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Sample records for dna crosslink repair

  1. DNA-Protein Crosslink Proteolysis Repair.

    Science.gov (United States)

    Vaz, Bruno; Popovic, Marta; Ramadan, Kristijan

    2017-06-01

    Proteins that are covalently bound to DNA constitute a specific type of DNA lesion known as DNA-protein crosslinks (DPCs). DPCs represent physical obstacles to the progression of DNA replication. If not repaired, DPCs cause stalling of DNA replication forks that consequently leads to DNA double-strand breaks, the most cytotoxic DNA lesion. Although DPCs are common DNA lesions, the mechanism of DPC repair was unclear until now. Recent work unveiled that DPC repair is orchestrated by proteolysis performed by two distinct metalloproteases, SPARTAN in metazoans and Wss1 in yeast. This review summarizes recent discoveries on two proteases in DNA replication-coupled DPC repair and establishes DPC proteolysis repair as a separate DNA repair pathway for genome stability and protection from accelerated aging and cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Understanding the molecular mechanism of formaldehyde-induced DNA-protein crosslink repair

    Science.gov (United States)

    Formaldehyde induces DNA-protein crosslinks (DPCs) in several experimental in vitro and in vivo test systems, as well as in exposed human workers. DPCs are repaired by several DNA repair pathways in different species, but the molecular understanding of DPC repair in human tissues...

  3. Understanding the Molecular Mechanism(s) of Formaldehyde-induced DNA-protein Crosslink Repair

    Science.gov (United States)

    Although formaldehyde has been shown to induce many kinds of DNA damage both in in vitro and in vivo assay systems, initial DNA-protein crosslink (DPC) formation might play a major role in FA-induced mutagenesis and carcinogenesis. Several DNA repair pathways, such as base excisi...

  4. Crosslinking of DNA repair and replication proteins to DNA in cells treated with 6-thioguanine and UVA.

    Science.gov (United States)

    Gueranger, Quentin; Kia, Azadeh; Frith, David; Karran, Peter

    2011-07-01

    The DNA of patients taking immunosuppressive and anti-inflammatory thiopurines contains 6-thioguanine (6-TG) and their skin is hypersensitive to ultraviolet A (UVA) radiation. DNA 6-TG absorbs UVA and generates reactive oxygen species that damage DNA and proteins. Here, we show that the DNA damage includes covalent DNA-protein crosslinks. An oligonucleotide containing a single 6-TG is photochemically crosslinked to cysteine-containing oligopeptides by low doses of UVA. Crosslinking is significantly more efficient if guanine sulphonate (G(SO3))--an oxidized 6-TG and a previously identified UVA photoproduct--replaces 6-TG, suggesting that G(SO3) is an important reaction intermediate. Crosslinking occurs via oligopeptide sulphydryl and free amino groups. The oligonucleotide-oligopeptide adducts are heat stable but are partially reversed by reducing treatments. UVA irradiation of human cells containing DNA 6-TG induces extensive heat- and reducing agent-resistant covalent DNA-protein crosslinks and diminishes the recovery of some DNA repair and replication proteins from nuclear extracts. DNA-protein crosslinked material has an altered buoyant density and can be purified by banding in cesium chloride (CsCl) gradients. PCNA, the MSH2 mismatch repair protein and the XPA nucleotide excision repair (NER) factor are among the proteins detectable in the DNA-crosslinked material. These findings suggest that the 6-TG/UVA combination might compromise DNA repair by sequestering essential proteins.

  5. Pathways of homologous recombinantion and DNA interstrand cross-link repair : roles of mammalian RAD54 and SNMI

    NARCIS (Netherlands)

    M.L.G. Dronkert (Mies)

    2002-01-01

    textabstractThe aim of this thesis is to investigate mammalian DNA interstrand cross-link (ICL) repair. ICLs are formed by a number of agents used in tumor therapy, like mitomycin C and cisplatin. They constitute one of the most toxic damages to DNA, as they inhibit DNA strand separation. However, l

  6. Orchestrating the nucleases involved in DNA interstrand cross-link (ICL) repair.

    Science.gov (United States)

    Sengerová, Blanka; Wang, Anderson T; McHugh, Peter J

    2011-12-01

    DNA interstrand cross-links (ICLs) pose a significant threat to genomic and cellular integrity by blocking essential cellular processes, including replication and transcription. In mammalian cells, much ICL repair occurs in association with DNA replication during S phase, following the stalling of a replication fork at the block caused by an ICL lesion. Here, we review recent work showing that the XPF-ERCC1 endonuclease and the hSNM1A exonuclease act in the same pathway, together with SLX4, to initiate ICL repair, with the MUS81-EME1 fork incision activity becoming important in the absence of the XPF-SNM1A-SLX4-dependent pathway. Another nuclease, the Fanconi anemia-associated nuclease (FAN1), has recently been implicated in the repair of ICLs, and we discuss the possible ways in which the activities of different nucleases at the ICL-stalled replication fork may be coordinated. In relation to this, we briefly speculate on the possible role of SLX4, which contains XPF and MUS81- interacting domains, in the coordination of ICL repair nucleases.

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

    DEFF Research Database (Denmark)

    Räschle, Markus; Smeenk, Godelieve; Hansen, Rebecca K

    2015-01-01

    a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we...

  8. Bloom syndrome complex promotes FANCM recruitment to stalled replication forks and facilitates both repair and traverse of DNA interstrand crosslinks.

