas RECQ1 was not, suggesting the two SF2 helicases interact with the cPu lesion by distinct mechanisms despite strand-specific inhibition for both. Using a protein trap to simulate single-turnover conditions, the rate of FANCJ or RECQ1 helicase activity was reduced 10-fold and 4.5-fold, respectively, by cPu in the translocating strand. In contrast, single-turnover rates of DNA unwinding by DDX11 and UvrD helicases were only modestly affected by the cPu lesion in the translocating strand. The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms. The apparent complexity of helicase encounters with an unusual form of oxidative damage is likely to have important consequences in the cellular response to DNA damage and DNA repair. PMID:25409515

  11. DNA repair enables sex identification in genetic material from human teeth

    PubMed Central

    Kovatsi, L; Nikou, D; Triantaphyllou, S; Njau, S N; Voutsaki, S; Kouidou, S

    2009-01-01

    Background: The purpose of this study was to test the effectiveness of a DNA repair protocol in improving genetic testing in compromised samples, frequently encountered in Forensic Medicine. Methods: In order to stretch the experiment conditions to the limits, as far as quality of samples and DNA is concerned, we tried the repair protocol on ten ancient human teeth obtained from an equal number of skeletons from a burial site in Lerna, Middle Helladic Greece (2100 - 1700 BC). For these samples, sex was previously determined morphologically, serving as a reference to compare our molecular data with. The samples were analysed using the DNA amelogenin sex test assay prior and after DNA polymerase repair. For every individual, two molecular sex determinations were obtained by visualising PCR products on an agarose gel. Results: DNA repair enabled genetic testing in these samples. Successful amplification of the amelogenin gene was obtained only from the repaired DNA in eight out of ten samples. Prior to the repair treatment, none of these samples yielded any PCR products, thus attesting to the authenticity of the amplified sequence. The concordance between morphological and molecular analysis was in reasonable agreement (71%). Conclusions: These results reveal the impact of the repair process in studying single copy genes from low quality DNA. This protocol could facilitate molecular analysis in compromised samples, encountered in forensic medicine, as well as enable genetic studies in ancient remnants. PMID:19918305

  12. Unusual DNA binding modes for metal anticancer complexes

    PubMed Central

    Pizarro, Ana M.; Sadler, Peter J.

    2010-01-01

    DNA is believed to be the primary target for many metal-based drugs. For example, platinum-based anticancer drugs can form specific lesions on DNA that induce apoptosis. New platinum drugs can be designed that have novel modes of interaction with DNA, such as the trinuclear platinum complex BBR3464. Also it is possible to design inert platinum(IV) pro-drugs which are non-toxic in the dark, but lethal when irradiated with certain wavelengths of light. This gives rise to novel DNA lesions which are not as readily repaired as those induced by cisplatin, and provides the basis for a new type of photoactivated chemotherapy. Finally, newly emerging ruthenium(II) organometallic complexes not only bind to DNA coordinatively, but also by H-bonding and hydrophibic interactions triggered by the introduction of extended arene rings into their versatile structures. Intriguingly osmium (the heavier congener of ruthenium) reacts differently with DNA but can also give rise to highly cytotoxic organometallic complexes. PMID:19344743

  13. The nuclear aryl hydocarbon receptor is involved in regulation of DNA repair and cell survival following treatment with ionizing radiation.

    PubMed

    Dittmann, K H; Rothmund, M C; Paasch, A; Mayer, C; Fehrenbacher, B; Schaller, M; Frauenstein, K; Fritsche, E; Haarmann-Stemmann, T; Braeuning, A; Rodemann, H P

    2016-01-01

    In the present study, we explored the role of the aryl hydrocarbon receptor (AhR) for ?-H2AX associated DNA repair in response to treatment with ionizing radiation. Ionizing radiation was able to stabilize AhR protein and to induce a nuclear translocation in a similar way as described for exposure to aromatic hydrocarbons. A comparable AhR protein stabilization was obtained by treatment with hydroxyl-nonenal-generated by radiation-induced lipid peroxidation. AhR knockdown resulted in significant radio-sensitization of both A549- and HaCaT cells. Under these conditions an increased amount of residual ?-H2AX foci and a delayed decline of ?-H2AX foci was observed. Knockdown of the co-activator ARNT, which is essential for transcriptional activation of AhR target genes, reduced AhR-dependent CYP1A expression in response to irradiation, but was without effect on the amount of residual ?-H2AX foci. Nuclear AhR was found in complex with ?-H2AX, DNA-PK, ATM and Lamin A. AhR and ?-H2AX form together nuclear foci, which disappear during DNA repair. Presence of nuclear AhR protein is associated with ATM activation and chromatin relaxation indicated by acetylation of histone H3. Taken together, we could show, that beyond the function as a transcription factor the nuclear AhR is involved in the regulation of DNA repair. Reduction of nuclear AhR inhibits DNA-double stand repair and radiosensitizes cells. First hints for its molecular mechanism suggest a role during ATM activation and chromatin relaxation, both essential for DNA repair. PMID:26520184

  14. Photoactivated excited states of DNA repair photolyase: Dynamical and semiempircal identification

    NASA Astrophysics Data System (ADS)

    Zheng, Xuehe; Ly, Ngan M.; Stuchebrukhov, Alexei A.

    DNA damage caused by UV light radiation is often naturally repaired in a process initiated by excited state electron transfer from the photoactivated photolyase enzyme to the DNA cyclobutane pyrimidine dimer lesion. The active cofactor in the excited state electron transfer in the photolyase is the two-electron fully reduced form of the flavin adenine dinucleotide (FADH-). To calculate electron tunneling matrix element and model the DNA binding with photolyase, the LUMO of the FADH- calculated using extended Huckel method was previously chosen from the SCF wavefunctions. Recently, the DNA-photolyase complex was crystallized in its bound form, in good agreement with our previous model in even minute details at the active site. Here we carry out molecular dynamics simulation of the entire complex using the new experimental structure of Anacystis nidulans and identify the low-lying photoactivated states of the enzyme for the dynamical confirmations. Our results from ZINDO/S CIS calculations are compared with experimental UV spectra, and their implications for excited state electron transfer and energy transfer are discussed.0

  15. Regulation of ATRIP protein abundance by RAD9 in the DNA damage repair pathway.

    PubMed

    Peng, X-J; Liu, S-J; Bao, C-M; Liu, Y-Z; Xie, H-W; Cai, Y-H; Li, B-M; Hang, H-Y; Ding, X

    2015-01-01

    Genotoxic stress activates checkpoint signaling pathways that activate the checkpoint kinases ATM and ATR, halt cell cycle progression, and promote DNA repair. A number of proteins act in concert with ATR to phosphorylate Chk1, including RAD17, the RAD9-RAD1-HUS1 complex, ATR/ATRIP and TopBp1. However, how these proteins involved act in concert with one another to propagate and maintain the checkpoint response is not well understood. Here, we reported that upregulation of RAD9 protein increased the quantity of ATRIP, suggesting that RAD9 activation will induce more efficient accumulation of ATRIP in vivo. Furthermore, the DNA damage-induced ATRIP foci formation was faster in the mRad9-/- ES cells. Also, ATRIP interacts specifically with RAD9, but not HUS1 and RAD1. Taken together, we suggested that RAD9 could affect both the ATRIP protein levels and DNA damage-induced ATRIP foci formation. Thus, we propose a role of RAD9 in the ATR-Chk1 pathway that is necessary for successful formation of the damage-sensing complex and DNA damage checkpoint signaling. PMID:26667770

  16. 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

  17. Interaction of DNA and DNA-anti-DNA complexes to fibronectin

    SciTech Connect

    Gupta, R.C.; Simpson, W.A.; Raghow, R.; Hasty, K.

    1986-03-01

    Fibronectin (Fn) is a large multidomain glycoprotein found in the basement membrane, on cell surface and in plasma. The interactions of Fn with DNA may be significant in glomerular deposition of DNA-anti-DNA complexes in patients with systemic lupus erythematosus (SLE). The authors examined the binding of DNA and DNA-anti-DNA complexes to Fn by a solid phase assay in which Fn was coated to microtiter plates and reacted with (/sup 3/H)DNA or DNA complexes with a monoclonal anti-DNA antibody. The optimal interaction of DNA with Fn occurs at <0.1M NaCl suggesting that the binding is charge dependent; the specificity of this binding was shown by competitive inhibition and locking experiments using anti-Fn. The binding was maximum at pH 6.5 and in the absence of Ca/sup 2 +/. The addition of Clq enhanced the binding of DNA and DNA-anti-DNA complexes to Fn, whereas heparan sulfate inhibited such binding. The monomeric or aggregated IgC did not bind Fn but aggregated IgG bound to Fn in the presence of Clq. Furthermore, DNA-anti-DNA complexes in sera from active SLE patients bound Fn which was enhanced in the presence of Clq; DNase abolished this binding indicating that the interaction of these complexes was mediated by DNA. These observations may partially explain the molecular mechanism(s) of the deposition of DNA-anti-DNA complexes in basement membrane.

  18. Selective recognition of uracil and its derivatives using a DNA repair enzyme structural mimic.

    PubMed

    Jiang, Yu Lin; Gao, Xiaonan; Zhou, Guannan; Patel, Arpit; Javer, Avani

    2010-01-15

    During DNA repair, uracil DNA glycosylase (UDG) pulls unwanted uracil into its active site through hydrogen bonding and pi-pi stacking interactions. The reason why UDG binds only uracil tightly--and not its derivatives, such as thymine--remains unclear. In this study, we synthesized the stable, water-soluble receptor 1a as a structural mimic of the active site in UDG. Compound 1a contains a 2,6-bis(glycylamino)pyridine group, which mimics the amino acid residues of UDG that interact with uracil through a hydrogen-bonding network; it also possesses a pyrene moiety as a pi-pi stacking interaction element and fluorescent probe that mimics the aromatic groups (phenyl and fluorescent indolyl units) found in the active site of UDG. Receptor 1a binds selectively to uracil and derivatives (including thymine, 5-formyluracil, 5-fluorouracil, and 5-nitrouracil) and some DNA and RNA nucleosides (including thymidine and uridine) through hydrogen bonding and pi-pi stacking interactions. Interestingly, a plot of log K(b) with respect to the values of pK(a) of the N(3)H units of uracil and its derivatives was linear, with a negative slope (beta) of -0.24 +/- 0.03. Thus, compounds featuring lower values of pK(a) for their N(3)H units provided greater apparent binding constants for their complexes with receptor 1a, suggesting acidity-dependent binding of uracil and its derivatives to this receptor; notably, uracil bound more tightly than did thymine. Our study provides some insight into how uracil and its derivatives in DNA are bound by DNA repair enzymes through hydrogen bonding and pi-pi stacking interactions. PMID:20017469

  19. Involvement of the Cohesin Cofactor PDS5 (SPO76) During Meiosis and DNA Repair in Arabidopsis thaliana

    PubMed Central

    Pradillo, Mónica; Knoll, Alexander; Oliver, Cecilia; Varas, Javier; Corredor, Eduardo; Puchta, Holger; Santos, Juan L.

    2015-01-01

    Maintenance and precise regulation of sister chromatid cohesion is essential for faithful chromosome segregation during mitosis and meiosis. Cohesin cofactors contribute to cohesin dynamics and interact with cohesin complexes during cell cycle. One of these, PDS5, also known as SPO76, is essential during mitosis and meiosis in several organisms and also plays a role in DNA repair. In yeast, the complex Wapl-Pds5 controls cohesion maintenance and colocalizes with cohesin complexes into chromosomes. In Arabidopsis, AtWAPL proteins are essential during meiosis, however, the role of AtPDS5 remains to be ascertained. Here we have isolated mutants for each of the five AtPDS5 genes (A–E) and obtained, after different crosses between them, double, triple, and even quadruple mutants (Atpds5a Atpds5b Atpds5c Atpds5e). Depletion of AtPDS5 proteins has a weak impact on meiosis, but leads to severe effects on development, fertility, somatic homologous recombination (HR) and DNA repair. Furthermore, this cohesin cofactor could be important for the function of the AtSMC5/AtSMC6 complex. Contrarily to its function in other species, our results suggest that AtPDS5 is dispensable during the meiotic division of Arabidopsis, although it plays an important role in DNA repair by HR. PMID:26648949

  20. Removal of oxygen free-radical-induced 5',8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells.

    PubMed

    Kuraoka, I; Bender, C; Romieu, A; Cadet, J; Wood, R D; Lindahl, T

    2000-04-11

    Exposure of cellular DNA to reactive oxygen species generates several classes of base lesions, many of which are removed by the base excision-repair pathway. However, the lesions include purine cyclodeoxynucleoside formation by intramolecular crosslinking between the C-8 position of adenine or guanine and the 5' position of 2-deoxyribose. This distorting form of DNA damage, in which the purine is attached by two covalent bonds to the sugar-phosphate backbone, occurs as distinct diastereoisomers. It was observed here that both diastereoisomers block primer extension by mammalian and microbial replicative DNA polymerases, using DNA with a site-specific purine cyclodeoxynucleoside residue as template, and consequently appear to be cytotoxic lesions. Plasmid DNA containing either the 5'R or 5'S form of 5',8-cyclo-2-deoxyadenosine was a substrate for the human nucleotide excision-repair enzyme complex. The R diastereoisomer was more efficiently repaired than the S isomer. No correction of the lesion by direct damage reversal or base excision repair was detected. Dual incision around the lesion depended on the core nucleotide excision-repair protein XPA. In contrast to several other types of oxidative DNA damage, purine cyclodeoxynucleosides are chemically stable and would be expected to accumulate at a slow rate over many years in the DNA of nonregenerating cells from xeroderma pigmentosum patients. High levels of this form of DNA damage might explain the progressive neurodegeneration seen in XPA individuals. PMID:10759556

  1. Statistical mechanics of topologically constrained DNA and nucleoprotein complexes

    NASA Astrophysics Data System (ADS)

    Giovan, Stefan Michael

    A complex connection exists between the 3 dimensional topological state of DNA in living organisms and biological processes including gene expression, DNA replication, recombination and repair. A significant limitation in developing a detailed, quantitative understanding of this connection is due to a lack of rigorous methods to calculate statistical mechanical properties of DNA molecules with complex topologies, including supercoiling, looping and knotting. This dissertation's main focus is on developing such methods and applying them to realistic DNA and nucleoprotein models. In chapter 2, a method is presented to calculate free energies and J factors of protein mediated DNA loops by normal mode analysis (NMA). This method is similar to calculations performed previously but with several significant advances. We apply the method to the specific case of DNA looping mediated by Cre recombinase protein. J factors calculated by our method are compared to experimental measurements to extract geometric and elastic properties of the Cre-DNA synaptic complex. In particular, the results suggest the existence of a synaptic complex that is more flexible than previously expected and may be explained by a stable intermediate in the reaction pathway that deviates significantly from the planar crystal structure. Calculating free energies of DNA looping is difficult in general, especially when considering intermediate length scales such as plasmid sized DNA which may readily adopt multiple topological states. In chapter 3, a novel method is presented to obtain free energies of semiflexible biopolymers with fixed topologies and arbitrary ratios of contour length L to persistence length P. High accuracy is demonstrated by calculating free energies of specific DNA knots with L/P = 20 and L/P = 40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex containing a pair of looped domains, revealing a bifurcation in the location of optimal synapse (crossover) sites. This transition is relevant to target-site selection by DNA-binding proteins that occupy multiple DNA sites separated by large linear distances along the genome, a problem that arises naturally in gene regulation, DNA recombination, and the action of type-II topoisomerases.

  2. Molecular targets, DNA breakage, DNA repair: Their roles in mutation induction in mammalian germ cells

    SciTech Connect

    Sega, G.A.

    1989-01-01

    Variability in genetic sensitivity among different germ-cell stages in the mammal to various mutagens could be the result of how much chemical reaches the different stages, what molecular targets may be affected in the different stages and whether or not repair of lesions occurs. Several chemicals have been found to bind very strongly to protamine in late-spermatid and early-spermatozoa stages in the mouse. The chemicals also produce their greatest genetic damage in these same germ-cell stages. While chemical binding to DNA has not been correlated with the level of induced genetic damage, DNA breakage in the sensitive stages has been shown to increase. This DNA breakage is believed to indirectly result from chemical binding to sulfhydryl groups in protamine which prevents normal chromatin condensation within the sperm nucleus. 22 refs., 5 figs.

  3. Cyclin D1-mediated inhibition of repair and replicative DNA synthesis in human fibroblasts.

    PubMed

    Pagano, M; Theodoras, A M; Tam, S W; Draetta, G F

    1994-07-15

    Cyclin D1 is a key regulator of the G1 phase of the cell cycle. Inhibition of cyclin D1 function results in cell cycle arrest, whereas unregulated expression of the protein accelerates G1. Cyclin D1 is localized to the nucleus during G1. We found that during repair DNA synthesis, subsequent to UV-induced DNA damage, G1 cells readily lost their cyclin D1 while the proliferating cell nuclear antigen (PCNA) tightly associated with nuclear structures. Microinjection of cyclin D1 antisense accelerated DNA repair, whereas overexpression of cyclin D1 prevented DNA repair and the relocation of PCNA after DNA damage. Coexpression of cyclin D1 with its primary catalytic subunit, Cdk4, or with Cdk2, also prevented repair. In contrast, coexpression of PCNA, which is also a cyclin D1-associated protein, restored the ability of cells to repair their DNA. Acute overexpression of cyclin D1 in fibroblasts prevented them from entering S phase. Again, these effects were abolished by coexpression of cyclin D1 together with PCNA, but not with Cdk4 or Cdk2. Altogether, these results indicate that down-regulation of cyclin D1 is necessary for PCNA relocation and repair DNA synthesis as well as for the start of DNA replication. Cyclin D1 appears to be an essential component of a G1-checkpoint. PMID:7958844

  4. DNA Damage Signalling and Repair Inhibitors: The Long-Sought-After Achilles' Heel of Cancer.

    PubMed

    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

  5. 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

  6. DNA damage action spectroscopy and DNA repair in intact organisms: Alfalfa seedlings

    SciTech Connect

    Sutherland, B.M.; Quaite, F.E.; Sutherland, J.C.

    1993-12-31

    Understanding the effects of UV, and increased levels of UV, on DNA in living organisms requires knowledge of both the frequency of damages induced by the quantities and quality (wavelength composition) of the damaging radiation, and of the capacity of the organisms to carry out efficient and accurate repair. The major levels of uncertainty in understanding the responses of intact organisms, both plant and animal, to UV indicates that we cannot assess accurately the impact of stratospheric ozone depletion without major increases in knowledge of DNA damage and repair. What repair paths does alfalfa use for dealing with UV damages? The rate of pyrimidine dimers induced at a low exposure of 280 nm radiation to alfalfa seedlings, was observed to be about 8 dimers/million bases. After UV exposure, the seedlings were kept in the dark or exposed to blue light filtered by a yellow. filter which excluded wavelengths shorter than about 405 nm. Seedlings so exposed carry out photorepair, but do not seem to remove dimers by excision.

  7. Repair of DNA damaged by ionizing radiation and other oxidative agents in yeast and human

    SciTech Connect

    Louise Prakash

    2000-01-15

    Treatment of cells with oxidative DNA damaging agents such as ionizing radiation and hydrogen peroxide produces .OH radicals which attack DNA, producing single strand breaks and double strand breaks that have a 3'-blocked terminus with a phosphoglycolate or a phosphate group attached to the 3'-terminus. While DNA strand breaks with 3'-blocked termini are the hallmark of oxidative DNA damage, the mechanisms by which such blocked 3'-termini are removed in eukaryotes remain poorly understood. The goals of this project were to identify the various genes that function in cleaning the blocked 3'-ends from DNA strand breaks generated by treatments with ionizing radiation and hydrogen peroxide, to purify the proteins encoded by these genes and to characterize their biochemical activities, and to determine the biological consequences when such damage is not repaired. Because of the high degree of conservation of DNA repair proteins between yeast and humans, and because of the ease of genetic manipulations, initial studies were to be carried out in Saccharomyces cerevisiae. The homologous genes and proteins would then be studied in humans. One aspect of our proposed research was to purify the Apn2 protein from yeast cells and to examine its AP endonuclease and 3'-phosphodiesterase activities. Apn2-like proteins have been identified in eukaryotes other than yeast, including humans, and these proteins form a distinct subfamily within the ExoIII/Ape1/Apn2 family of proteins. We purified the Apn2 protein from yeast and showed that it is a class II AP endonuclease. (Class II AP endonucleases cleave the phosphodiester backbone on the 5'-side of the AP site and produce a 3'-OH group and a 5'-baseless deoxyribose 5'-phosphate residue). Yeast Apn2 and its orthologs in higher eukaryotes differ from E. coli ExoIII and human Ape1 in possessing a C terminus that is absent from the ExoIII/Ape1 subfamily. We found that deletion of the carboxyl-terminus of yeast Apn2 protein does not affect the AP endonuclease activity of the protein, but this protein is defective in the removal of AP sites in vivo. The carboxyl-terminus may enable Apn2 to complex with other proteins, and such a multiprotein assembly may be necessary for the efficient recognition and cleavage of AP sites in vivo. We also carried out further biochemical characterization of the yeast Apn2 protein. As mentioned above, oxidative DNA damaging agents, such as hydrogen peroxide, produce DNA strand breaks which contain 3'-phosphate or 3'-phosphoglycolate termini. Such 3' termini are inhibitory to synthesis by DNA polymerases. We found that purified yeast Apn2 protein contains 3'-phosphodiesterase and 3'5' exonuclease activities, and mutation of the active site residue Glu59 to Ala in Apn2 inactivates both these activities. Consistent with these biochemical observations, our genetic studies indicate the involvement of APN2 in the repair of hydrogen peroxide induced DNA damage in a pathway alternate to APN1, and the Ala59 mutation inactivates this function of Apn2. From these results, we have concluded that the ability of Apn2 to remove 3'-end groups from DNA is paramount for the repair of strand breaks arising from the reaction of DNA with reactive oxygen species. Other studies from our laboratory indicate that the yeast APN1 and APN2 genes provide alternate pathways for the repair of abasic sites and for the repair of single strand breaks with 3'-blocked termini. The apn1 deletion apn2 deletion mutant is highly sensitive to both the alkylating agent methyl methanesulfonate and to the oxidizing agent hydrogen peroxide. While the apn1 deletion and apn2 deletion single mutants are proficient in repairing single strand breaks arising in DNA following treatment with hydrogen peroxide, the repair of abasic sites as well as of single strand DNA breaks with 3'-blocked termini is greatly reduced in the apn1 deletion.

  8. Repair of DNA damaged by ionizing radiation and other oxidative agents in yeast and human

    SciTech Connect

    Louisek Prakash

    2000-01-15

    OAK B202 Treatment of cells with oxidative DNA damaging agents such as ionizing radiation and hydrogen peroxide produces .OH radicals which attack DNA, producing single strand breaks and double strand breaks that have a 3'-blocked terminus with a phosphoglycolate or a phosphate group attached to the 3'-terminus. While DNA strand breaks with 3'-blocked termini are the hallmark of oxidative DNA damage, the mechanisms by which such blocked 3'-termini are removed in eukaryotes remain poorly understood. The goals of this project were to identify the various genes that function in cleaning the blocked 3'ends from DNA strand breaks generated by treatments with ionizing radiation and hydrogen peroxide, to purify the proteins encoded by these genes and to characterize their biochemical activities, and to determine the biological consequences when such damage is not repaired. Because of the high degree of conservation of DNA repair proteins between yeast and humans, and because of the ease of genetic manipulations, initial studies were to be carried out in Saccharomyces cerevisiae. The homologous genes and proteins would then be studied in humans. One aspect of our proposed research was to purify the Apn2 protein from yeast cells and to examine its AP endonuclease and 3'-phosphodiesterase activities. Apn2-like proteins have been identified in eukaryotes other than yeast, including humans, and these proteins form a distinct subfamily within the ExoIII/Ape1/Apn2 family of proteins. We purified the Apn2 protein from yeast and showed that it is a class II AP endonuclease. (Class II AP endonucleases cleave the phosphodiester backbone on the 5'-side of the AP site and produce a 3'-OH group and a 5'-baseless deoxyribose 5'-phosphate residue). Yeast Apn2 and its orthologs in higher eukaryotes differ from E. coli ExoIII and human Ape1 in possessing a C terminus that is absent from the ExoIII/Ape1 subfamily. We found that deletion of the carboxyl-terminus of yeast Apn2 protein does not affect the AP endonuclease activity of the protein, but this protein is defective in the removal of AP sites in vivo. The carboxyl-terminus may enable Apn2 to complex with other proteins, and such a multiprotein assembly may be necessary for the efficient recognition and cleavage of AP sites in vivo. We also carried out further biochemical characterization of the yeast Apn2 protein. As mentioned above, oxidative DNA damaging agents, such as hydrogen peroxide, produce DNA strand breaks which contain 3'-phosphate or 3'-phosphoglycolate termini. Such 3' termini are inhibitory to synthesis by DNA polymerases. We found that purified yeast Apn2 protein contains 3'-phosphodiesterase and 3'5' exonuclease activities, and mutation of the active site residue Glu59 to Ala in Apn2 inactivates both these activities. Consistent with these biochemical observations, our genetic studies indicate the involvement of APN2 in the repair of hydrogen peroxide induced DNA damage in a pathway alternate to APN1, and the Ala59 mutation inactivates this function of Apn2. From these results, we have concluded that the ability of Apn2 to remove 3'-end groups from DNA is paramount for the repair of strand breaks arising from the reaction of DNA with reactive oxygen species. Other studies from our laboratory indicate that the yeast APN1 and APN2 genes provide alternate pathways for the repair of abasic sites and for the repair of single strand breaks with 3'-blocked termini. The apn1 deletion apn2 deletion mutant is highly sensitive to both the alkylating agent methyl methanesulfonate and to the oxidizing agent hydrogen peroxide. While the apn1 deletion and apn2 deletion single mutants are proficient in repairing single strand breaks arising in DNA following treatment with hydrogen peroxide, the repair of abasic sites as well as of single strand DNA breaks with 3'-blocked termini is greatly reduced in the apn1 deletion.

  9. He Fei DNA Repair Mini-Symposium (April 19-20) Preliminary arrangement

    E-print Network

    Yu, Peter K.N.

    He Fei DNA Repair Mini-Symposium (April 19-20) Preliminary arrangement 1. Organizer Organized byCity of Hope Medical Center Coordination of enzyme switches in DNA replication fork via programmed posttranslational modifications 9:00-9:30 Junjie Chenthe University of Texas MD Anderson Cancer Center) #12;DNA

  10. 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...

  11. Genome analysis of DNA repair genes in the alpha proteobacterium Caulobacter crescentus

    PubMed Central

    Martins-Pinheiro, Marinalva; Marques, Regina CP; Menck, Carlos FM

    2007-01-01

    Background The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria. Results In this study, we employed a bioinformatic approach, to analyse and describe the open reading frames potentially related to DNA repair from the genome of the alpha-proteobacterium Caulobacter crescentus. This was performed by comparison with known DNA repair related genes found in public databases. As expected, although C. crescentus and E. coli bacteria belong to separate phylogenetic groups, many of their DNA repair genes are very similar. However, some important DNA repair genes are absent in the C. crescentus genome and other interesting functionally related gene duplications are present, which do not occur in E. coli. These include DNA ligases, exonuclease III (xthA), endonuclease III (nth), O6-methylguanine-DNA methyltransferase (ada gene), photolyase-like genes, and uracil-DNA-glycosylases. On the other hand, the genes imuA and imuB, which are involved in DNA damage induced mutagenesis, have recently been described in C. crescentus, but are absent in E. coli. Particularly interesting are the potential atypical phylogeny of one of the photolyase genes in alpha-proteobacteria, indicating an origin by horizontal transfer, and the duplication of the Ada orthologs, which have diverse structural configurations, including one that is still unique for C. crescentus. Conclusion The absence and the presence of certain genes are discussed and predictions are made considering the particular aspects of the C. crescentus among other known DNA repair pathways. The observed differences enlarge what is known for DNA repair in the Bacterial world, and provide a useful framework for further experimental studies in this organism. PMID:17352799

  12. 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

  13. Triple negative breast cancers have a reduced expression of DNA repair genes.

    PubMed

    Ribeiro, Enilze; Ganzinelli, Monica; 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

  14. The RECG1 DNA Translocase Is a Key Factor in Recombination Surveillance, Repair, and Segregation of the Mitochondrial DNA in Arabidopsis[OPEN

    PubMed Central

    Le Ret, Monique; Bergdoll, Marc; Bichara, Marc; Dietrich, André

    2015-01-01

    The mitochondria of flowering plants have considerably larger and more complex genomes than the mitochondria of animals or fungi, mostly due to recombination activities that modulate their genomic structures. These activities most probably participate in the repair of mitochondrial DNA (mtDNA) lesions by recombination-dependent processes. Rare ectopic recombination across short repeats generates new genomic configurations that contribute to mtDNA heteroplasmy, which drives rapid evolution of the sequence organization of plant mtDNAs. We found that Arabidopsis thaliana RECG1, an ortholog of the bacterial RecG translocase, is an organellar protein with multiple roles in mtDNA maintenance. RECG1 targets to mitochondria and plastids and can complement a bacterial recG mutant that shows defects in repair and replication control. Characterization of Arabidopsis recG1 mutants showed that RECG1 is required for recombination-dependent repair and for suppression of ectopic recombination in mitochondria, most likely because of its role in recovery of stalled replication forks. The analysis of alternative mitotypes present in a recG1 line and of their segregation following backcross allowed us to build a model to explain how a new stable mtDNA configuration, compatible with normal plant development, can be generated by stoichiometric shift. PMID:26462909

  15. 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.

  16. Evolutionary patterns of DNA base composition and correlation to polymorphisms in DNA repair systems.

    PubMed

    Li, Xianran; Scanlon, Michael J; Yu, Jianming

    2015-04-20

    DNA base composition is a fundamental genome feature. However, the evolutionary pattern of base composition and its potential causes have not been well understood. Here, we report findings from comparative analysis of base composition at the whole-genome level across 2210 species, the polymorphic-site level across eight population comparison sets, and the mutation-site level in 12 mutation-tracking experiments. We first demonstrate that base composition follows the individual-strand base equality rule at the genome, chromosome and polymorphic-site levels. More intriguingly, clear separation of base-composition values calculated across polymorphic sites was consistently observed between basal and derived groups, suggesting common underlying mechanisms. Individuals in the derived groups show an A&T-increase/G&C-decrease pattern compared with the basal groups. Spontaneous and induced mutation experiments indicated these patterns of base composition change can emerge across mutation sites. With base-composition across polymorphic sites as a genome phenotype, genome scans with human 1000 Genomes and HapMap3 data identified a set of significant genomic regions enriched with Gene Ontology terms for DNA repair. For three DNA repair genes (BRIP1, PMS2P3 and TTDN), ENCODE data provided evidence for interaction between genomic regions containing these genes and regions containing the significant SNPs. Our findings provide insights into the mechanisms of genome evolution. PMID:25765652

  17. Repair of UV damaged DNA, genes and proteins of yeast and human

    SciTech Connect

    Prakash, L.

    1991-04-01

    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, as well as studying the human homologs of yeast excision repair and postreplication repair proteins. In addition to its single-stranded DNA-dependent A TPase and DNA helicase activities, we have found that RAD3 protein also possesses DNA-RNA helicase activity, and that like RAD3, the Schizosaccharomyces pombe RAD3 homolog, rhp3{sup +}, is an essential gene. We have overexpressed the human RAD3 homolog, ERCC2, in yeast to facilitate its purification. The RAD10 protein was purified to homogeneity and shown to bind DNA. ERCC3y, the yeast homolog of the human ERCC-3/XP-B gene, has been sequenced and shown to be essential for viability. The Drosophila and human homologs of RAD6, required for postreplication repair and UV induced mutagenesis, were shown to complement the rad6 {Delta} mutation of yeast. Since defective DNA repair and enhanced neoplasia characterize several human genetic diseases, and repair proteins are highly conserved between yeast and man, a thorough understanding of the molecular mechanisms of DNA repir in yeast should provide a better understanding of the causes of carcinogenesis.

  18. The role of DNA damage and repair in atherosclerosis: A review.

    PubMed

    Shah, Nikunj R; Mahmoudi, Michael

    2015-09-01

    The global burden of cardiovascular disease is increasing despite therapeutic advances in medication and interventional technologies. Accumulated deoxyribonucleic acid (DNA) damage and subsequent repair pathways are now increasingly recognised as a causal factor in the initiation and progression of atherosclerosis. These molecular alterations have been shown to occur within affected vasculature, plaque microenvironment as well as in circulating cells. The DNA damage response (DDR) pathway is reliant on post-translational modification of sensing proteins which activate a signalling cascade to repair, if possible, DNA damaged sites in response to various environmental and physiological insults. This review summarises the current evidence for DNA damage in atherosclerosis, the key steps involved in the DDR pathway, DNA repair and their subsequent effects on atherosclerotic plaques, as well as the therapeutic options in managing DNA damage-induced atherosclerosis. PMID:26211712

  19. Chromatin dynamics during DNA repair revealed by pair correlation analysis of molecular flow in the nucleus.

    PubMed

    Hinde, Elizabeth; Kong, Xiangduo; Yokomori, Kyoko; Gratton, Enrico

    2014-07-01

    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

  20. DNA repair and induction of plasminogen activator in human fetal cells treated with ultraviolet light

    SciTech Connect

    Ben-Ishai, R.; Sharon, R.; Rothman, M.; Miskin, R.

    1984-03-01

    We have tested human fetal fibroblasts for development associated changes in DNA repair by utilizing nucleoid sedimentation as an assay for excision repair. Among skin fibroblasts the rate of excision repair was significantly higher in non-fetal cells than in fibroblasts derived from an 8 week fetus; this was evident by a delay in both the relaxation and the restoration of DNA supercoiling in nucleoids after irradiation. Skin fibroblasts derived at 12 week gestation were more repair proficient than those derived at 8 week gestation. However, they exhibited a somewhat lower rate of repair than non-fetal cells. The same fetal and non-fetal cells were also tested for induction of the protease plasminogen activator (PA) after u.v. irradiation. Enhancement of PA was higher in skin fibroblasts derived at 8 week than in those derived at 12 week gestation and was absent in non-fetal skin fibroblasts. These results are consistent with our previous findings that in human cells u.v. light-induced PA synthesis is correlated with reduced DNA repair capacity. Excision repair and PA inducibility were found to depend on tissue of origin in addition to gestational stage, as shown for skin and lung fibroblasts from the same 12 week fetus. Lung compared to skin fibroblasts exhibited lower repair rates and produced higher levels of PA after irradiation. The sedimentation velocity of nucleoids, prepared from unirradiated fibroblasts, in neutral sucrose gradients with or without ethidium bromide, indicated the presence of DNA strand breaks in fetal cells. It is proposed that reduced DNA repair in fetal cells may result from alterations in DNA supercoiling, and that persistent DNA strand breaks enhance transcription of PA gene(s).

  1. Monitoring DNA double strand breaks repair protiens before and after gamma-irradiation

    NASA Astrophysics Data System (ADS)

    Abdisalaam, Salim

    2011-04-01

    In this study, different fluorescence techniques (FCS, RICS and FRAP) are used to study kinetics and dynamics of DNA double stand breaks repair proteins before and after gamma-irradiation of mammalian cells. Diffusion and binding constants were obtained by fitting with different physical models. This work presents similarities and differences in double strand break repair response between gamma-irradiation versus laser damage.

  2. Analysis of NusA's roles in DNA repair and damage tolerance pathways in Escherichia coli

    E-print Network

    Zhao, Yimin, S.M. Massachusetts Institute of Technology

    2015-01-01

    NusA has long known to be a bona fide transcription factor that functions in both termination and antitermination. However, its role in DNA repair and damage tolerance has not been explored until recently. Cohen et al. ...

  3. The BER necessities: the repair of DNA damage in human-adapted bacterial pathogens.

    PubMed

    van der Veen, Stijn; Tang, Christoph M

    2015-02-01

    During colonization and disease, bacterial pathogens must survive the onslaught of the host immune system. A key component of the innate immune response is the generation of reactive oxygen and nitrogen species by phagocytic cells, which target and disrupt pathogen molecules, particularly DNA, and the base excision repair (BER) pathway is the most important mechanism for the repair of such oxidative DNA damage. In this Review, we discuss how the human-specific pathogens Mycobacterium tuberculosis, Helicobacter pylori and Neisseria meningitidis have evolved specialized mechanisms of DNA repair, particularly their BER pathways, compared with model organisms such as Escherichia coli. This specialization in DNA repair is likely to reflect the distinct niches occupied by these important human pathogens in the host. PMID:25578955

  4. 55K isoform of CDK9 associates with Ku70 and is involved in DNA repair

    SciTech Connect

    Liu, Hongbing; Herrmann, Christine H.; Chiang, Karen; Sung, Tzu-Ling; Moon, Sung-Hwan; Donehower, Lawrence A.; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 ; Rice, Andrew P.

    2010-06-25

    Positive elongation factor b (P-TEFb) is a cellular protein kinase that is required for RNA polymerase II (RNAP II) transcriptional elongation of protein coding genes. P-TEFb is a set of different molecular complexes, each containing CDK9 as the catalytic subunit. There are two isoforms of the CDK9 protein - the major 42 KDa CDK9 isoform and the minor 55KDa isoform that is translated from an in-frame mRNA that arises from an upstream transcriptional start site. We found that shRNA depletion of the 55K CDK9 protein in HeLa cells induces apoptosis and double-strand DNA breaks (DSBs). The levels of apoptosis and DSBs induced by the depletion were reduced by expression of a 55K CDK9 protein variant resistant to the shRNA, indicating that these phenotypes are the consequence of depletion of the 55K protein and not off-target effects. We also found that the 55K CDK9 protein, but not the 42K CDK9 protein, specifically associates with Ku70, a protein involved in DSB repair. Our findings suggest that the 55K CDK9 protein may function in repair of DNA through an association with Ku70.

  5. Nuclear and Mitochondrial DNA Repair in Selected Eukaryotic Aging Model Systems

    PubMed Central

    Gredilla, Ricardo; Garm, Christian; Stevnsner, Tinna

    2012-01-01

    Knowledge about the different mechanisms underlying the aging process has increased exponentially in the last decades. The fact that the basic mechanisms involved in the aging process are believed to be universal allows the use of different model systems, from the simplest eukaryotic cells such as fungi to the most complex organisms such as mice or human. As our knowledge on the aging mechanisms in those model systems increases, our understanding of human aging and the potential interventions that we could approach rise significantly. Among the different mechanisms that have been implicated in the aging process, DNA repair is one of the processes which have been suggested to play an important role. Here, we review the latest investigations supporting the role of these mechanisms in the aging process, stressing how beneficial the use of different model systems is. We discuss how human genetic studies as well as several investigations on mammalian models and simpler eukaryotic organisms have contributed to a better understanding of the involvement of DNA repair mechanisms in aging. PMID:23050036

  6. DNA repair and resistance to UV-B radiation in western spotted frogs

    USGS Publications Warehouse

    Blaustein, A.R.; Hays, J.B.; Hoffman, P.D.; Chivers, D.P.; Kiesecker, J.M.; Leonard, W.P.; Marco, A.; Olson, D.H.; Reaser, J.K.; Anthony, R.G.

    1999-01-01

    We assessed DNA repair and resistance to solar radiation in eggs of members of the western spotted frog complex (Rana pretiosa and R. luteiventris), species whose populations are suffering severe range reductions and declines. Specifically, we measured the activity of photoreactivating enzyme (photolyase) in oocytes of spotted frogs. In some species, photoreactivation is the most important mechanism for repair of UV-damaged DNA. Using field experiments, we also compared the hatching success of spotted frog embryos at natural oviposition sites at three elevations, where some embryos were subjected to ambient levels of UV-B radiation and others were shielded from UV-B radiation. Compared with other amphibians, photolyase activities in spotted frogs were relatively high. At all sites, hatching success was unaffected by UV-B. Our data support the interpretation that amphibian embryos with relatively high levels of photolyase are more resistant to UV-B radiation than those with lower levels of photolyase. At the embryonic stage, UV-B radiation does not presently seem to be contributing to the population declines of spotted frogs.

  7. The DNA Single-Strand Break Repair Machinery Facilitates CAF-1-Mediated Histone Deposition at Oxidative DNA Strand Breaks

    E-print Network

    Arman Nabatiyan; Zhihong Zeng; Keith W. Caldecott

    2012-02-01

    Oxidative DNA single strand breaks arise continuously in cells and defects in their repair have been implicated in neurological disease. While much progress has been made in understanding how chromosomal single strand breaks are repaired little is known about the changes chromatin structure that accompany this process. Here, we show that nascent recombinant histone H3.1 protein accumulates and is deposited into chromatin at sites of DNA strand breakage in quiescent human cells following oxidative stress, and that core components of the single-strand break repair machinery are required for this process. We show that the SSBR sensor and scaffold proteins poly (ADP-ribose) polymerase and XRCC1 facilitate accumulation of chromatin assembly factor-1 (CAF-1) at sites of oxidative DNA strand breakage, which in turn mediates the deposition of Histone H3.1. We also demonstrate that depletion of CAF-1 slows global rates of DNA strand break repair in quiescent cells following oxidative stress, demonstrating that single-strand break repair and histone deposition are tightly coordinated processes. These data describe a novel role for the DNA singlestrand break repair machinery and implicate histone turnover as a core component of the cellular response of quiescent cells to oxidative damage.

  8. The repair function of the multifunctional DNA repair/redox protein APE1 is neuroprotective after ionizing radiation

    PubMed Central

    Vasko, Michael R.; Guo, Chunlu; Thompson, Eric L.; Kelley, Mark R.

    2011-01-01

    Although exposure to ionizing radiation (IR) can produce significant neurotoxicity, the mechanisms mediating this toxicity remain to be determined. Previous studies using neurons isolated from the central nervous system show that IR produces reactive oxygen species and oxidative DNA damage in those cells. Because the base excision DNA repair pathway repairs single-base modifications caused by ROS, we asked whether manipulating this pathway by altering APE1 expression would affect radiation-induced neurotoxicity. In cultures of adult hippocampal and sensory neurons, IR produces DNA damage as measured by phosphorylation of histone H2A.X and results in dose-dependent cell death. In isolated sensory neurons, we demonstrate for the first time that radiation decreases the capsaicin-evoked release of the neuropeptide CGRP. Reducing APE1 expression in cultured cells augments IR-induced neurotoxicity, whereas overexpressing APE1 is neuroprotective. Using lentiviral constructs with a neuronal specific promoter that selectively expresses APE1’s different functions in neurons, we show that selective expression of the DNA repair competent (redox inactive) APE1 constructs in sensory neurons resurrects cell survival and neuronal function, whereas use of DNA-repair deficient (redox active) constructs is not protective. Use of an APE1 redox-specific inhibitor, APX3330, also facilitates neuronal protection against IR-induced toxicity. These results demonstrate for the first time that the repair function of APE1 is required to protect both hippocampal and DRG neuronal cultures—specifically neuronal cells—from IR-induced damage, while the redox activity of APE1 does not appear to be involved. PMID:21741887

  9. Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks.

    PubMed

    Langerak, Petra; Mejia-Ramirez, Eva; Limbo, Oliver; Russell, Paul

    2011-09-01

    The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5'-to-3' resection of DNA ends, generating 3' single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair. PMID:21931565

  10. [DNA-PKcs silencing inhibit the DNA repair induced by low dose radiation on human breast epithelial cells].

    PubMed

    Zou, Wei; Che, Jian; Wang, Chongjie; Cui, Yuying; Zhang, Qinming

    2009-05-01

    DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), plays an important role in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand breaks (DSBs) repair. To investigate the effects of DNA-PKcs down-regulation on cell growth and sensitization to low dose radiation (LDR), we transfected DNA-PKcs siRNA into human mammary epithelia cells MCF10F, then, detected the proliferation curve of the cells and the expression of protiens in DNA repair pathways. The results showed that DNA-PKcs gene silencing, induced by the transfection of DNA-PKcs siRNA could suppress significantly the cell proliferation and inhibit the expression of the DNA repair proteins, such as Ku80, ATM and P53 after 50 cGy 137Cs gamma-irradiation.The results suggested that DNA-PKcs gene silencing could increase the sensitivity of human breast epithelial cells to the LDR, which might be relative with the decrease of the proteins. PMID:19670642

  11. The Structure of DNA within Cationic Lipid/DNA Complexes

    E-print Network

    Braun, Chad S.; Jas, Gouri S.; Choosakoonriang, Sirirat; Koe, Gary S.; Smith, Janet G.; Middaugh, C. Russell

    2003-02-01

    using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue. CD spectra of supercoiled plasmid DNA undergo a significant loss of rotational strength in the signal near 275 nm upon interaction...