    Science.gov (United States)

    Ling, Chen; Huang, Jing; Yan, Zhijiang; Li, Yongjiang; Ohzeki, Mioko; Ishiai, Masamichi; Xu, Dongyi; Takata, Minoru; Seidman, Michael; Wang, Weidong

    2016-01-01

    The recruitment of FANCM, a conserved DNA translocase and key component of several DNA repair protein complexes, to replication forks stalled by DNA interstrand crosslinks (ICLs) is a step upstream of the Fanconi anemia (FA) repair and replication traverse pathways of ICLs. However, detection of the FANCM recruitment has been technically challenging so that its mechanism remains exclusive. Here, we successfully observed recruitment of FANCM at stalled forks using a newly developed protocol. We report that the FANCM recruitment depends upon its intrinsic DNA translocase activity, and its DNA-binding partner FAAP24. Moreover, it is dependent on the replication checkpoint kinase, ATR; but is independent of the FA core and FANCD2-FANCI complexes, two essential components of the FA pathway, indicating that the FANCM recruitment occurs downstream of ATR but upstream of the FA pathway. Interestingly, the recruitment of FANCM requires its direct interaction with Bloom syndrome complex composed of BLM helicase, Topoisomerase 3α, RMI1 and RMI2; as well as the helicase activity of BLM. We further show that the FANCM-BLM complex interaction is critical for replication stress-induced FANCM hyperphosphorylation, for normal activation of the FA pathway in response to ICLs, and for efficient traverse of ICLs by the replication machinery. Epistasis studies demonstrate that FANCM and BLM work in the same pathway to promote replication traverse of ICLs. We conclude that FANCM and BLM complex work together at stalled forks to promote both FA repair and replication traverse pathways of ICLs.

  9. Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair

    Directory of Open Access Journals (Sweden)

    Krisnamurthy Mahalakshmi

    2006-06-01

    Full Text Available Abstract Background Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs can be repaired through direct joining of broken ends (non homologous end joining, NHEJ or through recombination with the non broken sister chromosome (homologous recombination, HR. Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells. Results A deletion of the sbcC gene rendered Bacillus subtilis cells sensitive to DNA damage caused by Mitomycin C (MMC or by gamma irradiation. The deletion of the sbcC gene in a recN mutant background increased the sensitivity of the single recN mutant strain. SbcC was also non-epistatic with AddAB (analog of Escherichia coli RecBCD, but epistatic with RecA. A deletion of the ykoV gene encoding the B. subtilis Ku protein in a sbcC mutant strain did not resulted in an increase in sensitivity towards MMC and gamma irradiation, but exacerbated the phenotype of a recN or a recA mutant strain. In exponentially growing cells, SbcC-GFP was present throughout the cells, or as a central focus in rare cases. Upon induction of DNA damage, SbcC formed 1, rarely 2, foci on the nucleoids. Different to RecN protein, which forms repair centers at any location on the nucleoids, SbcC foci mostly co-localized with the DNA polymerase complex. In contrast to this, AddA-GFP or AddB-GFP did not form detectable foci upon addition of MMC. Conclusion Our experiments show that SbcC plays an important role in the repair of DNA inter-strand cross-links (induced by MMC, most likely

  10. Repair of 8-methoxypsoralen induced DNA interstrand cross-links in Tetrahymena thermophila. The effect of inhibitors of macromolecular synthesis

    DEFF Research Database (Denmark)

    Nielsen, P E; Køber, L

    1985-01-01

    The effect of several growth-inhibiting compounds on the repair of 8-methoxypsoralen-UVA-light-induced DNA interstrand cross-links has been studied in the protozoan Tetrahymena thermophila. The repair process was analyzed by the alkaline elution technique and could be divided into 3 phases......: a protein-DNA complexing phase, a DNA-incision phase and finally a DNA-ligation phase. The incision was found to be completely inhibited by novobiocin (50 micrograms/ml), nalidixic acid (150 micrograms/ml), n-butyrate (15 mM) and cycloheximide (1 microgram/ml), while no effect was observed for cytosine-1......-beta-D-arabinofuranoside (10 mM), puromycin (1 mM), hydroxyurea (5 mM) or 3-aminobenzamide (2.5 mM). None of the compounds showed any effect on the protein-DNA complexing step, and the ligation was partly inhibited only by nalidixic acid (150 micrograms/ml). The involvement of topoisomerases...

  11. Induction and repair of DNA cross-links induced by sulfur mustard in the A-549 cell line followed by a comet assay.

    Science.gov (United States)

    Jost, Petr; Svobodova, Hana; Stetina, Rudolf

    2015-07-25

    Sulfur mustard is a highly toxic chemical warfare agent with devastating impact on intoxicated tissues. DNA cross-links are probably the most toxic DNA lesions induced in the cell by sulfur mustard. The comet assay is a very sensitive method for measuring DNA damage. In the present study using the A-549 lung cell line, the comet assay protocol was optimized for indirect detection of DNA cross-links induced by sulfur mustard. The method is based on the additional treatment of the assayed cells containing cross-links with the chemical mutagen, styrene oxide. Alkali-labile adducts of styrene oxide cause DNA breaks leading to the formation of comets. A significant dose-dependent reduction of DNA migration of the comet's tail was found after exposing cells to sulfur mustard, indicative of the amount of sulfur mustard induced cross-links. The remarkable decrease of % tail DNA could be observed as early as 5min following exposure to sulfur mustard and the maximal effect was found after 30min, when DNA migration was reduced to the minimum. Sulfur mustard preincubated in culture medium without cells lost its ability to induce cross-links and had a half-life of about 15min. Pre-incubation longer than 30min does not lead to a significant increase in cross-links when applied to cells. However, the amount of cross-links is decreased during further incubation due to repair. The current modification of the comet assay provides a useful tool for detecting DNA cross-links induced by sulfur mustard and could be used for detection of other DNA cross-linking agents such as chemotherapeutic drugs.

  12. DNA single-strand breaks, double-strand breaks, and crosslinks in rat testicular germ cells: Measurements of their formation and repair by alkaline and neutral filter elution

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, M.O.; Dysart, G. (Merck Institute for Therapeutic Research, West Point, PA (USA))

    1985-06-01

    This work describes a neutral and alkaline elution method for measuring DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and DNA-DNA crosslinks in rat testicular germ cells after treatments in vivo or in vitro with both chemical mutagens and gamma-irradiation. The methods depend upon the isolation of testicular germ cells by collagenase and trypsin digestion, followed by filtration and centrifugation. {sup 137}Cs irradiation induced both DNA SSBs and DSBs in germ cells held on ice in vitro. Irradiation of the whole animal indicated that both types of DNA breaks are induced in vivo and can be repaired. A number of germ cell mutagens induced either DNA SSBs, DSBs, or cross-links after in vivo and in vitro dosing. These chemicals included methyl methanesulfonate, ethyl methanesulfonate, ethyl nitrosourea, dibromochlorpropane, ethylene dibromide, triethylene melamine, and mitomycin C. These results suggest that the blood-testes barrier is relatively ineffective for these mutagens, which may explain in part their in vivo mutagenic potency. This assay should be a useful screen for detecting chemical attack upon male germ-cell DNA and thus, it should help in the assessment of the mutagenic risk of chemicals. In addition, this approach can be used to study the processes of SSB, DSB, and crosslink repair in DNA of male germ cells, either from all stages or specific stages of development.

  13. Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5' flap DNA: basis of interstrand cross-link repair by FAN1

    Energy Technology Data Exchange (ETDEWEB)

    Gwon, Gwang Hyeon; Kim, Youngran; Liu, Yaqi; Watson, Adam T.; Jo, Aera; Etheridge, Thomas J.; Yuan, Fenghua; Zhang, Yanbin; Kim, YoungChang; Carr, Anthony M.; Cho, Yunje

    2014-10-15

    Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI–FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 5' flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domain playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 5' flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.

  14. The Fanconi anemia/BRCA pathway is involved in DNA interstrand cross-link repair of adriamycin-resistant leukemia cells.

    Science.gov (United States)

    Yao, Chenjiao; Du, Wei; Chen, Haibing; Xiao, Sheng; Huang, Lihua; Chen, Fangping

    2015-03-01

    The Fanconi anemia/BRCA (FA/BRCA) pathway plays a vital role in DNA damage repair induced by DNA cross-linking agents and is closely related to drug response in cancer treatment. Here we demonstrate that the FA/BRCA pathway contributes to acquired drug resistance in adriamycin (ADR)-resistant leukemia cell lines, and disruption of this pathway partially reverses the drug resistance. We observed that ADR-resistant cells have reduced DNA interstrand cross-links (ICL) compared with ADR-sensitive cells. Western blot studies demonstrated enhanced FA protein expression in ADR-resistant cells. Using siRNA to knock down FANCF in K562/R drug-resistant cells showed increases in sensitivity to ADR and ADR-induced DNA damage, and demonstrated a direct relationship between the FA/BRCA pathway and drug sensitivity. Overexpression of FANCF in K562 drug-sensitive cells partially reproduced the drug-resistant phenotype. These results show that the FA/BRCA pathway is involved in acquired ADR resistance of leukemia cells. The FA/BRCA pathway may be a new target to reverse ADR resistance in leukemia treatment.

  15. C. elegans ring finger protein RNF-113 is involved in interstrand DNA crosslink repair and interacts with a RAD51C homolog.

    Directory of Open Access Journals (Sweden)

    Hyojin Lee

    Full Text Available The Fanconi anemia (FA pathway recognizes interstrand DNA crosslinks (ICLs and contributes to their conversion into double-strand DNA breaks, which can be repaired by homologous recombination. Seven orthologs of the 15 proteins associated with Fanconi anemia are functionally conserved in the model organism C. elegans. Here we report that RNF-113, a ubiquitin ligase, is required for RAD-51 focus formation after inducing ICLs in C. elegans. However, the formation of foci of RPA-1 or FCD-2/FANCD2 in the FA pathway was not affected by depletion of RNF-113. Nevertheless, the RPA-1 foci formed did not disappear with time in the depleted worms, implying serious defects in ICL repair. As a result, RNF-113 depletion increased embryonic lethality after ICL treatment in wild-type worms, but it did not increase the ICL-induced lethality of rfs-1/rad51C mutants. In addition, the persistence of RPA-1 foci was suppressed in doubly-deficient rnf-113;rfs-1 worms, suggesting that there is an epistatic interaction between the two genes. These results lead us to suggest that RNF-113 and RFS-1 interact to promote the displacement of RPA-1 by RAD-51 on single-stranded DNA derived from ICLs.

  16. ATR-p53 restricts homologous recombination in response to replicative stress but does not limit DNA interstrand crosslink repair in lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Bianca M Sirbu

    Full Text Available Homologous recombination (HR is required for the restart of collapsed DNA replication forks and error-free repair of DNA double-strand breaks (DSB. However, unscheduled or hyperactive HR may lead to genomic instability and promote cancer development. The cellular factors that restrict HR processes in mammalian cells are only beginning to be elucidated. The tumor suppressor p53 has been implicated in the suppression of HR though it has remained unclear why p53, as the guardian of the genome, would impair an error-free repair process. Here, we show for the first time that p53 downregulates foci formation of the RAD51 recombinase in response to replicative stress in H1299 lung cancer cells in a manner that is independent of its role as a transcription factor. We find that this downregulation of HR is not only completely dependent on the binding site of p53 with replication protein A but also the ATR/ATM serine 15 phosphorylation site. Genetic analysis suggests that ATR but not ATM kinase modulates p53's function in HR. The suppression of HR by p53 can be bypassed under experimental conditions that cause DSB either directly or indirectly, in line with p53's role as a guardian of the genome. As a result, transactivation-inactive p53 does not compromise the resistance of H1299 cells to the interstrand crosslinking agent mitomycin C. Altogether, our data support a model in which p53 plays an anti-recombinogenic role in the ATR-dependent mammalian replication checkpoint but does not impair a cell's ability to use HR for the removal of DSB induced by cytotoxic agents.