  12. Defective DNA Mismatch Repair Influences Expression of Endometrial Carcinoma Biomarkers.

    PubMed

    Okoye, Ekene I; Bruegl, Amanda S; Fellman, Bryan; Luthra, Rajyalakshmi; Broaddus, Russell R

    2016-01-01

    Endometrial endometrioid carcinomas are related to estrogen excess and express estrogen and progesterone receptors. However, hormone receptor expression can be variable from tumor to tumor, and this variability is not always explained by differences in tumor grade. Variable expression of other biomarkers that may be used in the diagnostic work-up of endometrial cancer has also been noted. We hypothesized that mismatch repair (MMR) defects may contribute to this variability. A total of 411 unselected endometrial carcinomas were evaluated for immunohistochemical expression of DNA MMR proteins and MLH1 methylation. Loss of immunohistochemical expression of MLH1, MSH2, MSH6, or PMS2 was defined as MMR deficient; positive expression was defined as MMR intact. A case-control cohort of 80 Grade 2 endometrioid carcinomas was selected from this set (40 MMR deficient, 40 MMR intact). Cases were matched for histotype, grade, and age. Estrogen receptor, progesterone receptor, CK7, CK20, and Pax-8 immunohistochemistry was evaluated. The median percentage of CK7 tumor cells was significantly lower in the MMR deficient group compared with the MMR intact group. The mean percentage of tumor cells exhibiting estrogen receptor expression was similar in both the MMR-deficient and MMR intact groups. However, there was greater variability in the MMR-deficient group. Our study shows that MMR defects influence the expression of clinically important biomarkers for endometrioid-type endometrial carcinoma as decreased cytokeratin 7 expression is more commonly associated with MMR deficiency. PMID:25851713

  13. 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.

  14. Preventing Damage Limitation: Targeting DNA-PKcs and DNA Double-Strand Break Repair Pathways for Ovarian Cancer Therapy

    PubMed Central

    Dungl, Daniela A.; Maginn, Elaina N.; Stronach, Euan A.

    2015-01-01

    Platinum-based chemotherapy is the cornerstone of ovarian cancer treatment, and its efficacy is dependent on the generation of DNA damage, with subsequent induction of apoptosis. Inappropriate or aberrant activation of the DNA damage response network is associated with resistance to platinum, and defects in DNA repair pathways play critical roles in determining patient response to chemotherapy. In ovarian cancer, tumor cell defects in homologous recombination?–?a repair pathway activated in response to double-strand DNA breaks (DSB)?–?are most commonly associated with platinum-sensitive disease. However, despite initial sensitivity, the emergence of resistance is frequent. Here, we review strategies for directly interfering with DNA repair pathways, with particular focus on direct inhibition of non-homologous end joining (NHEJ), another DSB repair pathway. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a core component of NHEJ and it has shown considerable promise as a chemosensitization target in numerous cancer types, including ovarian cancer where it functions to promote platinum-induced survival signaling, via AKT activation. The development of pharmacological inhibitors of DNA-PKcs is on-going, and clinic-ready agents offer real hope to patients with chemoresistant disease. PMID:26579492

  15. Candidate DNA repair susceptibility genes identified by exome sequencing in high-risk pancreatic cancer.

    PubMed

    Smith, Alyssa L; Alirezaie, Najmeh; Connor, Ashton; Chan-Seng-Yue, Michelle; Grant, Robert; Selander, Iris; Bascuñana, Claire; Borgida, Ayelet; Hall, Anita; Whelan, Thomas; Holter, Spring; McPherson, Treasa; Cleary, Sean; Petersen, Gloria M; Omeroglu, Atilla; Saloustros, Emmanouil; McPherson, John; Stein, Lincoln D; Foulkes, William D; Majewski, Jacek; Gallinger, Steven; Zogopoulos, George

    2016-01-28

    The genetic basis underlying the majority of hereditary pancreatic adenocarcinoma (PC) is unknown. Since DNA repair genes are widely implicated in gastrointestinal malignancies, including PC, we hypothesized that there are novel DNA repair PC susceptibility genes. As germline DNA repair gene mutations may lead to PC subtypes with selective therapeutic responses, we also hypothesized that there is an overall survival (OS) difference in mutation carriers versus non-carriers. We therefore interrogated the germline exomes of 109 high-risk PC cases for rare protein-truncating variants (PTVs) in 513 putative DNA repair genes. We identified PTVs in 41 novel genes among 36 kindred. Additional genetic evidence for causality was obtained for 17 genes, with FAN1, NEK1 and RHNO1 emerging as the strongest candidates. An OS difference was observed for carriers versus non-carriers of PTVs with early stage (?IIB) disease. This adverse survival trend in carriers with early stage disease was also observed in an independent series of 130 PC cases. We identified candidate DNA repair PC susceptibility genes and suggest that carriers of a germline PTV in a DNA repair gene with early stage disease have worse survival. PMID:26546047

  16. Exome-Wide Somatic Microsatellite Variation Is Altered in Cells with DNA Repair Deficiencies

    PubMed Central

    Vaksman, Zalman; Fonville, Natalie C.; Tae, Hongseok; Garner, Harold R.

    2014-01-01

    Microsatellites (MST), tandem repeats of 1–6 nucleotide motifs, are mutational hot-spots with a bias for insertions and deletions (INDELs) rather than single nucleotide polymorphisms (SNPs). The majority of MST instability studies are limited to a small number of loci, the Bethesda markers, which are only informative for a subset of colorectal cancers. In this paper we evaluate non-haplotype alleles present within next-gen sequencing data to evaluate somatic MST variation (SMV) within DNA repair proficient and DNA repair defective cell lines. We confirm that alleles present within next-gen data that do not contribute to the haplotype can be reliably quantified and utilized to evaluate the SMV without requiring comparisons of matched samples. We observed that SMV patterns found in DNA repair proficient cell lines without DNA repair defects, MCF10A, HEK293 and PD20 RV:D2, had consistent patterns among samples. Further, we were able to confirm that changes in SMV patterns in cell lines lacking functional BRCA2, FANCD2 and mismatch repair were consistent with the different pathways perturbed. Using this new exome sequencing analysis approach we show that DNA instability can be identified in a sample and that patterns of instability vary depending on the impaired DNA repair mechanism, and that genes harboring minor alleles are strongly associated with cancer pathways. The MST Minor Allele Caller used for this study is available at https://github.com/zalmanv/MST_minor_allele_caller. PMID:25402475

  17. Enzymatic MPG DNA repair assays for two different oxidative DNA lesions reveal associations with increased lung cancer risk.

    PubMed

    Leitner-Dagan, Yael; Sevilya, Ziv; Pinchev, Mila; Kremer, Ran; Elinger, Dalia; Rennert, Hedy S; Schechtman, Edna; Freedman, Laurence; Rennert, Gad; Livneh, Zvi; Paz-Elizur, Tamar

    2014-12-01

    DNA repair is a major mechanism for minimizing mutations and reducing cancer risk. Here, we present the development of reproducible and specific enzymatic assays for methylpurine DNA glycosylase (MPG) repairing the oxidative lesions 1,N6-ethenoadenine (?A) and hypoxanthine (Hx) in peripheral blood mononuclear cells protein extracts. Association of these DNA repair activities with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean MPG-?A in case patients was 15.8 units/?g protein (95% CI 15.3-16.3), significantly higher than in control subjects-15.1 (14.6-15.5), *P = 0.011. The adjusted odds ratio for lung cancer associated with a one SD increase in MPG-?A activity (2.48 units) was significantly bigger than 1 (OR = 1.6, 95% CI = 1.1-2.4; *P = 0.013). When activity of OGG1, a different DNA repair enzyme for oxidative damage, was included in the model, the estimated odds ratio/SD for a combined MPG-?A-OGG1 score was 2.6 (95% CI 1.6-4.2) *P = 0.0001, higher than the odds ratio for each single assay. The MPG enzyme activity assays described provide robust functional risk biomarkers, with increased MPG-?A activity being associated with increased lung cancer risk, similar to the behavior of MPG-Hx. This underscores the notion that imbalances in DNA repair, including high DNA repair, usually perceived as beneficial, can cause cancer risk. Such DNA repair risk biomarkers may be useful for risk assessment of lung cancer and perhaps other cancer types, and for early detection techniques such as low-dose CT. PMID:25355292

  18. CUX2 protein functions as an accessory factor in the repair of oxidative DNA damage.

    PubMed

    Pal, Ranjana; Ramdzan, Zubaidah M; Kaur, Simran; Duquette, Philippe M; Marcotte, Richard; Leduy, Lam; Davoudi, Sayeh; Lamarche-Vane, Nathalie; Iulianella, Angelo; Nepveu, Alain

    2015-09-11

    CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair. PMID:26221032

  19. Repair of nonreplicating UV-irradiated DNA: cooperative dark repair by Escherichia coli uvr and phr functions.

    PubMed Central

    Hays, J B; Martin, S J; Bhatia, K

    1985-01-01

    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+ phr+ bacteria as in uvr+ delta phr bacteria. Similarly, UV-irradiated phages plated with higher efficiencies on phr+ than delta phr bacteria even under totally dark conditions. In uvr phr+ repressed infections, removal of dimers from nonreplicating DNA did not increase infectivity as much as in uvr+ infections, suggesting a requirement for repair of nondimer photoproducts by the uvrABC system. PMID:3881404

  20. 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 ?-phellandrene (?-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that ?-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of ?-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of ?-PA on total cell viability and the results indicated that ?-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 ?-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 ?-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 ?-PA increased p-p53, p-H2A.X, 14-3-3-?, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1322-1330, 2015. PMID:24861204

  1. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

    PubMed

    Zd?alik, Daria; Doma?ska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø; Rizzo, Carmelo J; Guengerich, F Peter; Tudek, Barbara

    2015-06-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(?)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ?-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 ?-adducts, 1,N(6)-ethenoadenine (?A), 3,N(4)-ethenocytosine (?C) and 1,N(2)-ethenoguanine (1,N(2)-?G) 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 ?-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ?A and ?C from ds and ssDNA but were inactive toward 1,N(2)-?G. SC-1A repaired only ?A with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ?-adducts in dsDNA, while only ?A and ?C in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ?C 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,N(2)-?G and that ALKBH3 removes only ?C 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

  2. Repair of a DNA-protein crosslink by replication-coupled proteolysis

    PubMed Central

    Duxin, Julien P.; Dewar, James M.; Yardimci, Hasan; Walter, Johannes C.

    2014-01-01

    Summary DNA-protein crosslinks (DPCs) are caused by environmental, endogenous, and chemotherapeutic agents and pose a severe threat to genome stability. We use Xenopus egg extracts to recapitulate DPC repair and show that this process is coupled to DNA replication. A DPC on the leading strand template arrests the replisome ahead of the lesion by stalling the CMG helicase. The DPC is then degraded on DNA, yielding a peptide-DNA adduct that is bypassed by CMG. The leading strand subsequently resumes synthesis, stalls again at the adduct, and then extends past it. Bypass of the peptide adduct requires DNA pol ?. A DPC on the lagging strand template only transiently stalls the replisome, but it too is degraded, allowing Okazaki fragment bypass. Our experiments describe a versatile, proteolysis-based mechanism of S phase DPC repair that avoids replication fork collapse as well as the need to induce double strand breaks in the repair process. PMID:25303529

  3. 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.

  4. Inducible SOS Response System of DNA Repair and Mutagenesis in Escherichia coli

    PubMed Central

    Janion, Celina

    2008-01-01

    Chromosomal DNA is exposed to continuous damage and repair. Cells contain a number of proteins and specific DNA repair systems that help maintain its correct structure. The SOS response was the first DNA repair system described in Escherichia coli induced upon treatment of bacteria with DNA damaging agents arrest DNA replication and cell division. Induction of the SOS response involves more than forty independent SOS genes, most of which encode proteins engaged in protection, repair, replication, mutagenesis and metabolism of DNA. Under normal growth conditions the SOS genes are expressed at a basal level, which increases distinctly upon induction of the SOS response. The SOS-response has been found in many bacterial species (e.g., Salmonella typhimurium, Caulobacter crescentus, Mycobacterium tuberculosis), but not in eukaryotic cells. However, species from all kingdoms contain some SOS-like proteins taking part in DNA repair that exhibit amino acid homology and enzymatic activities related to those found in E. coli. but are not organized in an SOS system. This paper presents a brief up-to-date review describing the discovery of the SOS system, the physiology of SOS induction, methods for its determination, and the role of some SOS-induced genes. PMID:18825275

  5. Gene-specific DNA repair of pyrimidine dimers does not decline during cellular aging in vitro.

    PubMed

    Christiansen, M; Stevnsner, T; Bohr, V A; Clark, B F; Rattan, S I

    2000-04-10

    A large number of studies have demonstrated that various kinds of DNA damage accumulate during aging and one of the causes for this could be a decrease in DNA repair capacity. However, the level of total genomic repair has not been strongly correlated with aging. DNA repair of certain kinds of damage is known to be closely connected to the transcription process; thus, we chose to investigate the level of gene-specific repair of UV-induced damage using in vitro aging of human diploid skin fibroblasts and trabecular osteoblasts as model systems for aging. We find that the total genomic repair is not significantly affected during cellular aging of cultures of both human skin fibroblasts and trabecular osteoblasts. Gene-specific repair was analyzed during cellular aging in the dihydrofolate reductase housekeeping gene, the p53 tumor suppressor gene, and the inactive region X(754). There was no clear difference in the capacity of young and old cells to repair UV-induced pyrimidine dimers in any of the analyzed genes. Thus, in vitro senescent cells can sustain the ability to repair externally induced damage. PMID:10739678

  6. Histone Deacetylase 10 Regulates DNA Mismatch Repair and May Involve the Deacetylation of MutS Homolog 2.

    PubMed

    Radhakrishnan, Rangasudhagar; Li, Yixuan; Xiang, Shengyan; Yuan, Fenghua; Yuan, Zhigang; Telles, Elphine; Fang, Jia; Coppola, Domenico; Shibata, David; Lane, William S; Zhang, Yanbin; Zhang, Xiaohong; Seto, Edward

    2015-09-11

    MutS homolog 2 (MSH2) is an essential DNA mismatch repair (MMR) protein. It interacts with MSH6 or MSH3 to form the MutS? or MutS? complex, respectively, which recognize base-base mispairs and insertions/deletions and initiate the repair process. Mutation or dysregulation of MSH2 causes genomic instability that can lead to cancer. MSH2 is acetylated at its C terminus, and histone deacetylase (HDAC6) deacetylates MSH2. However, whether other regions of MSH2 can be acetylated and whether other histone deacetylases (HDACs) and histone acetyltransferases (HATs) are involved in MSH2 deacetylation/acetylation is unknown. Here, we report that MSH2 can be acetylated at Lys-73 near the N terminus. Lys-73 is highly conserved across many species. Although several Class I and II HDACs interact with MSH2, HDAC10 is the major enzyme that deacetylates MSH2 at Lys-73. Histone acetyltransferase HBO1 might acetylate this residue. HDAC10 overexpression in HeLa cells stimulates cellular DNA MMR activity, whereas HDAC10 knockdown decreases DNA MMR activity. Thus, our study identifies an HDAC10-mediated regulatory mechanism controlling the DNA mismatch repair function of MSH2. PMID:26221039

  7. 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. PMID:26255934

  8. Telomere Regulation in Arabidopsis thaliana by the CST Capping Complex and DNA Damage Response Proteins 

    E-print Network

    Boltz, Kara A.

    2013-09-11

    The ends of chormosomes are capped by telomeres, which distinguish the termini from damaged DNA. Paradoxically, DNA repair proteins are also required for telomere maintenance. How DNA repair pathways are regulated to maintain telomeres while...

  9. Interaction of DNA repair gene polymorphisms and aflatoxin B1 in the risk of hepatocellular carcinoma

    PubMed Central

    Yao, Jin-Guang; Huang, Xiao-Ying; Long, Xi-Dai

    2014-01-01

    Aflatoxin B1 (AFB1) is an important environmental carcinogen and can induce DNA damage and involve in the carcinogenesis of hepatocellular carcinoma (HCC). The deficiency of DNA repair capacity related to the polymorphisms of DNA repair genes might play a central role in the process of HCC tumorigenesis. However, the interaction of DNA repair gene polymorphisms and AFB1 in the risk of hepatocellular carcinoma has not been elucidated. In this study, we investigated whether six polymorphisms (including rs25487, rs861539, rs7003908, rs28383151, rs13181, and rs2228001) in DNA repair genes (XPC, XRCC4, XRCC1, XRCC4, XPD, XRCC7, and XRCC3) interacted with AFB1, and the gene-environmental interactive role in the risk of HCC using hospital-based case-control study (including 1486 HCC cases and 1996 controls). Genotypes of DNA repair genes were tested using TaqMan-PCR technique. Higher AFB1 exposure was observed among HCC patients versus the control group [odds ratio (OR) = 2.08 for medium AFB1 exposure level and OR = 6.52 for high AFB1 exposure level]. Increasing risk of HCC was also observed in these with the mutants of DNA repair genes (risk values were from 1.57 to 5.86). Furthermore, these risk roles would be more noticeable under the conditions of two variables, and positive interactive effects were proved in the followed multiplicative interaction analysis. These results suggested that DNA repair risk genotypes might interact with AFB1 in the risk of HCC. PMID:25337275

  10. 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.

  11. 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. PMID:11459990

  12. 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. PMID:26052075

  13. Differential human nucleotide excision repair of paired and mispaired cisplatin-DNA adducts.

    PubMed Central

    Moggs, J G; Szymkowski, D E; Yamada, M; Karran, P; Wood, R D

    1997-01-01

    In order to understand the action of the chemotherapeutic drug cisplatin, it is necessary to determine why some types of cisplatin-DNA intrastrand crosslinks are repaired better than others. Using cell extracts and circular duplex DNA, we compared nucleotide excision repair of uniquely placed 1,2-GG, 1,2-AG, and 1,3-GTG cisplatin-crosslinks, and a 2-acetylaminofluorene lesion. The 1,3 crosslink and the acetylaminofluorene lesion were repaired by normal cell extracts approximately 15-20 fold better than the 1,2 crosslinks. No evidence was found for selective shielding of 1,2 cisplatin crosslinks from repair by cellular proteins. Fractionation of cell extracts to remove putative shielding proteins did not improve repair of the 1,2-GG crosslink, and cell extracts did not selectively inhibit access of UvrABC incision nuclease to 1,2-GG crosslinks. The poorer repair of 1,2 crosslinks in comparison to the 1,3 crosslink is more likely a consequence of different structural alterations of the DNA helix. In support of this, a 1,2-GG-cisplatin crosslink was much better repaired when it was opposite one or two non-complementary thymines. Extracts from cells defective in the hMutSalpha mismatch binding activity also showed preferential repair of the 1,3 crosslink over the 1,2 crosslink, and increased repair of the 1,2 adduct when opposite thymines, showing that hMutSalphais not involved in the differential NER of these substrates in vitro. Mismatched cisplatin adducts could arise by translesion DNA synthesis, and improved repair of such adducts could promote cisplatin-induced mutagenesis in some cases. PMID:9016585

  14. Strand specific DNA repair in different stages of the cell cycle

    SciTech Connect

    Lommel, L.; Crumpton, C.C.; Schimke, R.T.; Hanawalt, P.C. )

    1993-01-01

    The genomic location of a DNA lesions can dramatically influence the efficiency of repair. For example, in asynchronous human cells there is preferential repair of UV-induced cyclobutane pyrimidine dimers (CPDs) in the transcribed strands of active genes. The efficiencies of repair in the non-transcribed strands of the genes examined thus far are essentially equivalent to that in the genome overall, but significantly slower than that in the transcribed strands. The authors' current interest is how these DNA repair efficiencies might be influenced by the phase of the cell cycle. They developed a method to assay repair of specific DNA sequences in any phase of the cell cycle, using a synchronization procedure that does not perturb the cell cycle. Ethanol fixed cells were stained with chromomycin A3 and sorted on the basis of DNA content. Fractions were collected corresponding to G1; early, middle and late S; and G2/M. They are currently examining the initial frequency of CPDs and the efficiencies of repair in each strand of the dihydrofolate reductase gene in cultured human cells. These experiments are important to an understanding of how the heterogeneity in DNA damage processing in particular sequences and as a function of the cell cycle may be involved in biological endpoints such as mutagenesis and malignant transformation.

  15. Possible involvement of XPA in repair of oxidative DNA damage deduced from analysis of damage, repair and genotype in a human population study.

    PubMed

    Dusinská, Mária; Dzupinková, Zuzana; Wsólová, Ladislava; Harrington, Vikki; Collins, Andrew R

    2006-05-01

    Participants in a study of occupational exposure to mineral fibres in Slovakia were analysed for the polymorphism 23A-->G in the DNA repair gene XPA. Of the 388 subjects, 239 were exposed to asbestos, stonewool or glass fibre; the rest were unexposed controls. Levels of DNA base alterations (oxidation and alkylation) in lymphocytes were measured using the comet assay with lesion-specific endonucleases. 8-oxoguanine DNA glycosylase (OGG1) DNA repair activity was measured, as incision activity of a lymphocyte extract on DNA containing the OGG1 substrate 8-oxoguanine. Presence of the A allele was associated with higher levels of DNA damage (sites sensitive to formamidopyrimidine DNA glycosylase, endonuclease III or 3-methyladenine DNA glycosylase II) as well as with higher activity of OGG1 repair enzyme. DNA base damage increased with age, showing highly significant correlations when the whole population or subgroups of the population were analysed. OGG1 repair activity also increased with age, but when analysed according to XPA genotype, the increase was observed only in those individuals with an A allele. Although XPA is known as a protein involved in nucleotide excision repair of UV-induced damage and bulky DNA adducts, it may also have a role in the repair of oxidized bases. PMID:16613913

  16. MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5' end resection.

    PubMed

    Boersma, Vera; Moatti, Nathalie; Segura-Bayona, Sandra; Peuscher, Marieke H; van der Torre, Jaco; Wevers, Brigitte A; Orthwein, Alexandre; Durocher, Daniel; Jacobs, Jacqueline J L

    2015-05-28

    Appropriate repair of DNA lesions and the inhibition of DNA repair activities at telomeres are crucial to prevent genomic instability. By fuelling the generation of genetic alterations and by compromising cell viability, genomic instability is a driving force in cancer and ageing. Here we identify MAD2L2 (also known as MAD2B or REV7) through functional genetic screening as a novel factor controlling DNA repair activities at mammalian telomeres. We show that MAD2L2 accumulates at uncapped telomeres and promotes non-homologous end-joining (NHEJ)-mediated fusion of deprotected chromosome ends and genomic instability. MAD2L2 depletion causes elongated 3' telomeric overhangs, indicating that MAD2L2 inhibits 5' end resection. End resection blocks NHEJ while committing to homology-directed repair, and is under the control of 53BP1, RIF1 and PTIP. Consistent with MAD2L2 promoting NHEJ-mediated telomere fusion by inhibiting 5' end resection, knockdown of the nucleases CTIP or EXO1 partially restores telomere-driven genomic instability in MAD2L2-depleted cells. Control of DNA repair by MAD2L2 is not limited to telomeres. MAD2L2 also accumulates and inhibits end resection at irradiation-induced DNA double-strand breaks and promotes end-joining of DNA double-strand breaks in several settings, including during immunoglobulin class switch recombination. These activities of MAD2L2 depend on ATM kinase activity, RNF8, RNF168, 53BP1 and RIF1, but not on PTIP, REV1 and REV3, the latter two acting with MAD2L2 in translesion synthesis. Together, our data establish MAD2L2 as a crucial contributor to the control of DNA repair activity by 53BP1 that promotes NHEJ by inhibiting 5' end resection downstream of RIF1. PMID:25799990

  17. BRIT1/MCPH1 Is Essential for Mitotic and Meiotic Recombination DNA Repair and Maintaining Genomic Stability in Mice

    PubMed Central

    Liang, Yulong; Gao, Hong; Lin, Shiaw-Yih; Peng, Guang; Huang, Xingxu; Zhang, Pumin; Goss, John A.; Brunicardi, Francis C.; Multani, Asha S.; Chang, Sandy; Li, Kaiyi

    2010-01-01

    BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1?/? mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1?/? mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to ?-irradiation. BRIT1?/? MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1?/? mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2's function and as a result leads to infertility and genomic instability in mice. PMID:20107607

  18. [Individual radiosensitivity and DNA repair proficiency: the value of the comet assay].