  17. DNA repair. [UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, R.

    1978-01-01

    Some topics discussed are as follows: difficulty in extrapolating data from E. coli to mammalian systems; mutations caused by UV-induced changes in DNA; mutants deficient in excision repair; other postreplication mechanisms; kinds of excision repair systems; detection of repair by biochemical or biophysical means; human mutants deficient in repair; mutagenic effects of UV on XP cells; and detection of UV-repair defects among XP individuals. (HLW)

  18. Workshop on DNA repair.

    NARCIS (Netherlands)

    A.R. Lehmann (Alan); J.H.J. Hoeijmakers (Jan); A.A. van Zeeland (Albert); C.M.P. Backendorf (Claude); B.A. Bridges; A. Collins; R.P.D. Fuchs; G.P. Margison; R. Montesano; E. Moustacchi; A.T. Natarajan; M. Radman; A. Sarasin; E. Seeberg; C.A. Smith; M. Stefanini (Miria); L.H. Thompson; G.P. van der Schans; C.A. Weber (Christine); M.Z. Zdzienika

    1992-01-01

    textabstractA workshop on DNA repair with emphasis on eukaryotic systems was held, under the auspices of the EC Concerted Action on DNA Repair and Cancer, at Noordwijkerhout (The Netherlands) 14-19 April 1991. The local organization of the meeting was done under the auspices of the Medical Genetic C

  19. DNA repair protocols

    DEFF Research Database (Denmark)

    Bjergbæk, Lotte

    In its 3rd edition, this Methods in Molecular Biology(TM) book covers the eukaryotic response to genomic insult including advanced protocols and standard techniques in the field of DNA repair. Offers expert guidance for DNA repair, recombination, and replication. Current knowledge of the mechanisms...... that regulate DNA repair has grown significantly over the past years with technology advances such as RNA interference, advanced proteomics and microscopy as well as high throughput screens. The third edition of DNA Repair Protocols covers various aspects of the eukaryotic response to genomic insult including...... recent advanced protocols as well as standard techniques used in the field of DNA repair. Both mammalian and non-mammalian model organisms are covered in the book, and many of the techniques can be applied with only minor modifications to other systems than the one described. Written in the highly...

  20. Study on the Repair Ability of Ant Extract on DNA - Protein Crosslinks%蚂蚁提取液对DNA -蛋白质交联修复能力的研究

    Institute of Scientific and Technical Information of China (English)

    廖静; 周梦晨; 王娜娜; 杨旭

    2011-01-01

    目的 探讨蚂蚁提取液(ant extract,AE)对DNA -蛋白质交联(DPC)的修复能力.方法 以甲醛为染毒液,预处理小牛胸腺DNA和卵清蛋白混合液,使之形成DPC,再用不同浓度的AE处理,最后采用SDS - KCI沉淀法来检测不同处理组的DPC修复情况.结果 当甲醛染毒浓度为0.544mol/L时,10μg/ml的小牛胸腺DNA和10μg/ml的卵清蛋白DNA -蛋白质交联程度最高;制备2%的AE时,选择pH 7.6的磷酸氢二钠-磷酸二氢钠缓冲液作为组织匀浆液,AE具有最强的DPC修复能力,且用pH 3.6的柠檬酸-柠檬酸三钠缓冲液制备的AE也具有部分DPC修复能力;0.002%、0.02%、0.2%和2%的AE具有一定的DPC修复能力,且存在明显的浓度-效应关系.结论 本研究确定了蚂蚁提取液制备过程中缓冲液的最佳pH值,并证明了蚂蚁提取液具有一定的DPC修复能力.%Objective To investigate the repair capacity of ant extract (AE) on the DNA - protein crosslinks ( DPC). Methods Calf thymus DNA and ovalbumin mixture with formaldehyde solution were pretreated and they were made to form DPC. Then different concentrations of AE were used for DPC repair, and finally SDS - KC1 precipitation method was used to detect DPC repair capacity of different AE groups. Results When the formaldehyde exposure concentration was at 0.544mol/L for treating the mixture of 10μg/ml calf thymus DNA and 10μg/ml ovalbumin, the DNA - protein crosslink was up to the highest level. When 2% AE solution was made with pH =7. 6 disodium phosphate - sodium dihydrogen phosphate buffer, the AE had the highest capacity to repair the DPC, and when 2% AE solution was made with pH =3. 6 citric acid - sodium citrate buffer, the AE also had some capacity to repair the DPC. Along with AE concentration increasing,(0. 002% , 0.02% , 0. 2% and 2% ) the DPC repair capacity of AE solution increased, and an obvious dose - response effect between AE concentration and DPC repair capacity of AE can be found

  1. Optimality in DNA repair.

    Science.gov (United States)

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

    2012-01-07

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

  2. 8-Methoxypsoralen DNA interstrand cross-linking of the ribosomal RNA genes in Tetrahymena thermophila. Distribution, repair and effect on rRNA synthesis

    DEFF Research Database (Denmark)

    Fengquin, X; Nielsen, Henrik; Zhen, W;

    1993-01-01

    between three domains (terminal spacer, transcribed region and central spacer) as defined by restriction enzyme analysis (BamHI and ClaI). It is furthermore shown that a dosage resulting in approximately one cross-link per rDNA molecule (21 kbp, two genes) is sufficient to block RNA synthesis. Finally...

  3. Trans-dichlorooxovandium (IV) complex as a novel photoinducible DNA interstrand crosslinker for cancer therapy

    DEFF Research Database (Denmark)

    Somyajit, Kumar; Banik, Bhabatosh; Saxena, Sneha

    2016-01-01

    crosslinks. VDC efficiently activates Fanconi anemia (FA) pathway of DNA interstrand crosslink repair. Strikingly, photoinduction of VDC induces prolonged activation of cell cycle checkpoint and a high degree of cell death in homologous recombination (HR)/ICL repair defective cells. Moreover, VDC...

  4. DNA repair responses in human skin cells

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. The colibactin warhead crosslinks DNA

    Science.gov (United States)

    Vizcaino, Maria I.; Crawford, Jason M.

    2015-05-01

    Members of the human microbiota are increasingly being correlated to human health and disease states, but the majority of the underlying microbial metabolites that regulate host-microbe interactions remain largely unexplored. Select strains of Escherichia coli present in the human colon have been linked to the initiation of inflammation-induced colorectal cancer through an unknown small-molecule-mediated process. The responsible non-ribosomal peptide-polyketide hybrid pathway encodes ‘colibactin’, which belongs to a largely uncharacterized family of small molecules. Genotoxic small molecules from this pathway that are capable of initiating cancer formation have remained elusive due to their high instability. Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labelling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure. The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin's DNA-damaging activity. The data support unexpected models for both colibactin biosynthesis and its mode of action.

  6. Homologous recombination in DNA repair and DNA damage tolerance

    Institute of Scientific and Technical Information of China (English)

    Xuan Li; Wolf-Dietrich Heyer

    2008-01-01

    Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks (DSBs) and interstrand crosslinks (ICLs). In addition, recombination provides critical sup-port for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR: homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modaUties of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.

  7. Implication of the E. coli K12 uvrA and recA genes in the repair of 8-methoxypsoralen-induced mono adducts and crosslinks on plasmid DNA; Implicacion de los genes uvrA de E. coli K12 en la reparacion de monoaductos y entrecruzamien tos inducidos en DNA plasmidico por 8-metoxipso raleno mas luz ultravioleta A

    Energy Technology Data Exchange (ETDEWEB)

    Paramio, J.M.; Bauluz, C.; Vidania, R. de

    1986-07-01

    Genotoxicity of psoralen damages on plasmid DNA has been studied. pBR322 DNA was randomly modified with several concentrations of 8-methoxypsoralen plus 365 nm-UV light. After transformation into E. coli strains (wild-type, uvrA and recA) plasmid survival and mutagenesis were analyzed. To study the influence of the SOS response on plasmid recovery, preirradiation of the cells was performed. In absence of cell preirradiation, crosslinks were not repaired in any strain. Mono adducts were also lethal but in part removed by the excision-repair pathway. Preirradiation of the cells significantly. increased plasmid recovery in recA+ celia. In uvrA- only the mutagenic pathway seemed to be involved in the repair of the damaged DNA. Wild type strain showed the highest increase in plasmid survival, involving the repair of mono adducts and some fraction of crosslinks mainly through an error-free repair pathway. This suggests an enhancement of the excision repair promoted by the induction of SOS functions. (Author) 32 refs.

  8. The journey of DNA repair

    OpenAIRE

    Saini, Natalie

    2015-01-01

    21 years ago, the DNA Repair Enzyme was declared “Molecule of the Year”. Today, we are celebrating another “year of repair”, with the 2015 Nobel Prize in Chemistry being awarded to Aziz Sancar, Tomas Lindahl and Paul Modrich for their collective work on the different DNA repair pathways.

  9. Rethinking transcription coupled DNA repair.

    Science.gov (United States)

    Kamarthapu, Venu; Nudler, Evgeny

    2015-04-01

    Nucleotide excision repair (NER) is an evolutionarily conserved, multistep process that can detect a wide variety of DNA lesions. Transcription coupled repair (TCR) is a subpathway of NER that repairs the transcribed DNA strand faster than the rest of the genome. RNA polymerase (RNAP) stalled at DNA lesions mediates the recruitment of NER enzymes to the damage site. In this review we focus on a newly identified bacterial TCR pathway in which the NER enzyme UvrD, in conjunction with NusA, plays a major role in initiating the repair process. We discuss the tradeoff between the new and conventional models of TCR, how and when each pathway operates to repair DNA damage, and the necessity of pervasive transcription in maintaining genome integrity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Aging and DNA repair capability. [Review

    Energy Technology Data Exchange (ETDEWEB)

    Tice, R R

    1977-01-01

    A review of the literature on DNA repair processes in relation to aging is presented under the following headings: DNA repair processes; age-related occurrence of unrepaired DNA lesions; DNA repair capability as a function of age; tissue-specific DNA repair capability; acceleration of the aging process by exposure to DNA damaging agents; human genetic syndromes; and longevity and DNA repair processes. (HLW)

  11. The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks

    NARCIS (Netherlands)

    K. Hanada (Katsuhiro); M. Budzowska (Magdalena); M. Modesti (Mauro); A. Maas (Alex); C. Wyman (Claire); J. Essers (Jeroen); R. Kanaar (Roland)

    2006-01-01

    textabstractRepair of interstrand crosslinks (ICLs) requires multiple-strand incisions to separate the two covalently attached strands of DNA. It is unclear how these incisions are generated. DNA double-strand breaks (DSBs) have been identified as intermediates in ICL repair, but enzymes responsible

  12. DNA repair deficiency in neurodegeneration

    DEFF Research Database (Denmark)

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

    2011-01-01

    : 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......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...

  13. CtIP is required to initiate replication-dependent interstrand crosslink repair.

    Directory of Open Access Journals (Sweden)

    Michelle L Duquette

    Full Text Available DNA interstrand crosslinks (ICLs are toxic lesions that block the progression of replication and transcription. CtIP is a conserved DNA repair protein that facilitates DNA end resection in the double-strand break (DSB repair pathway. Here we show that CtIP plays a critical role during initiation of ICL processing in replicating human cells that is distinct from its role in DSB repair. CtIP depletion sensitizes human cells to ICL inducing agents and significantly impairs the accumulation of DNA damage response proteins RPA, ATR, FANCD2, γH2AX, and phosphorylated ATM at sites of laser generated ICLs. In contrast, the appearance of γH2AX and phosphorylated ATM at sites of laser generated double strand breaks (DSBs is CtIP-independent. We present a model in which CtIP functions early in ICL repair in a BRCA1- and FANCM-dependent manner prior to generation of DSB repair intermediates.

  14. DNA repair in Chromobacterium violaceum.

    Science.gov (United States)

    Duarte, Fábio Teixeira; Carvalho, Fabíola Marques de; Bezerra e Silva, Uaska; Scortecci, Kátia Castanho; Blaha, Carlos Alfredo Galindo; Agnez-Lima, Lucymara Fassarella; Batistuzzo de Medeiros, Silvia Regina

    2004-03-31

    Chromobacterium violaceum is a Gram-negative beta-proteobacterium that inhabits a variety of ecosystems in tropical and subtropical regions, including the water and banks of the Negro River in the Brazilian Amazon. This bacterium has been the subject of extensive study over the last three decades, due to its biotechnological properties, including the characteristic violacein pigment, which has antimicrobial and anti-tumoral activities. C. violaceum promotes the solubilization of gold in a mercury-free process, and has been used in the synthesis of homopolyesters suitable for the production of biodegradable polymers. The complete genome sequence of this organism has been completed by the Brazilian National Genome Project Consortium. The aim of our group was to study the DNA repair genes in this organism, due to their importance in the maintenance of genomic integrity. We identified DNA repair genes involved in different pathways in C. violaceum through a similarity search against known sequences deposited in databases. The phylogenetic analyses were done using programs of the PHILYP package. This analysis revealed various metabolic pathways, including photoreactivation, base excision repair, nucleotide excision repair, mismatch repair, recombinational repair, and the SOS system. The similarity between the C. violaceum sequences and those of Neisserie miningitidis and Ralstonia solanacearum was greater than that between the C. violaceum and Escherichia coli sequences. The peculiarities found in the C. violaceum genome were the absence of LexA, some horizontal transfer events and a large number of repair genes involved with alkyl and oxidative DNA damage.