    PubMed

    Alapetite, C

    1998-01-01

    Some rare hereditary syndromes demonstrate high cancer risk and hypersensitivity in response to exposures to agents such as ultraviolet or ionising radiation, and are characterised by a defective processing of DNA damage. They highlight the importance of the individual capacity of restoring the genomic integrity in the individual risk associated to exposures. The comet assay, a simple technique that detects DNA strand breaks, requires few cells and allows examination of DNA repair capacities in established cell lines, in blood samples or biopsies. The assay has been validated on cellular systems with known repair defects such as xeroderma pigmentosum defective in nucleotide excision repair, on mutant rodent cell lines defective in DNA single strand break rejoining (XRCC1) (alkaline version) or DNA double strand breaks rejoining (XRCC5/Ku80 and XRCC7/DNAPKcs) (neutral conditions). This assay does not allow to distinguish a defective phenotype in ataxia telangiectasia cells. It shows in homozygous mouse embryo fibroblasts Brca2-/- an impaired DNA double strand break rejoining. Simplicity, rapidity and sensitivity of the alkaline comet assay allow to examine the response of lymphocytes. It has been applied to the analysis of the role of DNA repair in the pathogenesis of collagen diseases, and the involvement of individual DNA repair proficiency in the thyroid tumorigenesis induced in some patients after therapeutic irradiation at childhood has been questioned. Preliminary results of these studies suggest that this type of approach could help for adapting treatment modalities and surveillance in subgroups of patients defective in DNA repair process. It could also have some incidence in the radioprotection field. PMID:9868398

  19. Protective roles of Gadd45 and MDM2 in blueberry anthocyanins mediated DNA repair of fragmented and non-fragmented DNA damage in UV-irradiated HepG2 cells.

    PubMed

    Liu, Wei; Lu, Xiangyi; He, Guangyang; Gao, Xiang; Xu, Maonian; Zhang, Jingkai; Li, Meiling; Wang, Lifeng; Li, Zhenjing; Wang, Likui; Luo, Cheng

    2013-01-01

    Growth Arrest and DNA Damage-inducible 45 (Gadd45) and MDM2 proteins, together with p21 and p53, play important roles in cell cycle checkpoints, DNA repair, and genome integrity maintenance. Gadd45 and MDM2 were activated and transcribed instantly by UV irradiation, whereas blueberry anthocyanins (BA) decreased the gene and protein expression levels in HepG2 cells for up to 24 h, and gradually restored the UV-induced fragmented and non-fragmented DNA damage of the nucleus at a time point of 12 h. Nevertheless, UV-irradiated HepG2 cell arrests occurred mainly in the G1 phase, which indicated G1 as a checkpoint. The proteins, p21 and p53, retain cellular integrity, suppressing the oncogenic transformation by interruption of the G1 phase of the cellular cycle, giving time for repairing the damage to DNA, or apoptosis induction if the damage is too severe to be repaired, while MDM2 and Gadd45 concomitantly ensure the presence of p53 and p21. Thus, we conclude that repair, together with Gadd45 and MDM2 genes, were involved in light and dark reaction mechanisms, however, BA could interfere and assist the repair through restoration, although further studies of the complex of the gene cascades triggered and responded to in BA-assisted DNA repair are needed. PMID:24177565

  20. Mitochondrial and nuclear DNA base excision repair are affected differently by caloric restriction1

    E-print Network

    Stuart, Jeffrey A.

    . Caloric restriction lowers DNA repair activity in brain and kidney but not liver mitochondria Most DNA was assessed by measuring BER activity in mito- chondrial extracts prepared from liver, brain, and kidney and kidney mitochon- dria, CR resulted in 30% reductions of BER activity (t test; P 0.06) compared with PF

  1. Having a direct look: Analysis of DNA damage and repair mechanisms by next generation sequencing

    PubMed Central

    Meier, Bettina; Gartner, Anton

    2014-01-01

    Genetic information is under constant attack from endogenous and exogenous sources, and the use of model organisms has provided important frameworks to understand how genome stability is maintained and how various DNA lesions are repaired. The advance of high throughput next generation sequencing (NGS) provides new inroads for investigating mechanisms needed for genome maintenance. These emerging studies, which aim to link genetic toxicology and mechanistic analyses of DNA repair processes in vivo, rely on defining mutational signatures caused by faulty replication, endogenous DNA damaging metabolites, or exogenously applied genotoxins; the analysis of their nature, their frequency and distribution. In contrast to classical studies, where DNA repair deficiency is assessed by reduced cellular survival, the localization of DNA repair factors and their interdependence as well as limited analysis of single locus reporter assays, NGS based approaches reveal the direct, quantal imprint of mutagenesis genome-wide, at the DNA sequence level. As we will show, such investigations require the analysis of DNA derived from single genotoxin treated cells, or DNA from cell populations regularly passaged through single cell bottlenecks when naturally occurring mutation accumulation is investigated. We will argue that the life cycle of the nematode Caenorhabditis elegans, its genetic malleability combined with whole genome sequencing provides an exciting model system to conduct such analysis. PMID:25131498

  2. Relevance of GC content to the conservation of DNA polymerase III/mismatch repair system in Gram-positive bacteria

    PubMed Central

    Akashi, Motohiro; Yoshikawa, Hirofumi

    2013-01-01

    The mechanism of DNA replication is one of the driving forces of genome evolution. Bacterial DNA polymerase III, the primary complex of DNA replication, consists of PolC and DnaE. PolC is conserved in Gram-positive bacteria, especially in the Firmicutes with low GC content, whereas DnaE is widely conserved in most Gram-negative and Gram-positive bacteria. PolC contains two domains, the 3?-5?exonuclease domain and the polymerase domain, while DnaE only possesses the polymerase domain. Accordingly, DnaE does not have the proofreading function; in Escherichia coli, another enzyme DnaQ performs this function. In most bacteria, the fidelity of DNA replication is maintained by 3?-5? exonuclease and a mismatch repair (MMR) system. However, we found that most Actinobacteria (a group of Gram-positive bacteria with high GC content) appear to have lost the MMR system and chromosomes may be replicated by DnaE-type DNA polymerase III with DnaQ-like 3?-5? exonuclease. We tested the mutation bias of Bacillus subtilis, which belongs to the Firmicutes and found that the wild type strain is AT-biased while the mutS-deletant strain is remarkably GC-biased. If we presume that DnaE tends to make mistakes that increase GC content, these results can be explained by the mutS deletion (i.e., deletion of the MMR system). Thus, we propose that GC content is regulated by DNA polymerase and MMR system, and the absence of polC genes, which participate in the MMR system, may be the reason for the increase of GC content in Gram-positive bacteria such as Actinobacteria. PMID:24062730

  3. DNA repair defects sensitize cells to anticodon nuclease yeast killer toxins.

    PubMed

    Klassen, Roland; Wemhoff, Sabrina; Krause, Jens; Meinhardt, Friedhelm

    2011-03-01

    Killer toxins from Kluyveromyces lactis (zymocin) and Pichia acaciae (PaT) were found to disable translation in target cells by virtue of anticodon nuclease (ACNase) activities on tRNA(Glu) and tRNA(Gln), respectively. Surprisingly, however, ACNase exposure does not only impair translation, but also affects genome integrity and concomitantly DNA damage occurs. Previously, it was shown that homologous recombination protects cells from ACNase toxicity. Here, we have analyzed whether other DNA repair pathways are functional in conferring ACNase resistance as well. In addition to HR, base excision repair (BER) and postreplication repair (PRR) promote clear resistance to either, PaT and zymocin. Comparative toxin sensitivity analysis of BER mutants revealed that its ACNase protective function is due to the endonucleases acting on apurinic (AP) sites, whereas none of the known DNA glycosylases is involved. Because PaT and zymocin require the presence of the ELP3/TRM9-dependent wobble uridine modification 5-methoxy-carbonyl-methyl (mcm(5)) for tRNA cleavage, we analyzed toxin response in DNA repair mutants additionally lacking such tRNA modifications. ACNase resistance caused by elp3 or trm9 mutations was found to rescue hypersensitivity of DNA repair defects, consistent with DNA damage to occur as a consequence of tRNA cleavage. The obtained genetic evidence promises to reveal new aspects into the mechanism linking translational fidelity and genome surveillance. PMID:21188417

  4. Regulation of the Rev1-pol ? complex during bypass of a DNA interstrand cross-link.

    PubMed

    Budzowska, Magda; Graham, Thomas G W; Sobeck, Alexandra; Waga, Shou; Walter, Johannes C

    2015-07-14

    DNA interstrand cross-links (ICLs) are repaired in S phase by a complex, multistep mechanism involving translesion DNA polymerases. After replication forks collide with an ICL, the leading strand approaches to within one nucleotide of the ICL ("approach"), a nucleotide is inserted across from the unhooked lesion ("insertion"), and the leading strand is extended beyond the lesion ("extension"). How DNA polymerases bypass the ICL is incompletely understood. Here, we use repair of a site-specific ICL in Xenopus egg extracts to study the mechanism of lesion bypass. Deep sequencing of ICL repair products showed that the approach and extension steps are largely error-free. However, a short mutagenic tract is introduced in the vicinity of the lesion, with a maximum mutation frequency of ~1%. Our data further suggest that approach is performed by a replicative polymerase, while extension involves a complex of Rev1 and DNA polymerase ?. Rev1-pol ? recruitment requires the Fanconi anemia core complex but not FancI-FancD2. Our results begin to illuminate how lesion bypass is integrated with chromosomal DNA replication to limit ICL repair-associated mutagenesis. PMID:26071591

  5. Hyperthermia, polyamine depletion, and inhibition of X-ray-induced DNA strand break repair

    SciTech Connect

    Snyder, R.D.; Lachmann, P.J. )

    1989-10-01

    We have recently demonstrated that HeLa cells that had been depleted of polyamines by treatment with inhibitors of polyamine biosynthesis were deficient in their ability to repair X-ray-induced DNA strand breaks. Since it had previously been demonstrated that hyperthermic shock also inhibited strand break repair following X irradiation and that hyperthermia resulted in a leakage of polyamines from cells, it seemed of interest to examine whether the inhibition of repair by hyperthermia was related to this loss of cellular polyamines. In the present paper it is demonstrated that both polyamine depletion and hyperthermia inhibit strand closure, and that a combined treatment further reduces the rate of repair. In cells not depleted of polyamines, repair is restored to normal levels if hyperthermia treatment is followed by a 4-h incubation at 37 degrees C before X irradiation. In polyamine-depleted cells, this 37 degrees C incubation does not result in a return of repair ability. Polyamine supplementation was not effective in reversing hyperthermia-dependent repair inhibition, and, in fact, restoration of repair in control cells following hyperthermic shock corresponded to a time at which polyamines show a maximum decrease in those cells. These results suggest that the inhibition of repair and the increased radiosensitivity observed in hyperthermically treated cells is not related to polyamine depletion. However, data further suggest that polyamine-depleted cells may have other alterations, perhaps in chromatin, which render them more sensitive to thermal inhibition of repair.

  6. Repair of cis-platinum-DNA adducts by ABC excinuclease in vivo and in vitro.

    PubMed Central

    Husain, I; Chaney, S G; Sancar, A

    1985-01-01

    cis-Platinum compounds, which are used in cancer chemotherapy, are thought to exert their effect by damaging DNA. It is known that this damage is partially repaired in Escherichia coli. Using cis-Pt-treated pBR322 DNA as a probe, we investigated the role of nucleotide excision repair in the removal of Pt-DNA adducts. We found that the nucleotide excision pathway was the major mechanism for repairing Pt adducts in transforming plasmid DNA but that a recA-dependent pathway also contributed to plasmid survival. When cis-Pt-damaged pBR322 was treated with the purified nucleotide excision enzyme ABC excinuclease in vitro, a fraction of the adducts was removed by the enzyme; this removal resulted in a corresponding increase in transformation efficiency. PMID:3897194

  7. Fission Yeast Pxd1 Promotes Proper DNA Repair by Activating Rad16XPF and Inhibiting Dna2

    PubMed Central

    Zhang, Jia-Min; Liu, Xiao-Man; Ding, Yue-He; Xiong, Liang-Yao; Ren, Jing-Yi; Zhou, Zhi-Xiong; Wang, Hai-Tao; Zhang, Mei-Jun; Yu, Yang; Dong, Meng-Qiu; Du, Li-Lin

    2014-01-01

    Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16XPF-Swi10ERCC1 and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 3? endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 5? endonuclease activity of Dna2. Pxd1 is required for Rad16-Swi10 to function in single-strand annealing, mating-type switching, and the removal of Top1-DNA adducts. Meanwhile, Pxd1 attenuates DNA end resection mediated by the Rqh1-Dna2 pathway. Disabling the Dna2-inhibitory activity of Pxd1 results in enhanced use of a break-distal repeat sequence in single-strand annealing and a greater loss of genetic information. We propose that Pxd1 promotes proper DNA repair by differentially regulating two structure-specific nucleases. PMID:25203555

  8. Evidence for the involvement of human DNA polymerase N in the repair of DNA interstrand cross-links

    PubMed Central

    Zietlow, Laura; Smith, Leigh Anne; Bessho, Mika; Bessho, Tadayoshi

    2009-01-01

    Human DNA polymerase N (PolN) is an A-family nuclear DNA polymerase whose function is unknown. This study examines the possible role of PolN in DNA repair in human cells treated with PolN-targeted siRNA. HeLa cells with siRNA-mediated knockdown of PolN were more sensitive than control cells to DNA cross-linking agent mitomycin C (MMC), but were not hyper-sensitive to UV irradiation. The MMC hyper-sensitivity of PolN knockdown cells was rescued by the overexpression of DNA polymerase-proficient PolN but not by DNA polymerase-deficient PolN. Furthermore, in vitro experiments showed that purified PolN conducts low efficiency non-mutagenic bypass of a psoralen DNA interstrand cross-link (ICL), whose structure resembles an intermediate in the proposed pathway of ICL repair. These results suggest that PolN might play a role in translesion DNA synthesis during ICL repair in human cells. PMID:19908865

  9. Activation of DNA damage repair pathways in response to nitrogen mustard-induced DNA damage and toxicity in skin keratinocytes

    PubMed Central

    Inturi, Swetha; Tewari-Singh, Neera; Agarwal, Chapla; White, Carl W.; Agarwal, Rajesh

    2014-01-01

    Nitrogen mustard (NM), a structural analog of chemical warfare agent sulfur mustard (SM), forms adducts and crosslinks with DNA, RNA and proteins. Here we studied the mechanism of NM-induced skin toxicity in response to double strand breaks (DSBs) resulting in cell cycle arrest to facilitate DNA repair, as a model for developing countermeasures against vesicant-induced skin injuries. NM exposure of mouse epidermal JB6 cells decreased cell growth and caused S-phase arrest. Consistent with these biological outcomes, NM exposure also increased comet tail extent moment and the levels of DNA DSB repair molecules phospho H2A.X Ser139 and p53 Ser15 indicating NM-induced DNA DSBs. Since DNA DSB repair occurs via non homologous end joining pathway (NHEJ) or homologous recombination repair (HRR) pathways, next we studied these two pathways and noted their activation as defined by an increase in phospho- and total DNA-PK levels, and the formation of Rad51 foci, respectively. To further analyze the role of these pathways in the cellular response to NM-induced cytotoxicity, NHEJ and HRR were inhibited by DNA-PK inhibitor NU7026 and Rad51 inhibitor BO2, respectively. Inhibition of NHEJ did not sensitize cells to NM-induced decrease in cell growth and cell cycle arrest. However, inhibition of the HRR pathway caused a significant increase in cell death, and prolonged G2M arrest following NM exposure. Together, our findings, indicating that HRR is the key pathway involved in the repair of NM-induced DNA DSBs, could be useful in developing new therapeutic strategies against vesicant-induced skin injury. PMID:24732344

  10. Histone H2A.Z controls a critical chromatin remodeling step required for DNA double-strand break repair

    PubMed Central

    Xu, Ye; Ayrapetov, Marina K.; Xu, Chang; Gursoy-Yuzugullu, Ozge; Hu, Yiduo; Price, Brendan D.

    2012-01-01

    Chromatin remodeling during DNA double-strand break (DSB) repair is required to facilitate access to and repair of DSBs. This remodeling requires increased acetylation of histones and a shift in nucleosome organization to create open, relaxed chromatin domains. However, the underlying mechanism driving changes in nucleosome structure at DSBs is poorly defined. Here, we demonstrate that histone H2A.Z is exchanged onto nucleosomes at DSBs by the p400 remodeling ATPase. H2A.Z exchange at DSBs shifts the chromatin to an open conformation, and is required for acetylation and ubiquitination of histones and for loading of the brca1 complex. H2A.Z exchange also restricts single-stranded DNA production by nucleases and is required for loading of the Ku70/80 DSB repair protein. H2A.Z exchange therefore promotes specific patterns of histone modification and reorganization of the chromatin architecture, leading to the assembly of a chromatin template which is an efficient substrate for the DSB repair machinery. PMID:23122415

  11. Dictyostelium discoideum, a lower eukaryote model for the study of DNA repair: Implications for the role of DNA-damaging chemicals in the evolution of repair proficient cells

    NASA Astrophysics Data System (ADS)

    Deering, R. A.

    1994-10-01

    The evolution of the ability of living cells to cope with stress is crucial for the maintenance of their genetic integrity. Yet low levels of mutation must remain to allow adaptation to environmental changes. The cellular slime mold D. discoideum is a good system for studying molecular aspects of the repair of lethal and mutagenic damage to DNA by radiation and chemicals. The wild-type strains of this soil microorganism are extremely resistant to DNA damaging agents. In nature the amoeboid cells in their replicative stage feed on soil bacteria and are exposed to numerous DNA-damaging chemicals produced by various soil microorganisms. It is probable that the evolution of repair systems in this organism and perhaps in others is a consequence of the necessity to cope with chemical damage which also confers resistance to radiation.

  12. Arsenic Biotransformation as a Cancer Promoting Factor by Inducing DNA Damage and Disruption of Repair Mechanisms

    PubMed Central

    Martinez, Victor D.; Vucic, Emily A.; Adonis, Marta; Gil, Lionel; Lam, Wan L.

    2011-01-01

    Chronic exposure to arsenic in drinking water poses a major global health concern. Populations exposed to high concentrations of arsenic-contaminated drinking water suffer serious health consequences, including alarming cancer incidence and death rates. Arsenic is biotransformed through sequential addition of methyl groups, acquired from s-adenosylmethionine (SAM). Metabolism of arsenic generates a variety of genotoxic and cytotoxic species, damaging DNA directly and indirectly, through the generation of reactive oxidative species and induction of DNA adducts, strand breaks and cross links, and inhibition of the DNA repair process itself. Since SAM is the methyl group donor used by DNA methyltransferases to maintain normal epigenetic patterns in all human cells, arsenic is also postulated to affect maintenance of normal DNA methylation patterns, chromatin structure, and genomic stability. The biological processes underlying the cancer promoting factors of arsenic metabolism, related to DNA damage and repair, will be discussed here. PMID:22091411

  13. OXIDATIVE DNA DAMAGE AND REPAIR IN RATS TREATED WITH POTASSIUM BROMATE AND A MIXTUE OF DRINKING WATER DISINFECTION BY-PRODUCTS

    EPA Science Inventory

    Oxidative DNA Damage and Repair in Rats Treated with Potassium Bromate and a Mixture of Drinking Water Disinfection By-Products

    Public drinking water treated with chemical disint'ectants contains a complex mixture of disinfection by-products (D BPs). There is a need for m...

  14. MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5? end-resection

    PubMed Central

    Segura-Bayona, Sandra; Peuscher, Marieke H.; van der Torre, Jaco; Wevers, Brigitte A.; Orthwein, Alexandre; Durocher, Daniel; Jacobs, Jacqueline J.L.

    2015-01-01

    Appropriate repair of DNA lesions and the inhibition of DNA repair activities at telomeres are critical to prevent genomic instability. By fuelling the generation of genetic alterations and by compromising cell viability, genomic instability is a driving force in cancer and aging1, 2. Here we identify MAD2L2 (also known as MAD2B or REV7) through functional genetic screening as a novel factor controlling DNA repair activities at mammalian telomeres. We show that MAD2L2 accumulates at uncapped telomeres and promotes non-homologous end-joining (NHEJ)-mediated fusion of deprotected chromosome ends and genomic instability. MAD2L2 depletion causes elongated 3? telomeric overhangs, implying that MAD2L2 inhibits 5? end-resection. End-resection blocks NHEJ while committing to homology-directed repair (HDR) and is under control of 53BP1, RIF1 and PTIP3. Consistent with MAD2L2 promoting NHEJ-mediated telomere fusion by inhibiting 5? end-resection, knockdown of the nucleases CTIP or EXO1 partially restores telomere-driven genomic instability in MAD2L2-depleted cells. Control of DNA repair by MAD2L2 is not limited to telomeres. MAD2L2 also accumulates and inhibits end-resection at irradiation (IR)-induced DNA double-strand breaks (DSBs) and promotes end-joining of DSBs in multiple settings, including during immunoglobulin class switch recombination (CSR). These activities of MAD2L2 depend on ATM kinase activity, RNF8, RNF168, 53BP1 and RIF1, but not on PTIP, REV1 and REV3, the latter two acting with MAD2L2 in translesion synthesis (TLS)4. Together our data establish MAD2L2 as a critical contributor to the control of DNA repair activity by 53BP1 that promotes NHEJ by inhibiting 5? end-resection downstream of RIF1. PMID:25799990

  15. DNA-dependent protein kinase catalytic subunit inhibitor reverses acquired radioresistance in lung adenocarcinoma by suppressing DNA repair.