  15. Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains.

    Science.gov (United States)

    Di Bernardo, Giovanni; Del Gaudio, Stefania; Cammarota, Marcella; Galderisi, Umberto; Cascino, Antonino; Cipollaro, Marilena

    2002-02-15

    Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (epsilon globin, p53 and gamma interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to alpha(lambda)mu(omicron)sigma(tau) 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed.

  16. Syntheses of DNA Duplexes That Contain a N4C-alkyl-N4C Interstrand Cross-Link

    Science.gov (United States)

    Miller, Paul S.

    2011-01-01

    A simple procedure is described for preparing short DNA duplexes that contain a single N4C-alkyl-N4C interstrand cross-link. The synthesis is carried out on an automated DNA synthesizer using standard phosphoramidite chemistry. The cross-link is introduced during the synthesis of the duplex. The method can be used to prepare mg quantities of cross-linked duplexes suitable for physical studies and for the preparation of larger DNA molecules that can be used as substrates to study DNA repair in whole cell extracts and in living cells in culture. PMID:21400705

  17. Disruption of mouse SNM1 causes increased sensitivity to the DNA interstrand cross-linking agent mitomycin C.

    NARCIS (Netherlands)

    M.L.G. Dronkert (Mies); J. de Wit (Jan); M. Boeve; M.L. Vasconcelos; H. van Steeg (Harry); T.L.R. Tan; J.H.J. Hoeijmakers (Jan); R. Kanaar (Roland)

    2000-01-01

    textabstractDNA interstrand cross-links (ICLs) represent lethal DNA damage, because they block transcription, replication, and segregation of DNA. Because of their genotoxicity, agents inducing ICLs are often used in antitumor therapy. The repair of ICLs is complex and involves proteins belonging to

  18. DNA damage by reactive species: Mechanisms, mutation and repair.

    Science.gov (United States)

    Jena, N R

    2012-07-01

    DNA is continuously attacked by reactive species that can affect its structure and function severely. Structural modifications to DNA mainly arise from modifications in its bases that primarily occur due to their exposure to different reactive species. Apart from this, DNA strand break, inter- and intra-strand crosslinks and DNA-protein crosslinks can also affect the structure of DNA significantly. These structural modifications are involved in mutation, cancer and many other diseases. As it has the least oxidation potential among all the DNA bases, guanine is frequently attacked by reactive species, producing a plethora of lethal lesions. Fortunately, living cells are evolved with intelligent enzymes that continuously protect DNA from such damages. This review provides an overview of different guanine lesions formed due to reactions of guanine with different reactive species. Involvement of these lesions in inter- and intra-strand crosslinks, DNA-protein crosslinks and mutagenesis are discussed. How certain enzymes recognize and repair different guanine lesions in DNA are also presented.

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

    Science.gov (United States)

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

    2016-04-07

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Stirling, Peter C; Hieter, Philip

    2016-07-22

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

  2. DNA replication, repair, and repair tests. [Rat; human leukocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lambert, B.

    1980-09-01

    The rate of inhibition and recovery of DNA synthesis can be used in a rapid assay system to detect genotoxic potentials of chemicals. Also, the observation that an agent stimulates DNA repair in a test system indicates its ability to cause damage in DNA. Different experimental approaches to the study of repair synthesis are discussed.

  3. DNA damage by reactive species: Mechanisms, mutation and repair

    Indian Academy of Sciences (India)

    N R Jena

    2012-07-01

    DNA is continuously attacked by reactive species that can affect its structure and function severely. Structural modifications to DNA mainly arise from modifications in its bases that primarily occur due to their exposure to different reactive species. Apart from this, DNA strand break, inter- and intra-strand crosslinks and DNA–protein crosslinks can also affect the structure of DNA significantly. These structural modifications are involved in mutation, cancer and many other diseases. As it has the least oxidation potential among all the DNA bases, guanine is frequently attacked by reactive species, producing a plethora of lethal lesions. Fortunately, living cells are evolved with intelligent enzymes that continuously protect DNA from such damages. This review provides an overview of different guanine lesions formed due to reactions of guanine with different reactive species. Involvement of these lesions in inter- and intra-strand crosslinks, DNA–protein crosslinks and mutagenesis are discussed. How certain enzymes recognize and repair different guanine lesions in DNA are also presented.

  4. UV-laser crosslinking of proteins to DNA.

    Science.gov (United States)

    Moss, T; Dimitrov, S I; Houde, D

    1997-02-01

    Photochemical crosslinking is now a powerful method for studying protein-nucleic acid interactions. UV light is a zero-length crosslinking agent that predominantly or exclusively crosslinks proteins to nucleic acids at their contact points. It can therefore provide strong evidence for close protein-nucleic acid interactions. However, to achieve an acceptable degree of crosslinking with conventional UV light sources, exposure times ranging from minutes to several hours are necessary. Such prolonged irradiation allows for the artifactual redistribution of proteins and precludes kinetic studies. The use of UV lasers overcomes these difficulties since the number of photons required for the crosslinking may be delivered in time intervals on the order of nano- or even picoseconds. We described detailed procedures for UV laser-induced protein-DNA crosslinking both in vivo and in vitro. Technical aspects, including the choice of UV laser for irradiation, the isolation of covalently crosslinked protein-DNA complexes, immunochemical techniques for both the identification and isolation of specific protein-DNA complexes and the identification of the crosslinked DNA sequences, are reviewed in detail. The application of UV laser crosslinking in kinetic studies is illustrated by the example of the TATA-binding protein (TBP) interaction with the adenovirus E4 promoter.