    PubMed

    Li, Yong; Li, Hang; Peng, Wen; He, Xin-Yun; Huang, Min; Qiu, Dong; Xue, Ying-Bo; Lu, Liang

    2015-07-01

    The mechanisms underlying lung cancer radioresistance remain to be fully elucidated. The DNA repair pathway is a predominant target of radiotherapy, which is considered to be involved in the acquired radioresistance of cancer cells. The present study aimed to establish a radioresistant cell model using the A549 human lung cancer cell line, and to further investigate the potential mechanisms underlying the radioresistance. The A549R radioresistant lung cancer cell variant was established by exposing the parental A549 cells to repeated ?-ray irradiation at a total dose of 60 Gy. Colony formation assays were then used to determine cell survival following ?-ray exposure. The established radioresistant cells were subsequently treated with or without the NU7026 DNA-PKcs inhibitor. The levels of DNA damage were determined by counting the number of fluorescent ?-H2AX foci in the cells. The cellular capacity for DNA repair was assessed using antibodies for the detection of various DNA repair pathway proteins. The radioresistant sub-clones exhibited significantly decreased survival following NU7026 treatment, compared with the parental cells, as determined by colony formation assays (P<0.05), and this finding was found to be dose-dependent. Treatment with the DNA-dependent protein kinase (DNA-PK) inhibitor significantly reduced ?-H2AX foci formation (P<0.05) following acute radiation exposure in the radioresistant sub-clones, compared with the parental control cells. The decreased levels of ?-H2AX were accompanied by an increase in the percentage of apoptotic cells in the radioresistant cell line following post-radiation treatment with the DNA-PKcs inhibitor. The expression levels of proteins associated with the DNA repair pathway were altered markedly in the cells treated with NU7026. The results of the present study suggested that radioresistance may be associated with enhanced DNA repair following exposure to radiation, resulting in reduced apoptosis. Therefore, the quantity of ?-H2AX determines the radioresistance of cells. The DNA repair pathway is important in mediating radioresistance, and treatment with the DNA-PKcs inhibitor, NU7026 restored the acquired radiation resistance. PMID:25815686

  16. Direct interaction between cohesin complex and DNA replication machinery

    SciTech Connect

    Ryu, Min-Jung; Kim, Beom-Jun; Lee, Jeong-Won; Lee, Min-Woo; Choi, Hyun-Kyung; Kim, Seong-Tae . E-mail: stkim@med.skku.ac.kr

    2006-03-17

    Structural maintenance of chromosome 1 (Smc1) is a multifunctional protein, which has been implicated in sister chromatid cohesion, DNA recombination and repair, and the activation of cell cycle checkpoints by ionizing radiation, ultraviolet light, and other genotoxic agents. In order to identify the proteins that interact with Smc1, we conducted the Tandem affinity purification (TAP) technique and analyzed the Smc1-interacting proteins via MALDI-TOF mass spectrometry. We identified minichromosome maintenance 7 (Mcm7), an essential component of the pre-replication complex, as a novel Smc1-interacting protein. Co-immunoprecipitation revealed an interaction occurring between Smc1 and Mcm7, both in vitro and in vivo. Using a GST pull-down assay, we determined that Smc1 interacts physically with Mcm7 via its N-terminal and hinge regions, and Mcm7 interacts with Smc1 via its middle region. Interestingly, we also discovered that Smc1 interacts with other DNA replication proteins, including Mcm6, RFC1, and DNA polymerase {alpha}. These results suggest that a functional link exists between the cohesin complex and DNA replication proteins.

  17. Variations on the standard protocol design of the hepatocyte DNA repair assay.

    PubMed

    Barfknecht, T R; Naismith, R W; Kornbrust, D J

    1987-06-01

    Several variations on the standard primary rat hepatocyte DNA/repair assay were evaluated for their ability to enhance the sensitivity of this genotoxicity test system. The use of hamster hepatocytes proved to be a much more sensitive system than rat hepatocytes for detecting the DNA repair inducing ability of the nitrosamines, dimethylnitrosamine and diethylnitrosamine, and the aromatic amines, 2-acetylaminofluorene, 9-aminoacridine, 1-naphthylamine and benzidine. In addition, hamster hepatocytes were a more sensitive indicator of the genotoxicity of the azo dyes, o-aminoazotoluene, Congo Red and Evans Blue. However, the azo reduction product of the azo dyes Congo Red, Trypan Blue and Evans Blue, benzidine and o-tolidine, respectively, were active in both rat and hamster hepatocytes at concentrations that were 10-100 fold lower than the parent dyes. This suggests that little or no azo reduction of the dyes occurred in the in vitro assay systems. The in vivo-in vitro variation of the rat hepatocytes DNA/repair assay exhibited a positive DNA repair response with the azo dye solvent Yellow 5, which was negative in the standard in vitro assay. The in vivo-in vitro hepatocyte DNA repair assay was also more sensitive for detecting the genotoxic activity of Evans Blue, which was positive in the in vivo-in vitro assay and equivocal in the standard in vitro assay. Also, Solvent Yellow 14 was negative in the in vitro assay, but induced an equivocal DNA repair response in the in vivo-in vitro assay system. A treatment/3H-thymidine labeling period of approximately 18 hours, compared to 4 hours, was demonstrated to be superior for detecting the DNA repair elicited by the mutagens 4-nitroquinoline-1-oxide, mitomycin C, dimethylnitrosamine and methyl methanesulfonate in the in vitro rat hepatocyte assay. There was little or no difference observed between the 4 hour and 18 hour treatment/labeling incubation periods for the detection of DNA repair induced by 2-acetylaminofluorene, aflatoxin B1, and benzidine. The data suggest that these several variations on the standard rat hepatocyte DNA/repair assay should be considered when evaluating the genotoxicity of chemicals for safety purposes. PMID:3507255

  18. Structure of the LexA?DNA complex and implications for SOS box measurement

    SciTech Connect

    Zhang, Adrianna P.P.; Pigli, Ying Z.; Rice, Phoebe A.

    2010-09-08

    The eubacterial SOS system is a paradigm of cellular DNA damage and repair, and its activation can contribute to antibiotic resistance. Under normal conditions, LexA represses the transcription of many DNA repair proteins by binding to SOS 'boxes' in their operators. Under genotoxic stress, accumulating complexes of RecA, ATP and single-stranded DNA (ssDNA) activate LexA for autocleavage. To address how LexA recognizes its binding sites, we determined three crystal structures of Escherichia coli LexA in complex with SOS boxes. Here we report the structure of these LexA-DNA complexes. The DNA-binding domains of the LexA dimer interact with the DNA in the classical fashion of a winged helix-turn-helix motif. However, the wings of these two DNA-binding domains bind to the same minor groove of the DNA. These wing-wing contacts may explain why the spacing between the two half-sites of E. coli SOS boxes is invariant.

  19. DNA repair genes are selectively mutated in diffuse large B cell lymphomas

    PubMed Central

    de Miranda, Noel FCC; Peng, Roujun; Georgiou, Konstantinos; Wu, Chenglin; Sörqvist, Elin Falk; Berglund, Mattias; Chen, Longyun; Gao, Zhibo; Lagerstedt, Kristina; Lisboa, Susana; Roos, Fredrik; van Wezel, Tom; Teixeira, Manuel R.; Rosenquist, Richard; Sundström, Christer; Enblad, Gunilla; Nilsson, Mats; Zeng, Yixin; Kipling, David

    2013-01-01

    DNA repair mechanisms are fundamental for B cell development, which relies on the somatic diversification of the immunoglobulin genes by V(D)J recombination, somatic hypermutation, and class switch recombination. Their failure is postulated to promote genomic instability and malignant transformation in B cells. By performing targeted sequencing of 73 key DNA repair genes in 29 B cell lymphoma samples, somatic and germline mutations were identified in various DNA repair pathways, mainly in diffuse large B cell lymphomas (DLBCLs). Mutations in mismatch repair genes (EXO1, MSH2, and MSH6) were associated with microsatellite instability, increased number of somatic insertions/deletions, and altered mutation signatures in tumors. Somatic mutations in nonhomologous end-joining (NHEJ) genes (DCLRE1C/ARTEMIS, PRKDC/DNA-PKcs, XRCC5/KU80, and XRCC6/KU70) were identified in four DLBCL tumors and cytogenetic analyses revealed that translocations involving the immunoglobulin-heavy chain locus occurred exclusively in NHEJ-mutated samples. The novel mutation targets, CHEK2 and PARP1, were further screened in expanded DLBCL cohorts, and somatic as well as novel and rare germline mutations were identified in 8 and 5% of analyzed tumors, respectively. By correlating defects in a subset of DNA damage response and repair genes with genomic instability events in tumors, we propose that these genes play a role in DLBCL lymphomagenesis. PMID:23960188

  20. Modeling the repair of DNA strand breaks caused by ?-radiation in a minichromosome

    NASA Astrophysics Data System (ADS)

    ?akomiec, K.; Kumala, S.; Hancock, R.; Rzeszowska-Wolny, J.; Fujarewicz, K.

    2014-08-01

    The objective of the studies described here was the development of a mathematical model which would fit experimental data for the repair of single and double strand breaks induced in DNA in living cells by exposure to ionizing radiation, and which would allow to better understand the processes of DNA repair. DNA breaks are believed to play the major role in radiation-induced lethality and formation of chromosome deletions, and are therefore crucial to the response of cells to radiotherapy. In an initial model which we reported on the basis of data for the repair of Epstein-Barr minichromosomes in irradiated Raji cells, we assumed that DNA breaks are induced only at the moment of irradiation and are later removed by repair systems. This work gives a development of that mathematical model which fits the experimental results more precisely and suggests strongly that DNA breaks are generated not only by direct irradiation but also later, probably by systems engaged in repair of oxidative damage.

  1. The role of the MORF/MRG family of genes in cell growth, differentiation, DNA repair, and thereby aging.

    PubMed

    Pena, Andreana N; Pereira-Smith, Olivia M

    2007-04-01

    The discovery that replicative cellular senescence is a dominant phenotype over immortality led to the discovery that there are at least four unique genetic subgroups of immortal cell lines that use distinct mechanistic pathways to evade cell cycle exit. Study of one of these genetic complementation groups demonstrated that one gene, MORF4, possessed the ability to induce senescence in group B cell lines. The MRG family of genes, of which MORF4 is a member, has since proven important for cellular aging, proliferation, positive and negative transcriptional regulation, and DNA damage repair. MRG15, the evolutionary ancestor of the family, is highly conserved in yeast, C. elegans, drosophila, plants, and mammals and has been implicated in chromatin remodeling in these species. Our proteomics studies have found that MRG15 is unique among mammalian genes in that it associates with both histone deacetylases and histone acetyl transferase complexes, and thus potentially plays a role in both transcriptional silencing and activation. Its knockout in mice is embryonic lethal, resulting in improper organogenesis, as well as cell proliferation and DNA damage repair defects. Future study of these genes will help clarify the role of chromatin remodeling in aging, cellular proliferation, and DNA damage repair. PMID:17460191

  2. Structure of DNA-liposome complexes

    SciTech Connect

    Lasic, D.D.; Strey, H.; Podgornik, R.; Stuart, M.C.A.; Frederik, P.M.

    1997-01-29

    Despite numerous studies and commericially available liposome kits, however, the structure of DNA-cationic liposome complexes is still not yet well understood. We have investigated the structure of these complexes using high-resolution cryo electron microscopy (EM) and small angle X-ray scattering (SAXS). 14 refs., 3 figs.

  3. Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair.

    PubMed

    Gursoy-Yuzugullu, Ozge; Ayrapetov, Marina K; Price, Brendan D

    2015-06-16

    The repair of DNA double-strand breaks (DSBs) requires open, flexible chromatin domains. The NuA4-Tip60 complex creates these flexible chromatin structures by exchanging histone H2A.Z onto nucleosomes and promoting acetylation of histone H4. Here, we demonstrate that the accumulation of H2A.Z on nucleosomes at DSBs is transient, and that rapid eviction of H2A.Z is required for DSB repair. Anp32e, an H2A.Z chaperone that interacts with the C-terminal docking domain of H2A.Z, is rapidly recruited to DSBs. Anp32e functions to remove H2A.Z from nucleosomes, so that H2A.Z levels return to basal within 10 min of DNA damage. Further, H2A.Z removal by Anp32e disrupts inhibitory interactions between the histone H4 tail and the nucleosome surface, facilitating increased acetylation of histone H4 following DNA damage. When H2A.Z removal by Anp32e is blocked, nucleosomes at DSBs retain elevated levels of H2A.Z, and assume a more stable, hypoacetylated conformation. Further, loss of Anp32e leads to increased CtIP-dependent end resection, accumulation of single-stranded DNA, and an increase in repair by the alternative nonhomologous end joining pathway. Exchange of H2A.Z onto the chromatin and subsequent rapid removal by Anp32e are therefore critical for creating open, acetylated nucleosome structures and for controlling end resection by CtIP. Dynamic modulation of H2A.Z exchange and removal by Anp32e reveals the importance of the nucleosome surface and nucleosome dynamics in processing the damaged chromatin template during DSB repair. PMID:26034280

  4. Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

    PubMed Central

    Han, Chunhua; Wani, Gulzar; Zhao, Ran; Qian, Jiang; Sharma, Nidhi; He, Jinshan; Zhu, Qianzheng; Wang, Qi-En; Wani, Altaf A

    2015-01-01

    Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3? side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4Cdt2. Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase ? and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4Cdt2 for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase ? to the damage site and completion of gap-filling DNA synthesis during the final stage of NER. PMID:25483071

  5. Uracil in duplex DNA is a substrate for the nucleotide incision repair pathway in human cells.

    PubMed

    Prorok, Paulina; Alili, Doria; Saint-Pierre, Christine; Gasparutto, Didier; Zharkov, Dmitry O; Ishchenko, Alexander A; Tudek, Barbara; Saparbaev, Murat K

    2013-09-24

    Spontaneous hydrolytic deamination of cytosine to uracil (U) in DNA is a constant source of genome instability in cells. This mutagenic process is greatly enhanced at high temperatures and in single-stranded DNA. If not repaired, these uracil residues give rise to C ? T transitions, which are the most common spontaneous mutations occurring in living organisms and are frequently found in human tumors. In the majority of species, uracil residues are removed from DNA by specific uracil-DNA glycosylases in the base excision repair pathway. Alternatively, in certain archaeal organisms, uracil residues are eliminated by apurinic/apyrimidinic (AP) endonucleases in the nucleotide incision repair pathway. Here, we characterized the substrate specificity of the major human AP endonuclease 1, APE1, toward U in duplex DNA. APE1 cleaves oligonucleotide duplexes containing a single U?G base pair; this activity depends strongly on the sequence context and the base opposite to U. The apparent kinetic parameters of the reactions show that APE1 has high affinity for DNA containing U but cleaves the DNA duplex at an extremely low rate. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of a U?G duplex generates the expected DNA fragments containing a 5'-terminal deoxyuridine monophosphate. The fact that U in duplex DNA is recognized and cleaved by APE1 in vitro suggests that this property of the exonuclease III family of AP endonucleases is remarkably conserved from Archaea to humans. We propose that nucleotide incision repair may act as a backup pathway to base excision repair to remove uracils arising from cytosine deamination. PMID:24023064

  6. Profiling DNA damage and repair capacity in sea urchin larvae and coelomocytes exposed to genotoxicants.

    PubMed

    Reinardy, Helena C; Bodnar, Andrea G

    2015-11-01

    The ability to protect the genome from harmful DNA damage is critical for maintaining genome stability and protecting against disease, including cancer. Many echinoderms, including sea urchins, are noted for the lack of neoplastic disease, but there are few studies investigating susceptibility to DNA damage and capacity for DNA repair in these animals. In this study, DNA damage was induced in adult sea urchin coelomocytes and larvae by exposure to a variety of genotoxicants [UV-C (0-3000 J/m(2)), hydrogen peroxide (0-10mM), bleomycin (0-300 µM) and methylmethanesulfonate (MMS, 0-30mM)] and the capacity for repair was measured over a 24-h period of recovery. Larvae were more sensitive than coelomocytes, with higher levels of initial DNA damage (fast micromethod) for all genotoxicants except MMS and increased levels of mortality 24h following treatment for all genotoxicants. The larvae that survived were able to efficiently repair damage within 24-h recovery. The ability to repair DNA damage differed depending on treatments, but both larvae and coelomocytes were able to most efficiently repair H2O2-induced damage. Time profiles of expression of a panel of DNA repair genes (ddb1, ercc1, xpc, xrcc1, pcna, ogg1, parp1, parp2, ape, brca1, rad51, xrcc2, xrcc3, xrcc4, xrcc5, xrcc6 and gadd45), throughout the period of recovery, showed greater gene induction in coelomocytes compared with larvae, with particularly high expression of xrcc1, ercc1, parp2 and pcna. The heterogeneous response of larvae to DNA damage may reflect a strategy whereby a subset of the population is equipped to withstand acute genotoxic stress, while the ability of coelomocytes to resist and repair DNA damage confirm their significant role in protection against disease. Consideration of DNA repair capacity is critical for understanding effects of genotoxicants on organisms, in addition to shedding light on life strategies and disease susceptibility. PMID:26175033

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

    PubMed Central

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

    2015-01-01

    Ionizing radiation generates DNA double-strand breaks (DSB) which, unless faithfully repaired, can generate chromosomal rearrangements in hematopoietic stem and/or progenitor cells (HSPC), potentially priming the cells towards a leukemic phenotype. Using an enhanced green fluorescent protein (EGFP)-based reporter system, we recently identified differences in the removal of enzyme-mediated DSB in human HSPC versus mature peripheral blood lymphocytes (PBL), particularly regarding homologous DSB repair (HR). Assessment of chromosomal breaks via premature chromosome condensation or ?H2AX foci indicated similar efficiency and kinetics of radiation-induced DSB formation and rejoining in PBL and HSPC. Prolonged persistence of chromosomal breaks was observed for higher LET charged particles which are known to induce more complex DNA damage compared to X-rays. Consistent with HR deficiency in HSPC observed in our previous study, we noticed here pronounced focal accumulation of 53BP1 after X-ray and carbon ion exposure (intermediate LET) in HSPC versus PBL. For higher LET, 53BP1 foci kinetics was similarly delayed in PBL and HSPC suggesting similar failure to repair complex DNA damage. Data obtained with plasmid reporter systems revealed a dose- and LET-dependent HR increase after X-ray, carbon ion and higher LET exposure, particularly in HR-proficient immortalized and primary lymphocytes, confirming preferential use of conservative HR in PBL for intermediate LET damage repair. HR measured adjacent to the leukemia-associated MLL breakpoint cluster sequence in reporter lines revealed dose dependency of potentially leukemogenic rearrangements underscoring the risk of leukemia-induction by radiation treatment. PMID:26618143

  8. Mitochondrial DNA damage and repair during ischemia-reperfusion injury of the heart.

    PubMed

    Bliksøen, M; Baysa, A; Eide, L; Bjørås, M; Suganthan, R; Vaage, J; Stensløkken, K O; Valen, G

    2015-01-01

    Ischemia-reperfusion (IR) injury of the heart generates reactive oxygen species that oxidize macromolecules including mitochondrial DNA (mtDNA). The 8-oxoguanine DNA glycosylase (OGG1) works synergistically with MutY DNA glycosylase (MYH) to maintain mtDNA integrity. Our objective was to study the functional outcome of lacking the repair enzymes OGG1 and MYH after myocardial IR and we hypothesized that OGG1 and MYH are important enzymes to preserve mtDNA and heart function after IR. Ex vivo global ischemia for 30min followed by 10min of reperfusion induced mtDNA damage that was removed within 60min of reperfusion in wild-type mice. After 60min of reperfusion the ogg1(-/-) mice demonstrated increased mtDNA copy number and decreased mtDNA damage removal suggesting that OGG1 is responsible for removal of IR-induced mtDNA damage and copy number regulation. mtDNA damage was not detected in the ogg1(-/-)/myh(-/-), inferring that adenine opposite 8-oxoguanine is an abundant mtDNA lesion upon IR. The level and integrity of mtDNA were restored in all genotypes after 35min of regional ischemia and six week reperfusion with no change in cardiac function. No consistent upregulation of other mitochondrial base excision repair enzymes in any of our knockout models was found. Thus repair of mtDNA oxidative base lesions may not be important for maintenance of cardiac function during IR injury in vivo. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease." PMID:25446179

  9. Reactive oxygen species are involved in nickel inhibition of dna repair

    SciTech Connect

    Lynn, S.; Yew, F.H.; Chen, K.S.; Jan, K.Y.

    1997-06-01

    Nickel has been shown to inhibit DNA repair in a way that may play a role in its toxicity. Since nickel treatment increases cellular reactive oxygen species (ROS), we have investigated the involvement of ROS in nickel inhibition of DNA repair. Inhibition of glutathione synthesis or catalase activity increased the enhancing effect of nickel on the cytotoxicity of ultraviolet (UV) light. Inhibition of catalase and glutathione peroxidase activities also enhanced the retardation effect of nickel on the rejoining of DNA strand breaks accumulated by hydroxyurea plus cytosine-{beta}-D-arabinofuranoside in UV-irradiated cells. Since DNA polymerization and ligation are involved in the DNA-break rejoining, we have investigated the effect of ROS on these two steps in an extract of Chinese hamster ovary cells. Nickel inhibition of the incorporation of ({sup 3}H)dTTP into the DNase l-activated calf thymus DNA was stronger than the ligation of poly(dA){center_dot}oligo(dT), whereas H{sub 2}O{sub 2} was more potent in inhibiting DNA ligation than DNA polymerization. Nickel, in the presence of H{sub 2}O{sub 2}, exhibited a synergistic inhibition on both DNA polymerization and ligation and caused protein fragmentation. In addition, glutathione could completely recover the inhibition by nickel or H{sub 2}O{sub 2} alone but only partially recover the inhibition by nickel plus H{sub 2}O{sub 2}. Therefore, nickel may bind to DNA-repair enzymes and generate oxygen-free radicals to cause protein degradation in situ. This irreversible damage to the proteins involved in DNA repair, replication, recombination, and transcription could be important for the toxic effects of nickel. 60 refs., 6 figs., 4 tabs.

  10. Two separable functions of Ctp1 in the early steps of meiotic DNA double-strand break repair

    PubMed Central

    Ma, Lijuan; Milman, Neta; Nambiar, Mridula; Smith, Gerald R.