  5. Assessment of DNA interstrand crosslinks using the modified alkaline comet assay.

    Science.gov (United States)

    Wu, Jian Hong; Jones, Nigel J

    2012-01-01

    The single cell gel electrophoresis (SCGE) assay, more commonly known as the comet assay, due to the "comet-like" appearance of the cells, was originally developed as a technique to measure the presence of DNA single-strand breaks. The assay is performed on single cells embedded in agar and placed in an electrical field at alkaline pH, so that fragments of negatively charged single-stranded DNA move through the gel toward the positively charged anode. Undamaged DNA moves relatively slowly, forming the head of the comet, while DNA fragmented due to the presence of single-strand breaks, moves more quickly giving the appearance of the tail. The extent of DNA migration is a measure of the DNA damage present. Since it was first developed, the comet assay has been adapted for measuring other types of DNA damage. The neutral comet assay has been employed for DNA double-strand breaks, while techniques using DNA repair enzymes to cleave specific adducts, UvrABC for ultraviolet radiation induced adducts, for example, have also been described. Here, we describe a modified version of the comet assay for the measurement of interstrand crosslinks (ICLs). Interstrand crosslinking agents include the chemotherapeutic agents mitomycin C and cis-platin, psoralen plus UVA light (PUVA) used to treat hyperproliferative skin disorders and diepoxybutane, a metabolite of 1,3-butadiene used in industrial processes and an environmental pollutant. ICLs are a potent and cytotoxic form of DNA damage as they prevent DNA strand separation, thereby preventing DNA replication. Their removal requires several different DNA repair processes including translesion synthesis and homologous recombination. As ICLs prevent separation of the DNA strands, their presence results in less DNA migration in the comet assay. To successfully measure ICLs, it is necessary to incorporate a step that induces single-strand breaks (using a defined dose of ionizing radiation) that allows the crosslinked DNA to migrate.

  6. FANCM interacts with PCNA to promote replication traverse of DNA interstrand crosslinks.

    Science.gov (United States)

    Rohleder, Florian; Huang, Jing; Xue, Yutong; Kuper, Jochen; Round, Adam; Seidman, Michael; Wang, Weidong; Kisker, Caroline

    2016-04-20

    FANCM is a highly conserved DNA remodeling enzyme that promotes the activation of the Fanconi anemia DNA repair pathway and facilitates replication traverse of DNA interstrand crosslinks. However, how FANCM interacts with the replication machinery to promote traverse remains unclear. Here, we show that FANCM and its archaeal homolog Hef from Thermoplasma acidophilum interact with proliferating cell nuclear antigen (PCNA), an essential co-factor for DNA polymerases in both replication and repair. The interaction is mediated through a conserved PIP-box; and in human FANCM, it is strongly stimulated by replication stress. A FANCM variant carrying a mutation in the PIP-box is defective in promoting replication traverse of interstrand crosslinks and is also inefficient in promoting FANCD2 monoubiquitination, a key step of the Fanconi anemia pathway. Our data reveal a conserved interaction mode between FANCM and PCNA during replication stress, and suggest that this interaction is essential for FANCM to aid replication machines to traverse DNA interstrand crosslinks prior to post-replication repair.

  7. Extreme dryness and DNA-protein cross-links

    Science.gov (United States)

    Bieger-Dose, A.; Dose, K.; Meffert, R.; Mehler, M.; Risi, S.

    Exposure of fungal conidia (Aspergillus ochraceus) or spores of Bacillus subtilis to extreme dryness or vacuum induces DNA lesions, including strand breaks and the formation of DNA-protein cross-links. In wet cells only a small amount of protein is bound to DNA, but exposure to conditions of lowered water activity results in an increasing number of cross-links between DNA and proteins. In fungal conidia these cross-links are detected after selective iodination (125J) of the DNA-bound proteins followed by gel electrophoresis and subsequent autoradiography. Another approach is the labelling of DNA with 32p by means of nick translation and the detection of differences in the electrophoretic mobility of DNA before and after digestion with proteinase K of proteins bound to DNA.

  8. Formaldehyde catabolism is essential in cells deficient for the Fanconi anemia DNA-repair pathway.

    Science.gov (United States)

    Rosado, Ivan V; Langevin, Frédéric; Crossan, Gerry P; Takata, Minoru; Patel, Ketan J

    2011-11-13

    Metabolism is predicted to generate formaldehyde, a toxic, simple, reactive aldehyde that can damage DNA. Here we report a synthetic lethal interaction in avian cells between ADH5, encoding the main formaldehyde-detoxifying enzyme, and the Fanconi anemia (FA) DNA-repair pathway. These results define a fundamental role for the combined action of formaldehyde catabolism and DNA cross-link repair in vertebrate cell survival.

  9. DNA repair mechanisms in C. elegans

    NARCIS (Netherlands)

    Brouwer, K.|info:eu-repo/dai/nl/336462557

    2009-01-01

    DNA is the carrier of genetic information. DNA is constantly damaged by, for example, UV light and X-rays. Cells can utilize a large number of proteins that can repair the damages, thereby avoiding changes in the DNA sequence. Damages that are not repaired result in an increase in the number of muta

  10. DNA repair phenotype and dietary antioxidant supplementation

    DEFF Research Database (Denmark)

    Guarnieri, Serena; Loft, Steffen; Riso, Patrizia

    2008-01-01

    -release vitamin C tablets had increased DNA repair activity (27 (95 % CI 12, 41) % higher incision activity). These subjects also benefited from the supplementation by reduced levels of oxidised guanines in MNBC. In conclusion, nutritional status, DNA repair activity and DNA damage are linked, and beneficial...

  11. ROLE OF MISMATCH REPAIR PROTEINS IN THE PROCESSING OF CISPLATIN INTERSTRAND CROSS-LINKS

    Science.gov (United States)

    Sawant, Akshada; Kothandapani, Anbarasi; Zhitkovich, Anatoly; Sobol, Robert W.; Patrick, Steve M.