    2015-01-01

    Meiotic programmed DNA double-strand break (DSB) repair is essential for crossing-over and viable gamete formation and requires removal of Spo11-oligonucleotide complexes from 5? ends (clipping) and their resection to generate invasive 3?-end single-stranded DNA (resection). Ctp1 (Com1, Sae2, CtIP homolog) acting with the Mre11-Rad50-Nbs1 (MRN) complex is required in both steps. We isolated multiple S. pombe ctp1 mutants deficient in clipping but proficient in resection during meiosis. Remarkably, all of the mutations clustered in or near the conserved CxxC or RHR motif in the C-terminal portion. The mutants tested, like ctp1?, were clipping-deficient by both genetic and physical assays­. But, unlike ctp1?, these mutants were recombination-proficient for Rec12 (Spo11 homolog)-independent break-repair and resection-proficient by physical assay. We conclude that the intracellular Ctp1 C-terminal portion is essential for clipping, while the N-terminal portion is sufficient for DSB end-resection. This conclusion agrees with purified human CtIP resection and endonuclease activities being independent. Our mutants provide intracellular evidence for separable functions of Ctp1. Some mutations truncate Ctp1 in the same region as one of the CtIP mutations linked to the Seckel and Jawad severe developmental syndromes, suggesting that these syndromes are caused by a lack of clipping at DSB ends that require repair. PMID:26130711

  11. DNA oxidation and DNA repair in gills of zebra mussels exposed to cadmium and benzo(a)pyrene.

    PubMed

    Michel, Cécile; Vincent-Hubert, Françoise

    2015-11-01

    Freshwater bivalve molluscs are considered as effective indicators of environmental pollution. The comet assay allows the detection of DNA damage such as DNA strand breaks and alkali-labile sites. The main oxidative lesion, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is a pre-mutagenic lesion, can be detected by the comet assay coupled with the hOGG1 DNA repair enzyme. With this modified assay we recently observed that BaP induced 8-oxodG lesions and with the modified comet-Fpg assay we observed that Cd induced oxidative DNA damage. The aim of this study was to determine the stability of DNA lesions in Cd and BaP exposed zebra mussels using the comet-hOGG1 assay. Mussels were exposed for 24 h to these two chemicals and then placed in clean water for 6 days. We observed that BaP (7, 12 and 18 µg/L) induced an increase of DNA strand break levels as soon as 6 h of exposure and that the two highest concentrations of BaP induced a low level of hOGG1-sensitive sites. After 2 days of depuration, BaP induced DNA lesions returned to the basal level, indicating an effective DNA repair. Cd (3, 32 and 81 µg/L) induced an increase of the DNA strand break levels and a low level of hOGG1-sensitive sites. This study revealed that BaP-induced DNA lesions are repaired more efficiently than Cd-induced DNA lesions. As the level of hOGG1 sensitive sites was increased in Cd and BaP exposed mussels, it seems that these chemicals induce 8-oxo-dG. PMID:26438356

  12. Reconstitution and structure of a bacterial Pnkp1RnlHen1 RNA repair complex

    SciTech Connect

    Wang, Pei; Selvadurai, Kiruthika; Huang, Raven H.

    2015-04-17

    Ribotoxins cleave essential RNAs for cell killing, and RNA repair neutralizes the damage inflicted by ribotoxins for cell survival. We report a new bacterial RNA repair complex that performs RNA repair linked to immunity. This new RNA repair complex is a 270-kDa heterohexamer composed of three proteins—Pnkp1, Rnl and Hen1—that are required to repair ribotoxin-cleaved RNA in vitro. The crystal structure of the complex reveals the molecular architecture of the heterohexamer as two rhomboid-shaped ring structures of Pnkp1–Rnl–Hen1 heterotrimer fused at the Pnkp1 dimer interface. The four active sites required for RNA repair are located on the inner rim of each ring. Furthermore, the architecture and the locations of the active sites of the Pnkp1–Rnl–Hen1 heterohexamer suggest an ordered series of repair reactions at the broken RNA ends that confer immunity to recurrent damage.

  13. Reconstitution and structure of a bacterial Pnkp1–Rnl–Hen1 RNA repair complex

    PubMed Central

    Wang, Pei; Selvadurai, Kiruthika; Huang, Raven H.

    2015-01-01

    Ribotoxins cleave essential RNAs for cell killing, and RNA repair neutralizes the damage inflicted by ribotoxins for cell survival. Here we report a new bacterial RNA repair complex that performs RNA repair linked to immunity. This new RNA repair complex is a 270-kDa heterohexamer composed of three proteins—Pnkp1, Rnl and Hen1—that are required to repair ribotoxin-cleaved RNA in vitro. The crystal structure of the complex reveals the molecular architecture of the heterohexamer as two rhomboid-shaped ring structures of Pnkp1–Rnl–Hen1 heterotrimer fused at the Pnkp1 dimer interface. The four active sites required for RNA repair are located on the inner rim of each ring. The architecture and the locations of the active sites of the Pnkp1–Rnl–Hen1 heterohexamer suggest an ordered series of repair reactions at the broken RNA ends that confer immunity to recurrent damage. PMID:25882814

  14. Ubiquitin-SUMO circuitry controls activated fanconi anemia ID complex dosage in response to DNA damage.

    PubMed

    Gibbs-Seymour, Ian; Oka, Yasuyoshi; Rajendra, Eeson; Weinert, Brian T; Passmore, Lori A; Patel, Ketan J; Olsen, Jesper V; Choudhary, Chunaram; Bekker-Jensen, Simon; Mailand, Niels

    2015-01-01

    We show that central components of the Fanconi anemia (FA) DNA repair pathway, the tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The ID complex is SUMOylated in a manner that depends on the ATR kinase, the FA ubiquitin ligase core complex, and the SUMO E3 ligases PIAS1/PIAS4 and is antagonized by the SUMO protease SENP6. SUMOylation of the ID complex drives substrate selectivity by triggering its polyubiquitylation by the SUMO-targeted ubiquitin ligase RNF4 to promote its removal from sites of DNA damage via the DVC1-p97 ubiquitin segregase complex. Deregulation of ID complex SUMOylation compromises cell survival following replication stress. Our results uncover a regulatory role for SUMOylation in the FA pathway, and we propose that ubiquitin-SUMO signaling circuitry is a mechanism that contributes to the balance of activated ID complex dosage at sites of DNA damage. PMID:25557546

  15. Characterization of DNA Repair Deficient Strains of Chlamydomonas reinhardtii Generated by Insertional Mutagenesis

    PubMed Central

    Plecenikova, Andrea; Slaninova, Miroslava; Riha, Karel

    2014-01-01

    While the mechanisms governing DNA damage response and repair are fundamentally conserved, cross-kingdom comparisons indicate that they differ in many aspects due to differences in life-styles and developmental strategies. In photosynthetic organisms these differences have not been fully explored because gene-discovery approaches are mainly based on homology searches with known DDR/DNA repair proteins. Here we performed a forward genetic screen in the green algae Chlamydomonas reinhardtii to identify genes deficient in DDR/DNA repair. We isolated five insertional mutants that were sensitive to various genotoxic insults and two of them exhibited altered efficiency of transgene integration. To identify genomic regions disrupted in these mutants, we established a novel adaptor-ligation strategy for the efficient recovery of the insertion flanking sites. Four mutants harbored deletions that involved known DNA repair factors, DNA Pol zeta, DNA Pol theta, SAE2/COM1, and two neighbouring genes encoding ERCC1 and RAD17. Deletion in the last mutant spanned two Chlamydomonas-specific genes with unknown function, demonstrating the utility of this approach for discovering novel factors involved in genome maintenance. PMID:25144319

  16. [Application of repair enzymes to improve the quality of degraded DNA templates for PCR amplification].

    PubMed

    Dovgerd, A P; Zharkov, D O

    2014-01-01

    PCR amplification of severely degraded DNA, including ancient DNA, forensic samples, and preparations from deeply processed foodstuffs, is a serious problem. Living organisms have a set of enzymes to repair lesions in their DNA. In this work, we have developed and characterized model systems of degraded high-molecular-weight DNA with a predominance of different types of damage. It was shown that depurination and oxidation of the model plasmid DNA template led to a decrease in the PCR efficiency. A set of enzymes performing a full cycle of excision repair of some lesions was determined. The treatment of model-damaged substrates with this set of enzymes resulted in an increased PCR product yield as compared with that of the unrepaired samples. PMID:25757334

  17. Both base excision repair and O-6-methylguanine-DNA methyltransferase protect against methylation-induced colon carcinogenesis

    E-print Network

    Samson, Leona D.

    Methylating agents are widely distributed environmental carcinogens. Moreover, they are being used in cancer chemotherapy. The primary target of methylating agents is DNA, and therefore, DNA repair is the first-line barrier ...

  18. Differential expression of DNA repair genes in Hispanic women with breast cancer.

    PubMed

    Matta, Jaime; Morales, Luisa; Dutil, Julie; Bayona, Manuel; Alvarez, Carolina; Suarez, Erick

    2013-02-01

    Previous studies have found a link between a low DNA repair capacity (DRC) level and increased risk for breast cancer (BC). A recent study by Matta et al. 2012 showed that women with BC have an average reduction of 60% in DRC compared to controls (P < 0.001). Using the same group of Hispanic women, we selected a subgroup of cases (n=35) and controls (n=2) who donated their tumors and normal tissue for performing molecular studies in order to 1) compare the expression of DNA repair genes in breast tissue between BC cases and controls without this disease, 2) assess the correlation between gene expression and DRC levels, 3) examine whether DRC levels are associated with tumor DNA repair gene expression profiling when women were stratified according to their hormone receptor status. DRC levels were measured in lymphocytes by means of a host-cell reactivation assay. Gene expression levels were measured in tumors by means of DNA microarray analysis. Twenty-one DNA repair genes were found to be differentially and significantly expressed in women with BC. Those candidate genes were CHEK2, EME1 (MMS4L), ERCC3 (XPB), FANCM, H2AFX (H2AX), HMGB1, HUS1, MBD4, NEIL3, PCNA, RAD1, RAD23B, RAD51, RAD54B, RDM1 (RAD52B), SHFM1 (DSS1), TP1, UBE2N (UBC13) and XRCC5 (Ku80). Most DNA repair genes (n=18 or 82%) were overexpressed, ranging from 3.76-fold (RDM1) to 1.47-fold (XRCC5). Only 4 genes (18%) were underexpressed, ranging from 62% (SAPCD1) to 25% (RAD23B). Statistically significant positive correlations between DRC level and gene expression were found for the RAD51, FANCB and FANCA genes. We discuss the clinical and translational significance of these findings. Our results support the usefulness of studying DNA repair as a measure of BC risk. This study also provides a list of candidate DNA repair genes that might be associated with dysregulation of DNA repair in breast cancer. PMID:25309843

  19. Opposing ISWI- and CHD-class chromatin remodeling activities orchestrate heterochromatic DNA repair

    PubMed Central

    Klement, Karolin; Luijsterburg, Martijn S.; Pinder, Jordan B.; Cena, Chad S.; Del Nero, Victor; Wintersinger, Christopher M.; Dellaire, Graham; van Attikum, Haico

    2014-01-01

    Heterochromatin is a barrier to DNA repair that correlates strongly with elevated somatic mutation in cancer. CHD class II nucleosome remodeling activity (specifically CHD3.1) retained by KAP-1 increases heterochromatin compaction and impedes DNA double-strand break (DSB) repair requiring Artemis. This obstruction is alleviated by chromatin relaxation via ATM-dependent KAP-1S824 phosphorylation (pKAP-1) and CHD3.1 dispersal from heterochromatic DSBs; however, how heterochromatin compaction is actually adjusted after CHD3.1 dispersal is unknown. In this paper, we demonstrate that Artemis-dependent DSB repair in heterochromatin requires ISWI (imitation switch)-class ACF1–SNF2H nucleosome remodeling. Compacted chromatin generated by CHD3.1 after DNA replication necessitates ACF1–SNF2H–mediated relaxation for DSB repair. ACF1–SNF2H requires RNF20 to bind heterochromatic DSBs, underlies RNF20-mediated chromatin relaxation, and functions downstream of pKAP-1–mediated CHD3.1 dispersal to enable DSB repair. CHD3.1 and ACF1–SNF2H display counteractive activities but similar histone affinities (via the plant homeodomains of CHD3.1 and ACF1), which we suggest necessitates a two-step dispersal and recruitment system regulating these opposing chromatin remodeling activities during DSB repair. PMID:25533843

  20. The DNA damage/repair cascade in glioblastoma cell lines after chemotherapeutic agent treatment.

    PubMed

    Annovazzi, Laura; Caldera, Valentina; Mellai, Marta; Riganti, Chiara; Battaglia, Luigi; Chirio, Daniela; Melcarne, Antonio; Schiffer, Davide

    2015-01-01

    Therapeutic resistance in glioblastoma multiforme (GBM) has been linked to a subpopulation of cells with stem cell-like properties, the glioma stem cells (GSCs), responsible for cancer progression and recurrence. This study investigated the in vitro cytotoxicity of three chemotherapeutics, temozolomide (TMZ), doxorubicin (Dox) and paclitaxel (PTX) on glioma cell lines, by analyzing the molecular mechanisms leading to DNA repair and cell resistance, or to cell death. The drugs were tested on 16 GBM cell lines, grown as neurospheres (NS) or adherent cells (AC), by studying DNA damage occurrence by Comet assay, the expression by immunofluorescence and western blotting of checkpoint/repair molecules and apoptosis. The three drugs were able to provoke a genotoxic injury and to inhibit dose- and time-dependently cell proliferation, more evidently in AC than in NS. The first cell response to DNA damage was the activation of the damage sensors (p-ATM, p-53BP1, ?-H2AX), followed by repair effectors; the expression of checkpoint/repair molecules appeared higher in NS than in AC. The non-homologous repair pathway (NHEJ) seemed more involved than the homologous one (HR). Apoptosis occurred after long treatment times, but only a small percentage of cells in NS underwent death, even at high drug concentration, whereas most cells survived in a quiescent state and resumed proliferation after drug removal. In tumor specimens, checkpoint/repair proteins were constitutively expressed in GBMs, but not in low-grade gliomas. PMID:25892134

  1. The DNA damage/repair cascade in glioblastoma cell lines after chemotherapeutic agent treatment

    PubMed Central

    ANNOVAZZI, LAURA; CALDERA, VALENTINA; MELLAI, MARTA; RIGANTI, CHIARA; BATTAGLIA, LUIGI; CHIRIO, DANIELA; MELCARNE, ANTONIO; SCHIFFER, DAVIDE

    2015-01-01

    Therapeutic resistance in glioblastoma multiforme (GBM) has been linked to a subpopulation of cells with stem cell-like properties, the glioma stem cells (GSCs), responsible for cancer progression and recurrence. This study investigated the in vitro cytotoxicity of three chemotherapeutics, temozolomide (TMZ), doxorubicin (Dox) and paclitaxel (PTX) on glioma cell lines, by analyzing the molecular mechanisms leading to DNA repair and cell resistance, or to cell death. The drugs were tested on 16 GBM cell lines, grown as neurospheres (NS) or adherent cells (AC), by studying DNA damage occurrence by Comet assay, the expression by immunofluorescence and western blotting of checkpoint/repair molecules and apoptosis. The three drugs were able to provoke a genotoxic injury and to inhibit dose- and time-dependently cell proliferation, more evidently in AC than in NS. The first cell response to DNA damage was the activation of the damage sensors (p-ATM, p-53BP1, ?-H2AX), followed by repair effectors; the expression of checkpoint/repair molecules appeared higher in NS than in AC. The non-homologous repair pathway (NHEJ) seemed more involved than the homologous one (HR). Apoptosis occurred after long treatment times, but only a small percentage of cells in NS underwent death, even at high drug concentration, whereas most cells survived in a quiescent state and resumed proliferation after drug removal. In tumor specimens, checkpoint/repair proteins were constitutively expressed in GBMs, but not in low-grade gliomas. PMID:25892134

  2. RNF20-SNF2H Pathway of Chromatin Relaxation in DNA Double-Strand Break Repair

    PubMed Central

    Kato, Akihiro; Komatsu, Kenshi

    2015-01-01

    Rapid progress in the study on the association of histone modifications with chromatin remodeling factors has broadened our understanding of chromatin dynamics in DNA transactions. In DNA double-strand break (DSB) repair, the well-known mark of histones is the phosphorylation of the H2A variant, H2AX, which has been used as a surrogate marker of DSBs. The ubiquitylation of histone H2B by RNF20 E3 ligase was recently found to be a DNA damage-induced histone modification. This modification is required for DSB repair and regulated by a distinctive pathway from that of histone H2AX phosphorylation. Moreover, the connection between H2B ubiquitylation and the chromatin remodeling activity of SNF2H has been elucidated. In this review, we summarize the current knowledge of RNF20-mediated processes and the molecular link to H2AX-mediated processes during DSB repair. PMID:26184323

  3. Beyond xeroderma pigmentosum: DNA damage and repair in an ecological context. A tribute to James E. Cleaver.

    PubMed

    Karentz, Deneb

    2015-01-01

    The ability to repair DNA is a ubiquitous characteristic of life on Earth and all organisms possess similar mechanisms for dealing with DNA damage, an indication of a very early evolutionary origin for repair processes. James E. Cleaver's career (initiated in the early 1960s) has been devoted to the study of mammalian ultraviolet radiation (UVR) photobiology, specifically the molecular genetics of xeroderma pigmentosum and other human diseases caused by defects in DNA damage recognition and repair. This work by Jim and others has influenced the study of DNA damage and repair in a variety of taxa. Today, the field of DNA repair is enhancing our understanding of not only how to treat and prevent human disease, but is providing insights on the evolutionary history of life on Earth and how natural populations are coping with UVR-induced DNA damage from anthropogenic changes in the environment such as ozone depletion. PMID:25395165

  4. Excision repair of UV-damaged plasmid DNA in Xenopus oocytes is mediated by DNA polymerase alpha (and/or delta).

    PubMed Central

    Saxena, J K; Hays, J B; Ackerman, E J

    1990-01-01

    We studied DNA repair by injecting plasmids containing random pyrimidine dimers into Xenopus oocytes. We demonstrated excision repair by recovering plasmids and analyzing them with T4 UV endonuclease treatment and alkaline agarose gel electrophoresis. The mechanism for excision repair of these plasmids appears to be processive, rather than distributive, since repair occurs in 'all or none' fashion. At less than 4-5 dimers/plasmid, nearly all repair occurs within 4-6 hours (approximately 10(10) dimers repaired per oocyte); the oocyte, therefore, has abundant repair activity. Specific antibodies and inhibitors were used to determine enzymes involved in repair. We conclude that DNA polymerase alpha (and/or delta) is required because repair is inhibited by antibodies to human DNA polymerase alpha, as well as by aphidicolin, an inhibitor of polymerases alpha (and/or delta). Repair was not inhibited by hydroxyurea, cytosine beta-D-arabinofuranoside, or inhibitors of topoisomerase II (novobiocin). Oocyte repair does not activate semi-conservative DNA replication, nor is protein synthesis required. Photoreactivation cannot account for repair because dimer removal is independent of exogenous light. Images PMID:2175436

  5. Repair of uv damaged DNA: Genes and proteins of yeast and human. Progress report, November 1, 1991--April 15, 1992

    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.

  6. [Role of DNA repair genes in radiation-induced changes of lifespan of Drosophila melanogaster].

    PubMed

    Shilova, L A; Pliusnina, E N; Zemskaia, N V; Moskalev, A A

    2014-01-01

    One of the main effects of various stress factors, including ionizing radiation, is DNA damage. Accumulation of DNA damage and somatic mutations in the somatic tissues is regarded as one of the basic mechanisms of aging. We have developed an approach to the study of molecular and genetic mechanisms of radioadaptation, which is based on the analysis of changes in the lifespan of Drosophila with a transformed genotype. In this study we investigated the radioadaptive response and hormesis by radiation-induced changed of the lifespan of different strains of Drosophila melanogaster, such as a wild type strain Canton-Sand strains with mutations in DNA damage response gene (homologue of GADD45), excision repair genes (homologues of XPF, XPC, PCNA) and double-strand breaks repair genes (homologues of RAD54, XRCC3, BLM). The exposure to irradiation at the dose rate of 40 cGy was performed chronically through the stages of fly development; an acute exposure at the dose rate of 30 Gy was applied to the adult stages of flies. Also, we investigated the resistance to acute gamma-radiation of Drosophila with conditional ubiquitous overexpression of genes that are involved in DNA damage recognition (homologues of GADD45, HUS1, CHK2), excision repair (homologues of XPF, XPC, AP-endonuclease-1) and double-strand break repair (homologues of BRCA2, XRCC3, KU80, WRNexo). In the wild type strain Canton-S, manifestation of the radioadaptive response and radiation hormesis were observed. In individuals with DNA repair gene mutations, no radioadaptive response was observed, or observed to a lesser extent than in wild type flies. Mifepristone--inducible transgene activation does not lead to an increase in resistance to acute irradiation by the parameters of lifespan of Drosophila. Overexpression of DNA repair genes led to a sharp decline in lifespan also in the absence of irradiation. PMID:25775840

  7. The prostate cancer risk locus at 10q11 is associated with DNA repair capacity.

    PubMed

    Rinckleb, Antje E; Surowy, Harald M; Luedeke, Manuel; Varga, Dominic; Schrader, Mark; Hoegel, Josef; Vogel, Walther; Maier, Christiane

    2012-08-01

    Genome-wide association studies (GWAS) have identified several single nucleotide polymorphisms (SNPs) that mildly predict prostate cancer risk. These SNPs are local tagging markers for causal gene alterations. Consideration of candidate genes in the tagged regions would be facilitated by additional information on the particular pathomechanisms which contribute to the observed risk increase. In this study we test for an association of prostate cancer tagging SNPs with alterations in DNA repair capacity, a phenotype that is frequently involved in cancer predisposition. DNA repair capacity was assessed on blood lymphocytes from 128 healthy probands after ionizing irradiation. We used the micronucleus (MN) assay to determine the cellular DNA double-strand break repair capacity and flow cytometry to measure damage induced mitotic delay (MD). Probands were genotyped for a panel of 14 SNPs, each representing an independent prostate cancer risk locus previously identified by GWAS. Associations between germline variants and DNA repair capacity were found for the SNPs rs1512268 (8p21), rs6983267 (8q24) and rs10993994 (10q11). The most significant finding was an association of homozygous rs10993994 T-allele carriers with a lower MN frequency (p=0.0003) and also a decreased MD index (p=0.0353). Cells with prostate cancer risk alleles at rs10993994 seem to cope more efficiently with DNA double strand breaks (less MN) in a shorter time (decreased MD index). This intriguing finding imposes concern about the accuracy of repair, with respect to the cancer risk that is mediated by T genotypes. To date, MSMB (microseminoprotein ?) is favored as the causal gene at the 10q11 risk locus, since it was the first candidate gene known to be expressionally altered by rs10993994. Based on the present observation, candidate genes from the contexts of DNA repair and apoptosis may be more promising targets for expression studies with respect to the rs10993994 genotype. PMID:22677538

  8. Recent progress in characterizing human DNA repair genes cloned using rodent cell lines

    SciTech Connect

    Thompson, L.H.; Caldecott, K.W.; Brookman, K.W.

    1991-07-15

    Knowledge of DNA repair processes in human cells is essential for developing rational approaches toward the treatment of cancer as well as understanding its origin and that of human heritable disorders. A detailed knowledge of the individual repair genes and their encoded proteins should eventually result in our understanding the action of repair pathways on various classes of DNA damage. In mammalian cells as in other organisms, the characterization of repair genes requires having mutations affecting those genes. A considerable number of mutant cell lines have been produced, mainly in hamster cells that have altered responses to ultraviolet (UV) and/or ionizing radiation damage. The use of rodent mutant cell lines has greatly accelerated progress in isolating characterizing human DNA repair genes. This advance results from several genetic properties of the hamster cell lines (particularly CHO cells), including their ability to integrate relatively large quantities of foreign DNA. A considerable variety of interesting mutants have been obtained in V79 hamster cells. However, as yet there is insufficient knowledge of this cell line to know whether mutant derivatives will be efficient recipients for gene transfer.

  9. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation.

    PubMed

    Blunt, T; Finnie, N J; Taccioli, G E; Smith, G C; Demengeot, J; Gottlieb, T M; Mizuta, R; Varghese, A J; Alt, F W; Jeggo, P A; Jackson, S P

    1995-03-10

    Murine cells homozygous for the severe combined immune deficiency mutation (scid) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair. Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PKcs, the catalytic subunit of this enzyme. Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PKcs gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PKcs is encoded by the XRCC7 gene. As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes. PMID:7889575

  10. Atomic Simulation of Complex DNA DSBs and the Interactions with the Ku70/80 Heterodimer

    NASA Technical Reports Server (NTRS)

    Hu, Shaowen; Cucinotta, Francis A.

    2011-01-01

    DNA double strand breaks (DSBs) induced by ionizing radiation (IR) usually contain modified bases such as 8-oxo-7,8-dihydroguanine (8-oxoG) and thymine glycol, apurinic/apyrimidinic (AP) sites, 2-deoxyribonolactone, or single-strand breaks (SSBs). The presence of such lesions in close proximity to the DSB terminus makes the DNA nicks more difficult to repair and rejoin than endogenously induced simple DSBs, and as such a major determinant of the biological effects of high linear energy transfer (LET) radiation as encountered in space travel. In this study we conducted molecular dynamics simulations on a series of DNA duplexes with various complex lesions of 8-oxoG and AP sites, in an effort to investigate the effects of such lesions to the structural integrity and stability of DNA after insulted by IR. We also simulated the interaction of such complex DSBs with the Ku70/80 heterodimer, the first protein in mammalian cells to embark the non-homologous end joining (NHEJ) DNA repair pathway. The results indicate, compared to DNA with simple DSBs, the complex lesions can enhance the hydrogen bonds opening rate at the DNA terminus, and increase the mobility of the whole duplex, thus they present more deleterious effects to the genome integrity if not captured and repaired promptly in cells. Simulations also demonstrate the binding of Ku drastically reduces structural disruption and flexibility caused by the complex lesions, and the interactions of Ku with complex DSBs have a different potential energy landscape from the bound structure with simple DSB. In all complex DSBs systems, the binding of DSB terminus with Ku70 is softened while the binding of the middle duplex with Ku80 is tightened. This energy shift may help the Ku protein to secure at the DSB terminus for a longer time, so that other end processing factors or repair pathways can proceed at the lesions before NHEJ repair process starts. These atomic simulations may provide valuable new insight into the selective action of repair proteins on damaged DNA.

  11. SWI/SNF complex deficiency and mismatch repair protein expression in undifferentiated and dedifferentiated endometrial carcinoma.

    PubMed

    Stewart, Colin J R; Crook, Maxine L

    2015-08-01

    Undifferentiated endometrial carcinoma (UEC) is a relatively uncommon but clinically aggressive uterine malignancy. In common with a subset of poorly differentiated carcinomas arising in other sites, UEC may exhibit rhabdoid morphology and be associated with a low-grade tumour component (dedifferentiated carcinoma). Recent studies have implicated inactivation of the SWI/SNF complex subunits in the aforementioned extrauterine tumours. Therefore we have examined INI1 (SMARCB1), BRG1 (SMARCA4), and BAF250a (ARID1A) immunostaining, and also expression of the DNA mismatch repair (MMR) proteins MLH1, PMS2, MSH2 and MSH6 in 22 UEC, seventeen of which were dedifferentiated. Abnormal SWI/SNF subunit expression was detected in four dedifferentiated carcinomas including three with loss of BRG1 staining limited to the undifferentiated tumour component and one case with loss of INI1 expression in both low- and high-grade elements; the latter case also showed BAF250a deficiency in the undifferentiated tumour cells. Abnormal MMR protein expression was identified in 13 tumours (59%) including nine with concurrent loss of MLH1 and PMS2. These findings suggest that SWI/SNF subunit alterations may play a role in the progression/ dedifferentiation of endometrial carcinoma, and that SWI/SNF and MMR protein deficiencies may act synergistically in deregulating DNA repair mechanisms in these tumours. PMID:26126041

  12. Surgical technique for repair of complex anterior skull base defects

    PubMed Central

    Reinard, Kevin; Basheer, Azam; Jones, Lamont; Standring, Robert; Lee, Ian; Rock, Jack

    2015-01-01

    Background: Modern microsurgical techniques enable en bloc resection of complex skull base tumors. Anterior cranial base surgery, particularly, has been associated with a high rate of postoperative cerebrospinal fluid (CSF) leak, meningitis, intracranial abscess, and pneumocephalus. We introduce simple modifications to already existing surgical strategies designed to minimize the incidence of postoperative CSF leak and associated morbidity and mortality. Methods: Medical records from 1995 to 2013 were reviewed in accordance with the Institutional Review Board. We identified 21 patients who underwent operations for repair of large anterior skull base defects following removal of sinonasal or intracranial pathology using standard craniofacial procedures. Patient charts were screened for CSF leak, meningitis, or intracranial abscess formation. Results: A total of 15 male and 6 female patients with an age range of 26–89 years were included. All patients were managed with the same operative technique for reconstruction of the frontal dura and skull base defect. Spinal drainage was used intraoperatively in all cases but the lumbar drain was removed at the end of each case in all patients. Only one patient required re-operation for repair of persistent CSF leak. None of the patients developed meningitis or intracranial abscess. There were no perioperative mortalities. Median follow-up was 10 months. Conclusion: The layered reconstruction of large anterior cranial fossa defects resulted in postoperative CSF leak in only 5% of the patients and represents a simple and effective closure option for skull base surgeons. PMID:25722926

  13. DNA polymerases ? and ? cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae.

    PubMed

    Meyer, Damon; Fu, Becky Xu Hua; Heyer, Wolf-Dietrich

    2015-12-15

    Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol ?) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol ? proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol ? and DNA polymerase 4 (Pol ?), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol ? showed faster kinetics associating with MMEJ substrates following DSB induction than Pol ?. The association of Pol ? depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol ? recruitment was diminished in cells lacking Pol ?. These data suggest cooperative involvement of both polymerases in MMEJ. PMID:26607450

  14. DNA polymerases ? and ? cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

    PubMed Central

    Meyer, Damon; Fu, Becky Xu Hua; Heyer, Wolf-Dietrich

    2015-01-01

    Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol ?) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol ? proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol ? and DNA polymerase 4 (Pol ?), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol ? showed faster kinetics associating with MMEJ substrates following DSB induction than Pol ?. The association of Pol ? depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol ? recruitment was diminished in cells lacking Pol ?. These data suggest cooperative involvement of both polymerases in MMEJ. PMID:26607450

  15. Modulation of DNA Damage and Repair Pathways by Human Tumour Viruses

    PubMed Central

    Hollingworth, Robert; Grand, Roger J

    2015-01-01

    With between 10% and 15% of human cancers attributable to viral infection, there is great interest, from both a scientific and clinical viewpoint, as to how these pathogens modulate host cell functions. Seven human tumour viruses have been identified as being involved in the development of specific malignancies. It has long been known that the introduction of chromosomal aberrations is a common feature of viral infections. Intensive research over the past two decades has subsequently revealed that viruses specifically interact with cellular mechanisms responsible for the recognition and repair of DNA lesions, collectively known as the DNA damage response (DDR). These interactions can involve activation and deactivation of individual DDR pathways as well as the recruitment of specific proteins to sites of viral replication. Since the DDR has evolved to protect the genome from the accumulation of deleterious mutations, deregulation is inevitably associated with an increased risk of tumour formation. This review summarises the current literature regarding the complex relationship between known human tumour viruses and the DDR and aims to shed light on how these interactions can contribute to genomic instability and ultimately the development of human cancers. PMID:26008701

  16. DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae

    SciTech Connect

    Budd, M.E.; Wittrup, K.D.; Bailey, J.E.; Campbell, J.L.

    1989-02-01

    We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

  17. Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

    PubMed Central

    Reid, Dylan A.; Keegan, Sarah; Leo-Macias, Alejandra; Watanabe, Go; Strande, Natasha T.; Chang, Howard H.; Oksuz, Betul Akgol; Fenyo, David; Lieber, Michael R.; Ramsden, Dale A.; Rothenberg, Eli

    2015-01-01

    Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation. PMID:25941401

  18. Interactome analysis identifies a new paralogue of XRCC4 in non-homologous end joining DNA repair pathway

    PubMed Central

    Xing, Mengtan; Yang, Mingrui; Huo, Wei; Feng, Feng; Wei, Leizhen; Jiang, Wenxia; Ning, Shaokai; Yan, Zhenxin; Li, Wen; Wang, Qingsong; Hou, Mei; Dong, Chunxia; Guo, Rong; Gao, Ge; Ji, Jianguo; Zha, Shan; Lan, Li; Liang, Huanhuan; Xu, Dongyi

    2015-01-01

    Non-homologous end joining (NHEJ) is a major pathway to repair DNA double-strand breaks (DSBs), which can display different types of broken ends. However, it is unclear how NHEJ factors organize to repair diverse types of DNA breaks. Here, through systematic analysis of the human NHEJ factor interactome, we identify PAXX as a direct interactor of Ku. The crystal structure of PAXX is similar to those of XRCC4 and XLF. Importantly, PAXX-deficient cells are sensitive to DSB-causing agents. Moreover, epistasis analysis demonstrates that PAXX functions together with XLF in response to ionizing radiation-induced complex DSBs, whereas they function redundantly in response to Topo2 inhibitor-induced simple DSBs. Consistently, PAXX and XLF coordinately promote the ligation of complex but not simple DNA ends in vitro. Altogether, our data identify PAXX as a new NHEJ factor and provide insight regarding the organization of NHEJ factors responding to diverse types of DSB ends. PMID:25670504

  19. Hepatitis B virus X protein interacts with a probable cellular DNA repair protein.

    PubMed Central

    Lee, T H; Elledge, S J; Butel, J S

    1995-01-01

    The mechanism of action of hepatitis B virus (HBV) X protein in transcriptional transactivation and in tumorigenesis remains obscure. We have used the yeast two-hybrid system to identify a cellular protein that can interact with HBV X protein. This protein, designated X-associated protein 1 (XAP-1), is a human homolog of the UV-damaged DNA-binding protein (UV-DDB) recovered from a monkey cell cDNA library. UV-DDB is presumed to be involved in DNA repair. The interaction between X protein and XAP-1 protein was verified by immunoprecipitation of yeast cell lysates expressing both proteins and by in vitro mixing with X protein expressed as a glutathione S-transferase fusion protein and XAP-1 protein either in HeLa cell extracts or synthesized by in vitro translation. We speculate that the interaction of X protein with a DNA repair protein may recruit cellular proteins to repair the partially double-stranded HBV genome or may modify cellular transcription processes. An effect on the cellular DNA repair system may explain a cofactor role for HBV in liver cancer development. PMID:7815490

  20. Low doses of ochratoxin A induce micronucleus formation and delay DNA repair in human lymphocytes.

    PubMed

    González-Arias, Cyndia A; Benitez-Trinidad, Alma B; Sordo, Monserrat; Robledo-Marenco, Lourdes; Medina-Díaz, Irma M; Barrón-Vivanco, Briscia S; Marín, Sonia; Sanchis, Vicente; Ramos, Antonio J; Rojas-García, Aurora E

    2014-12-01

    The contamination of food commodities by fungal toxins has attracted great interest because many of these mycotoxins are responsible for different diseases, including cancer and other chronic illnesses. Ochratoxin A (OTA) is a mycotoxin naturally present in food, and long-term exposure to food contaminated with low levels of OTA has been associated with renal cancer. In the present study, the cytotoxicity, cytostaticity, and genotoxicity of OTA (0.075-15?µM) in human lymphocytes were evaluated. A comet assay, a modified comet assay (DNA repair assay), which uses N-hydroxyurea (NHU) to detect non-repaired lesions produced by OTA, and a cytokinesis-blocked micronucleus assay were used. Treatments with OTA were not cytotoxic, but OTA caused a cytostatic effect in human lymphocytes at a concentration of 15?µM. OTA (0.075-5?µM) produced a slight increase in the percentage of DNA in the comets and a delay in the DNA repair capacity of the lymphocytes. Micronucleus (MN) induction was observed at OTA concentrations of 1.5 and 5?µM. Our results indicate that OTA induces DNA stable damage at low doses that are neither cytotoxic nor cytostatic, and OTA delays the DNA repair kinetics. These findings indicate that OTA affects two pivotal events in the carcinogenesis pathway. PMID:25455892

  1. Single nucleotide polymorphisms in DNA repair genes and prostate cancer risk

    PubMed Central

    Park, Jong; Huang, Yifan; Sellers, Thomas A.

    2014-01-01

    The specific causes of prostate cancer are not known. However, multiple etiological factors, including genetic profile, metabolism of steroid hormones, nutrition, chronic inflammation, family history of prostate cancer, and environmental exposures are thought to play significant roles. Variations in exposure to these risk factors may explain inter-individual differences in prostate cancer risk. However, regardless of the precise mechanism(s), a robust DNA repair capacity may mitigate any risks conferred by mutations from these risk factors. Numerous single nucleotide polymorphisms (SNPs) in DNA repair genes have been found, and studies of these SNPs and prostate cancer risk are critical to understanding the response of prostate cells to DNA damage. A few SNPs in DNA repair genes cause significantly increased risk of prostate cancer, however, in most cases, the effects are moderate and often depend upon interactions among the risk alleles of several genes in a pathway or with other environmental risk factors. This report reviews the published epidemiologic literature on the association of SNPs in genes involved in DNA repair pathways and prostate cancer risk. PMID:19109789

  2. Ada response – a strategy for repair of alkylated DNA in bacteria

    PubMed Central

    Mielecki, Damian; Grzesiuk, El?bieta

    2014-01-01

    Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N3-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N1-methyladenine (1meA) and N3-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O6-methylguanine (O6meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA. PMID:24810496

  3. Enzymology of repair of DNA adducts produced by N-nitroso compounds

    SciTech Connect

    Setlow, R.B.; Cao, E.H.; Delihas, N.C.

    1983-01-01

    The biological effects of DNA adducts depend on their nature, and on their half-lives relative to the rates of DNA replication and transcription. Their half-lives are determined by the rates of spontaneous decay, such as depurination, and the rates of enzymatic repair of the adducts or their decay products. The principle modes of repair of methylating and ethylating agents are by glycosylase catalyzed depurination of 7-alkylguanine and 3-alkyladenine and by the dealkalation of O/sup 6/-alkylguanine. Repair by dealkylation cannot be detected by the standard methods used to measure DNA repair, but it is easy to estimate the acceptor activity in cell extracts by measuring the transfer of radioactive O/sup 6/-alkyl groups in an exogenous DNA to protein. In extracts of cells treated with alkylating agents the activity is depressed because the endogenous DNA is rapidly dealkylated, using up the acceptor activity. In many cell types the decrease in activity is followed by an increase to the normal constitutive level. In other cells there is no such adaptive response. Differences in constitutive levels of methyl accepting activity in extracts of human lymphocytes and on the acceptor activity in lung macrophages from smokers (low activity) and non-smokers (high activity) have been observed. 46 references.

  4. Intercalation processes of copper complexes in DNA.

    PubMed

    Galindo-Murillo, Rodrigo; García-Ramos, Juan Carlos; Ruiz-Azuara, Lena; Cheatham, Thomas E; Cortés-Guzmán, Fernando

    2015-06-23

    The family of anticancer complexes that include the transition metal copper known as Casiopeínas® shows promising results. Two of these complexes are currently in clinical trials. The interaction of these compounds with DNA has been observed experimentally and several hypotheses regarding the mechanism of action have been developed, and these include the generation of reactive oxygen species, phosphate hydrolysis and/or base-pair intercalation. To advance in the understanding on how these ligands interact with DNA, we present a molecular dynamics study of 21 Casiopeínas with a DNA dodecamer using 10 ?s of simulation time for each compound. All the complexes were manually inserted into the minor groove as the starting point of the simulations. The binding energy of each complex and the observed representative type of interaction between the ligand and the DNA is reported. With this extended sampling time, we found that four of the compounds spontaneously flipped open a base pair and moved inside the resulting cavity and four compounds formed stacking interactions with the terminal base pairs. The complexes that formed the intercalation pocket led to more stable interactions. PMID:25958394

  5. Intercalation processes of copper complexes in DNA

    PubMed Central

    Galindo-Murillo, Rodrigo; García-Ramos, Juan Carlos; Ruiz-Azuara, Lena; Cheatham, Thomas E.; Cortés-Guzmán, Fernando

    2015-01-01

    The family of anticancer complexes that include the transition metal copper known as Casiopeínas® shows promising results. Two of these complexes are currently in clinical trials. The interaction of these compounds with DNA has been observed experimentally and several hypotheses regarding the mechanism of action have been developed, and these include the generation of reactive oxygen species, phosphate hydrolysis and/or base-pair intercalation. To advance in the understanding on how these ligands interact with DNA, we present a molecular dynamics study of 21 Casiopeínas with a DNA dodecamer using 10 ?s of simulation time for each compound. All the complexes were manually inserted into the minor groove as the starting point of the simulations. The binding energy of each complex and the observed representative type of interaction between the ligand and the DNA is reported. With this extended sampling time, we found that four of the compounds spontaneously flipped open a base pair and moved inside the resulting cavity and four compounds formed stacking interactions with the terminal base pairs. The complexes that formed the intercalation pocket led to more stable interactions. PMID:25958394

  6. Unstable Simple Elbow Dislocation Treated with the Repair of Lateral Collateral Ligament Complex

    PubMed Central

    Yi, Jin Woong; Lee, Jung Bum; Lee, Dae Hee; Park, Won Keun; Kim, Sun Joong

    2015-01-01

    Background Unstable simple elbow dislocation (USED) repair is challenged by the maintenance of joint reduction; hence, primary repair or reconstruction of disrupted ligaments is required to maintain the congruency and allow early motion of the elbow. We evaluated the effectiveness and the outcome of lateral collateral ligament (LCL) complex repair with additional medial collateral ligament (MCL) repair in cases of USED. Methods We retrospectively reviewed 21 cases of diagnosed USED without fractures around the elbow that were treated with primary ligament repair. In all cases, anatomical repair of LCL complex with or without common extensor origin was performed using suture anchor and the bone tunnel method. Next, the instability and congruency of elbow for a full range of motion were evaluated under the image intensifier. MCL was repaired only if unstable or incongruent elbow was observed. Clinical outcomes were evaluated using the Mayo elbow performance score (MEPS) and radiographic outcomes on last follow-up images. Results All cases achieved a stable elbow on radiographic and clinical results. LCL complex repair alone was sufficient to obtain the stable elbow in 17 of 21 cases. Four cases required additional MCL repair after restoration of the LCL complex. The overall mean MEPS was 91 (range, 70 to 100): excellent in 12 cases, good in 7 cases, and fair in 2 cases. All 17 cases with LCL complex repair only and 2 of 4 cases with additional MCL repair had excellent or good results by MEPS. Conclusions USED requires surgical treatment to achieve a congruent and stable joint. If the repair of lateral stabilizer such as LCL complex acquires enough joint stability to maintain a full range of motion, it may not be necessary to repair the medial stabilizer in all cases of USED. PMID:26217472

  7. DNA repair efficiency in germ cells and early mouse embryos and consequences for radiation-induced transgenerational genomic damage

    SciTech Connect

    Marchetti, Francesco; Wyrobek, Andrew J.

    2009-01-18

    Exposure to ionizing radiation and other environmental agents can affect the genomic integrity of germ cells and induce adverse health effects in the progeny. Efficient DNA repair during gametogenesis and the early embryonic cycles after fertilization is critical for preventing transmission of DNA damage to the progeny and relies on maternal factors stored in the egg before fertilization. The ability of the maternal repair machinery to repair DNA damage in both parental genomes in the fertilizing egg is especially crucial for the fertilizing male genome that has not experienced a DNA repair-competent cellular environment for several weeks prior to fertilization. During the DNA repair-deficient period of spermatogenesis, DNA lesions may accumulate in sperm and be carried into the egg where, if not properly repaired, could result in the formation of heritable chromosomal aberrations or mutations and associated birth defects. Studies with female mice deficient in specific DNA repair genes have shown that: (i) cell cycle checkpoints are activated in the fertilized egg by DNA damage carried by the sperm; and (ii) the maternal genotype plays a major role in determining the efficiency of repairing genomic lesions in the fertilizing sperm and directly affect the risk for abnormal reproductive outcomes. There is also growing evidence that implicates DNA damage carried by the fertilizing gamete as a mediator of postfertilization processes that contribute to genomic instability in subsequent generations. Transgenerational genomic instability most likely involves epigenetic mechanisms or error-prone DNA repair processes in the early embryo. Maternal and embryonic DNA repair processes during the early phases of mammalian embryonic development can have far reaching consequences for the genomic integrity and health of subsequent generations.

  8. The Eukaryotic Mismatch Recognition Complexes Track with the Replisome during DNA Synthesis

    PubMed Central

    Haye, Joanna E.; Gammie, Alison E.

    2015-01-01

    During replication, mismatch repair proteins recognize and repair mispaired bases that escape the proofreading activity of DNA polymerase. In this work, we tested the model that the eukaryotic mismatch recognition complex tracks with the advancing replisome. Using yeast, we examined the dynamics during replication of the leading strand polymerase Pol? using Pol2 and the eukaryotic mismatch recognition complex using Msh2, the invariant protein involved in mismatch recognition. Specifically, we synchronized cells and processed samples using chromatin immunoprecipitation combined with custom DNA tiling arrays (ChIP-chip). The Pol? signal was not detectable in G1, but was observed at active origins and replicating DNA throughout S-phase. The Pol? signal provided the resolution to track origin firing timing and efficiencies as well as replisome progression rates. By detecting Pol? and Msh2 dynamics within the same strain, we established that the mismatch recognition complex binds origins and spreads to adjacent regions with the replisome. In mismatch repair defective PCNA mutants, we observed that Msh2 binds to regions of replicating DNA, but the distribution and dynamics are altered, suggesting that PCNA is not the sole determinant for the mismatch recognition complex association with replicating regions, but may influence the dynamics of movement. Using biochemical and genomic methods, we provide evidence that both MutS complexes are in the vicinity of the replisome to efficiently repair the entire spectrum of mutations during replication. Our data supports the model that the proximity of MutS?/? to the replisome for the efficient repair of the newly synthesized strand before chromatin reassembles. PMID:26684201

  9. The Eukaryotic Mismatch Recognition Complexes Track with the Replisome during DNA Synthesis.

    PubMed

    Haye, Joanna E; Gammie, Alison E

    2015-12-01

    During replication, mismatch repair proteins recognize and repair mispaired bases that escape the proofreading activity of DNA polymerase. In this work, we tested the model that the eukaryotic mismatch recognition complex tracks with the advancing replisome. Using yeast, we examined the dynamics during replication of the leading strand polymerase Pol? using Pol2 and the eukaryotic mismatch recognition complex using Msh2, the invariant protein involved in mismatch recognition. Specifically, we synchronized cells and processed samples using chromatin immunoprecipitation combined with custom DNA tiling arrays (ChIP-chip). The Pol? signal was not detectable in G1, but was observed at active origins and replicating DNA throughout S-phase. The Pol? signal provided the resolution to track origin firing timing and efficiencies as well as replisome progression rates. By detecting Pol? and Msh2 dynamics within the same strain, we established that the mismatch recognition complex binds origins and spreads to adjacent regions with the replisome. In mismatch repair defective PCNA mutants, we observed that Msh2 binds to regions of replicating DNA, but the distribution and dynamics are altered, suggesting that PCNA is not the sole determinant for the mismatch recognition complex association with replicating regions, but may influence the dynamics of movement. Using biochemical and genomic methods, we provide evidence that both MutS complexes are in the vicinity of the replisome to efficiently repair the entire spectrum of mutations during replication. Our data supports the model that the proximity of MutS?/? to the replisome for the efficient repair of the newly synthesized strand before chromatin reassembles. PMID:26684201

  10. Microtubule-targeting agents augment the toxicity of DNA-damaging agents by disrupting intracellular trafficking of DNA repair proteins.

    PubMed

    Poruchynsky, Marianne S; Komlodi-Pasztor, Edina; Trostel, Shana; Wilkerson, Julia; Regairaz, Marie; Pommier, Yves; Zhang, Xu; Kumar Maity, Tapan; Robey, Robert; Burotto, Mauricio; Sackett, Dan; Guha, Udayan; Fojo, Antonio Tito

    2015-02-01

    The paradigm that microtubule-targeting agents (MTAs) cause cell death via mitotic arrest applies to rapidly dividing cells but cannot explain MTA activity in slowly growing human cancers. Many preferred cancer regimens combine a MTA with a DNA-damaging agent (DDA). We hypothesized that MTAs synergize with DDAs by interfering with trafficking of DNA repair proteins on interphase microtubules. We investigated nine proteins involved in DNA repair: ATM, ATR, DNA-PK, Rad50, Mre11, p95/NBS1, p53, 53BP1, and p63. The proteins were sequestered in the cytoplasm by vincristine and paclitaxel but not by an aurora kinase inhibitor, colocalized with tubulin by confocal microscopy and coimmunoprecipitated with the microtubule motor dynein. Furthermore, adding MTAs to radiation, doxorubicin, or etoposide led to more sustained ?-H2AX levels. We conclude DNA damage-repair proteins traffic on microtubules and addition of MTAs sequesters them in the cytoplasm, explaining why MTA/DDA combinations are common anticancer regimens. PMID:25605897

  11. Microtubule-targeting agents augment the toxicity of DNA-damaging agents by disrupting intracellular trafficking of DNA repair proteins

    PubMed Central

    Poruchynsky, Marianne S.; Komlodi-Pasztor, Edina; Trostel, Shana; Wilkerson, Julia; Regairaz, Marie; Pommier, Yves; Zhang, Xu; Kumar Maity, Tapan; Robey, Robert; Burotto, Mauricio; Sackett, Dan; Guha, Udayan; Fojo, Antonio Tito

    2015-01-01

    The paradigm that microtubule-targeting agents (MTAs) cause cell death via mitotic arrest applies to rapidly dividing cells but cannot explain MTA activity in slowly growing human cancers. Many preferred cancer regimens combine a MTA with a DNA-damaging agent (DDA). We hypothesized that MTAs synergize with DDAs by interfering with trafficking of DNA repair proteins on interphase microtubules. We investigated nine proteins involved in DNA repair: ATM, ATR, DNA-PK, Rad50, Mre11, p95/NBS1, p53, 53BP1, and p63. The proteins were sequestered in the cytoplasm by vincristine and paclitaxel but not by an aurora kinase inhibitor, colocalized with tubulin by confocal microscopy and coimmunoprecipitated with the microtubule motor dynein. Furthermore, adding MTAs to radiation, doxorubicin, or etoposide led to more sustained ?-H2AX levels. We conclude DNA damage-repair proteins traffic on microtubules and addition of MTAs sequesters them in the cytoplasm, explaining why MTA/DDA combinations are common anticancer regimens. PMID:25605897

  12. Structure of Bacterial LigD -phosphoesterase Unveils a DNA Repair Superfamily

    SciTech Connect

    Nair, P.; Smith, P; Shuman, S

    2010-01-01

    The DNA ligase D (LigD) 3{prime}-phosphoesterase (PE) module is a conserved component of the bacterial nonhomologous end-joining (NHEJ) apparatus that performs 3{prime} end-healing reactions at DNA double-strand breaks. Here we report the 1.9 {angstrom} crystal structure of Pseudomonas aeruginosa PE, which reveals that PE exemplifies a unique class of DNA repair enzyme. PE has a distinctive fold in which an eight stranded {beta} barrel with a hydrophobic interior supports a crescent-shaped hydrophilic active site on its outer surface. Six essential side chains coordinate manganese and a sulfate mimetic of the scissile phosphate. The PE active site and mechanism are unique vis a vis other end-healing enzymes. We find PE homologs in archaeal and eukaryal proteomes, signifying that PEs comprise a DNA repair superfamily.

  13. Theory uncovers an unusual mechanism of DNA repair of a lesioned adenine by AlkB enzymes.

    PubMed

    Wang, Binju; Usharani, Dandamudi; Li, Chunsen; Shaik, Sason

    2014-10-01

    DNA-base lesions cause cancer and propagate into the genome. We use in-protein QM/MM calculations to study the repair of etheno-bridged adenine (?A) by the iron(IV)-oxo species of AlkB enzymes. Recent experimental investigations, using mass-spectrometry and in crystallo isolation, suggested that ?A was repaired by formation of an epoxide (?A-ep) that further transforms to a glycol (?A-gl), ending finally in adenine and glyoxal. Theory reproduces the experimentally observed barrier for the rate-determining step and its pH dependence. However, as we show, the mass-spectrometrically identified species are side-byproducts unassociated with the repair mechanism. The repair is mediated by a zwitterionic species, of the same molecular mass as the epoxide, which transforms to an intermediate that matches the in crystallo trapped species in structure and mass, but is NOT the assumed ?A-gl iron-glycol complex. Verifiable/falsifiable predictions, regarding the key protein residues, follow. The paper underscores the indispensable role of theory by providing atomistic descriptions of this vital mechanism, and guiding further experimental investigations. PMID:25203306

  14. A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines.

    PubMed

    Bouley, Julien; Saad, Lina; Grall, Romain; Schellenbauer, Amelie; Biard, Denis; Paget, Vincent; Morel-Altmeyer, Sandrine; Guipaud, Olivier; Chambon, Christophe; Salles, Bernard; Maloum, Karim; Merle-Béral, Hélène; Chevillard, Sylvie; Delic, Jozo

    2015-09-29

    Ku70-dependent canonical nonhomologous end-joining (c-NHEJ) DNA repair system is fundamental to the genome maintenance and B-cell lineage. c-NHEJ is upregulated and error-prone in incurable forms of chronic lymphocytic leukemia which also displays telomere dysfunction, multiple chromosomal aberrations and the resistance to DNA damage-induced apoptosis. We identify in these cells a novel DNA damage inducible form of phospho-Ku70. In vitro in different cancer cell lines, Ku70 phosphorylation occurs in a heterodimer Ku70/Ku80 complex within minutes of genotoxic stress, necessitating its interaction with DNA damage-induced kinase pS2056-DNA-PKcs and/or pS1981-ATM. The mutagenic effects of phospho-Ku70 are documented by a defective S/G2 checkpoint, accelerated disappearance of ?-H2AX foci and kinetics of DNA repair resulting in an increased level of genotoxic stress-induced chromosomal aberrations. Together, these data unveil an involvement of phospho-Ku70 in fast but inaccurate DNA repair; a new paradigm linked to both the deregulation of c-NHEJ and the resistance of malignant cells. PMID:26337656

  15. A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines.

    TOXLINE Toxicology Bibliographic Information

    Bouley J; Saad L; Grall R; Schellenbauer A; Biard D; Paget V; Morel-Altmeyer S; Guipaud O; Chambon C; Salles B; Maloum K; Merle-Béral H; Chevillard S; Delic J

    2015-09-29

    Ku70-dependent canonical nonhomologous end-joining (c-NHEJ) DNA repair system is fundamental to the genome maintenance and B-cell lineage. c-NHEJ is upregulated and error-prone in incurable forms of chronic lymphocytic leukemia which also displays telomere dysfunction, multiple chromosomal aberrations and the resistance to DNA damage-induced apoptosis. We identify in these cells a novel DNA damage inducible form of phospho-Ku70. In vitro in different cancer cell lines, Ku70 phosphorylation occurs in a heterodimer Ku70/Ku80 complex within minutes of genotoxic stress, necessitating its interaction with DNA damage-induced kinase pS2056-DNA-PKcs and/or pS1981-ATM. The mutagenic effects of phospho-Ku70 are documented by a defective S/G2 checkpoint, accelerated disappearance of ?-H2AX foci and kinetics of DNA repair resulting in an increased level of genotoxic stress-induced chromosomal aberrations. Together, these data unveil an involvement of phospho-Ku70 in fast but inaccurate DNA repair; a new paradigm linked to both the deregulation of c-NHEJ and the resistance of malignant cells.

  16. Partial reconstitution of DNA large loop repair with purified proteins from Saccharomyces cerevisiae

    PubMed Central

    Sommer, Debbie; Stith, Carrie M.; Burgers, Peter M. J.; Lahue, Robert S.

    2008-01-01

    Small looped mispairs are corrected by DNA mismatch repair. In addition, a distinct process called large loop repair (LLR) corrects heteroduplexes up to several hundred nucleotides in bacteria, yeast and human cells, and in cell-free extracts. Only some LLR protein components are known, however. Previous studies with neutralizing antibodies suggested a role for yeast DNA polymerase ? (Pol ?), RFC and PCNA in LLR repair synthesis. In the current study, biochemical fractionation studies identified FEN1 (Rad27) as another required LLR component. In the presence of purified FEN1, Pol ?, RFC and PCNA, repair occurred on heteroduplexes with loops ranging from 8 to 216 nt. Repair utilized a 5? nick, with correction directed to the nicked strand, irrespective of which strand contained the loop. In contrast, repair of a G/T mismatch occurred at low levels, suggesting specificity of the reconstituted system for looped mispairs. The presence of RPA enhanced reactivity on some looped substrates, but RPA was not required for activity. Although additional LLR factors remain to be identified, the excision and resynthesis steps of LLR from a 5? nick can be reconstituted in a purified system with FEN1 and Pol ?, together with PCNA and its loader RFC. PMID:18628298

  17. Germ cell DNA-repair systems-possible tools in cancer research?

    PubMed

    Helle, F

    2012-04-01

    A major dogma in cancer research is that cancer begins at the cellular level. Because of this single-cell origin, evolutionary principles have often been used to explain how somatic cancer cells are selected at a sub-individual level. The traditional application of Darwinian theory, however, in which the colony of cells constituting an individual is regarded as a whole, has not been applied extensively to the understanding of cancer until recently. Two proponents for this view, Breivik and Gaudernack, have suggested that in certain situations the cost of DNA repair might exceed the cost of errors. This model predicts that genetic stability is configured for an optimal cost-benefit relationship. Natural selection is not expected to have produced the best genetic stability available in the human body, merely the best compromise of DNA repair and costs. Repair and maintenance of the vast human genome is thermodynamically expensive, and an optimal balance between DNA repair and dietary needs is likely to have originated. Furthermore, fast growth conveys significant advantages such as early maturation or cognitive development, but usually at the expense of replication accuracy. Thus, a compromise between growth speed and cancer risk is likely to have taken place. These and other ecological mechanisms have probably prevented genomic stability to reach its full potential in the human body. In contrast, germ lines express near perfect DNA maintenance. Although germ cells are specialized DNA-conserving cells with few other functions, it's not given that their proteins will all be incompatible with the somatic cell. One approach to study this would be to systematically explore which DNA-stability and -repair systems are unique in germ cells, and induce their expression in invertebrate and mammalian model organisms. This could unveil which DNA-repair systems are switched off in the somatic cell lines, as they are incompatible, and which are absent due to evolution. The present review discuss different DNA-repair systems and cell cycle check point control mechanisms shown to be different or unique in the germ cell, and how they may be utilized in cancer therapy. PMID:22322886

  18. DNA repair of clustered lesions in mammalian cells: involvement of non-homologous end-joining

    PubMed Central

    Malyarchuk, Svitlana; Castore, Reneau; Harrison, Lynn

    2008-01-01

    Clustered lesions are defined as ?two lesions within 20 bps and are generated in DNA by ionizing radiation. In vitro studies and work in bacteria have shown that attempted repair of two closely opposed lesions can result in the formation of double strand breaks (DSBs). Since mammalian cells can repair DSBs by non-homologous end-joining (NHEJ), we hypothesized that NHEJ would repair DSBs formed during the removal of clustered tetrahydrofurans (furans). However, two opposing furans situated 2, 5 or 12 bps apart in a firefly luciferase reporter plasmid caused a decrease in luciferase activity in wild-type, Ku80 or DNA-PKcs-deficient cells, indicating the generation of DSBs. Loss of luciferase activity was maximal at 5 bps apart and studies using siRNA implicate the major AP endonuclease in the initial cleavage. Since NHEJ-deficient cells had equivalent luciferase activity to their isogenic wild-type cells, NHEJ was not involved in accurate repair of clustered lesions. However, quantitation and examination of re-isolated DNA showed that damage-containing plasmids were inaccurately repaired by Ku80-dependent, as well as Ku80-independent mechanisms. This work indicates that not even NHEJ can completely prevent the conversion of clustered lesions to potentially lethal DSBs, so demonstrating the biological relevance of ionizing radiation-induced clustered damage. PMID:18653525

  19. Altered spatio-temporal dynamics of RNase H2 complex assembly at replication and repair sites in Aicardi-Goutières syndrome.

    PubMed

    Kind, Barbara; Muster, Britta; Staroske, Wolfgang; Herce, Henry D; Sachse, René; Rapp, Alexander; Schmidt, Franziska; Koss, Sarah; Cardoso, M Cristina; Lee-Kirsch, Min Ae

    2014-11-15

    Ribonuclease H2 plays an essential role for genome stability as it removes ribonucleotides misincorporated into genomic DNA by replicative polymerases and resolves RNA/DNA hybrids. Biallelic mutations in the genes encoding the three RNase H2 subunits cause Aicardi-Goutières syndrome (AGS), an early-onset inflammatory encephalopathy that phenotypically overlaps with the autoimmune disorder systemic lupus erythematosus. Here we studied the intracellular dynamics of RNase H2 in living cells during DNA replication and in response to DNA damage using confocal time-lapse imaging and fluorescence cross-correlation spectroscopy. We demonstrate that the RNase H2 complex is assembled in the cytosol and imported into the nucleus in an RNase H2B-dependent manner. RNase H2 is not only recruited to DNA replication foci, but also to sites of PCNA-dependent DNA repair. By fluorescence recovery after photobleaching, we demonstrate a high mobility and fast exchange of RNase H2 at sites of DNA repair and replication. We provide evidence that recruitment of RNase H2 is not only PCNA-dependent, mediated by an interaction of the B subunit with PCNA, but also PCNA-independent mediated via the catalytic domain of the A subunit. We found that AGS-associated mutations alter complex formation, recruitment efficiency and exchange kinetics at sites of DNA replication and repair suggesting that impaired ribonucleotide removal contributes to AGS pathogenesis. PMID:24986920

  20. Reduced cellular DNA repair capacity after environmentally relevant arsenic exposure. Influence of Ogg1 deficiency.

    PubMed

    Bach, Jordi; Peremartí, Jana; Annangi, Balasubramnayam; Marcos, Ricard; Hernández, Alba

    2015-09-01

    Inorganic arsenic (i-As) is a genotoxic and carcinogenic environmental contaminant known to affect millions of people worldwide. Our previous work demonstrated that chronic sub-toxic i-As concentrations were able to induce biologically significant levels of genotoxic and oxidative DNA damage that were strongly influenced by the Ogg1 genotype. In order to study the nature of the observed levels of damage and the observed differences between MEF Ogg1(+/+) and Ogg1(-/-) genetic backgrounds, the genotoxic and oxidative DNA repair kinetics of 18-weeks exposed MEF cells were evaluated by the comet assay. Results indicate that MEF Ogg1(+/+) and Ogg1(-/-) cells chronically exposed to i-As repair the DNA damage induced by arsenite, potassium bromide and UVC radiation less efficiently than control cells, being that observation clearly more pronounced in MEF Ogg1(-/-) cells. Consequently, exposed cells accumulate a higher percentage of unrepaired DNA damage at the end of the repair period. As an attempt to eliminate i-As associated toxicity, chronically exposed MEF Ogg1(-/-) cells overexpress the arsenic metabolizing enzyme As3mt. This adaptive response confers cells a significant resistance to i-As-induced cell death, but at expenses of accumulating high levels of DNA damage due to their repair impairment. Overall, the work presented here evidences that i-As chronic exposure disrupts the normal cellular repair function, and that oxidative DNA damage-and Ogg1 deficiency-exacerbates this phenomenon. The observed cell death resistance under a chronic scenario of genotoxic and oxidative stress may in turn contribute to the carcinogenic effects of i-As. PMID:26210637

  1. E2F1 responds to ultraviolet radiation by directly stimulating DNA repair and suppressing carcinogenesis.

    PubMed

    Biswas, Anup Kumar; Mitchell, David L; Johnson, David G

    2014-06-15

    In response to DNA damage, the E2F1 transcription factor is phosphorylated at serine 31 (serine 29 in mouse) by the ATM or ATR kinases, which promotes E2F1 protein stabilization. Phosphorylation of E2F1 also leads to the recruitment of E2F1 to sites of DNA damage, where it functions to enhance DNA repair. To study the role of this E2F1 phosphorylation event in vivo, a knock-in mouse model was generated, in which serine 29 was mutated to alanine. The S29A mutation impairs E2F1 stabilization in response to ultraviolet (UV) radiation and doxorubicin treatment, but has little effect on the expression of E2F target genes. The apoptotic and proliferative responses to acute UV radiation exposure are also similar between wild-type and E2f1(S29A/) (S29A) mice. As expected, the S29A mutation prevents E2F1 association with damaged DNA and reduces DNA repair efficiency. Moreover, E2f1(S29A/) (S29A) mice display increased sensitivity to UV-induced skin carcinogenesis. This knock-in mouse model thus links the ability of E2F1 to directly promote DNA repair with the suppression of tumor development. PMID:24741006

  2. Anhydrobiosis-Associated Nuclear DNA Damage and Repair in the Sleeping Chironomid: Linkage with Radioresistance

    PubMed Central

    Vanyagina, Veronica; Malutina, Ludmila; Cornette, Richard; Sakashita, Tetsuya; Hamada, Nobuyuki; Kikawada, Takahiro; Kobayashi, Yasuhiko; Okuda, Takashi

    2010-01-01

    Anhydrobiotic chironomid larvae can withstand prolonged complete desiccation as well as other external stresses including ionizing radiation. To understand the cross-tolerance mechanism, we have analyzed the structural changes in the nuclear DNA using transmission electron microscopy and DNA comet assays in relation to anhydrobiosis and radiation. We found that dehydration causes alterations in chromatin structure and a severe fragmentation of nuclear DNA in the cells of the larvae despite successful anhydrobiosis. Furthermore, while the larvae had restored physiological activity within an hour following rehydration, nuclear DNA restoration typically took 72 to 96 h. The DNA fragmentation level and the recovery of DNA integrity in the rehydrated larvae after anhydrobiosis were similar to those of hydrated larvae irradiated with 70 Gy of high-linear energy transfer (LET) ions (4He). In contrast, low-LET radiation (gamma-rays) of the same dose caused less initial damage to the larvae, and DNA was completely repaired within within 24 h. The expression of genes encoding the DNA repair enzymes occurred upon entering anhydrobiosis and exposure to high- and low-LET radiations, indicative of DNA damage that includes double-strand breaks and their subsequent repair. The expression of antioxidant enzymes-coding genes was also elevated in the anhydrobiotic and the gamma-ray-irradiated larvae that probably functions to reduce the negative effect of reactive oxygen species upon exposure to these stresses. Indeed the mature antioxidant proteins accumulated in the dry larvae and the total activity of antioxidants increased by a 3–4 fold in association with anhydrobiosis. We conclude that one of the factors explaining the relationship between radioresistance and the ability to undergo anhydrobiosis in the sleeping chironomid could be an adaptation to desiccation-inflicted nuclear DNA damage. There were also similarities in the molecular response of the larvae to damage caused by desiccation and ionizing radiation. PMID:21103355