    2015-01-01

    Mismatch repair (MMR) deficiency gives rise to cisplatin resistance and can lead to poor prognosis in cancers. Various models have been proposed to explain this low level of resistance caused due to loss of MMR proteins. We have shown that MMR proteins are required to maintain cisplatin interstrand cross-links (ICLs) on the DNA leading to increased cellular sensitivity. In our previous studies, we have shown that BER processing of the cisplatin ICLs is mutagenic. Polymerase β (Polβ) can generate mismatches which leads to the activation and the recruitment of mismatch repair proteins. In this paper, we distinguished between the requirement of different downstream MMR proteins for maintaining cisplatin sensitivity. We show that the MutSα (MSH2-MSH6) heterocomplex is required to maintain cisplatin sensitivity, whereas the Mutsβ complex has no effect. These results can be correlated with the increased repair of cisplatin ICLs and ICL induced DNA double strand breaks (DSBs) in the resistant cells. Moreover, we show that MLH1 proficient cells displayed a cisplatin sensitive phenotype when compared with the MLH1 deficient cells and the ATPase activity of MLH1 is essential to mediate this effect. Based on these results, we propose that MutSα as well as the downstream MMR pathway proteins are essential to maintain a cisplatin sensitive phenotype as a consequence of processing Polβ induced mismatches at sites flanking cisplatin ICLs. PMID:26519826

  12. 1,2,3,4-Diepoxybutane-induced DNA-protein cross-linking in human fibrosarcoma (HT1080) cells.

    Science.gov (United States)

    Gherezghiher, Teshome B; Ming, Xun; Villalta, Peter W; Campbell, Colin; Tretyakova, Natalia Y

    2013-05-03

    1,2,3,4-Diepoxybutane (DEB) is the key carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is a genotoxic bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs). In the present work, mass spectrometry-based proteomics was employed to characterize DEB-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. Over 150 proteins including histones, high mobility group proteins, transcription factors, splicing factors, and tubulins were found among those covalently cross-linked to chromosomal DNA in the presence of DEB. A large portion of the cross-linked proteins are known factors involved in DNA binding, transcriptional regulation, cell signaling, DNA repair, and DNA damage response. HPLC-ESI(+)-MS/MS analysis of total proteolytic digests revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol conjugates, confirming that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA. However, relatively high concentrations of DEB were required to achieve significant DPC formation, indicating that it is a poor cross-linking agent as compared to antitumor nitrogen mustards and platinum compounds.

  13. Characterization of Interstrand DNA-DNA Cross-Links Using the α-Hemolysin Protein Nanopore.

    Science.gov (United States)

    Zhang, Xinyue; Price, Nathan E; Fang, Xi; Yang, Zhiyu; Gu, Li-Qun; Gates, Kent S

    2015-12-22

    Nanopore-based sensors have been studied extensively as potential tools for DNA sequencing, characterization of epigenetic modifications such as 5-methylcytosine, and detection of microRNA biomarkers. In the studies described here, the α-hemolysin protein nanopore embedded in a lipid bilayer was used for the detection and characterization of interstrand cross-links in duplex DNA. Interstrand cross-links are important lesions in medicinal chemistry and toxicology because they prevent the strand separation that is required for read-out of genetic information from DNA in cells. In addition, interstrand cross-links are used for the stabilization of duplex DNA in structural biology and materials science. Cross-linked DNA fragments produced unmistakable current signatures in the nanopore experiment. Some cross-linked substrates gave irreversible current blocks of >10 min, while others produced long current blocks (10-100 s) before the double-stranded DNA cross-link translocated through the α-hemolysin channel in a voltage-driven manner. The duration of the current block for the different cross-linked substrates examined here may be dictated by the stability of the duplex region left in the vestibule of the nanopore following partial unzipping of the cross-linked DNA. Construction of calibration curves measuring the frequency of cross-link blocking events (1/τon) as a function of cross-link concentration enabled quantitative determination of the amounts of cross-linked DNA present in samples. The unique current signatures generated by cross-linked DNA in the α-HL nanopore may enable the detection and characterization of DNA cross-links that are important in toxicology, medicine, and materials science.

  14. Structure of a DNA glycosylase that unhooks interstrand cross-links

    Energy Technology Data Exchange (ETDEWEB)

    Mullins, Elwood A.; Warren, Garrett M.; Bradley, Noah P.; Eichman, Brandt F. (Vanderbilt)

    2017-04-10

    DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.

  15. Chromatin structure and DNA damage repair

    Directory of Open Access Journals (Sweden)

    Dinant Christoffel

    2008-11-01

    Full Text Available Abstract 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.

  16. Role of Deubiquitinating Enzymes in DNA Repair

    OpenAIRE

    2016-01-01

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

  17. Changes in DNA repair during aging

    Science.gov (United States)

    Gorbunova, Vera; Seluanov, Andrei; Mao, Zhiyong; Hine, Christpher

    2007-01-01

    DNA is a precious molecule. It encodes vital information about cellular content and function. There are only two copies of each chromosome in the cell, and once the sequence is lost no replacement is possible. The irreplaceable nature of the DNA sets it apart from other cellular molecules, and makes it a critical target for age-related deterioration. To prevent DNA damage cells have evolved elaborate DNA repair machinery. Paradoxically, DNA repair can itself be subject to age-related changes and deterioration. In this review we will discuss the changes in efficiency of mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER) and double-strand break (DSB) repair systems during aging, and potential changes in DSB repair pathway usage that occur with age. Mutations in DNA repair genes and premature aging phenotypes they cause have been reviewed extensively elsewhere, therefore the focus of this review is on the comparison of DNA repair mechanisms in young versus old. PMID:17913742

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

    Directory of Open Access Journals (Sweden)

    Kate eColeman

    2016-04-01

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

  19. p53 downregulates the Fanconi anaemia DNA repair pathway.

    Science.gov (United States)

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

    2016-04-01

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

  20. Swelling properties of cross-linked DNA gels.

    Science.gov (United States)

    Costa, Diana; Miguel, M Graça; Lindman, Björn

    2010-07-12

    This work represents our contribution to the field of physical chemistry of DNA gels, and concerns the synthesis and study of novel chemically cross-linked DNA gels. The use of covalent DNA gels is a very promising way to study DNA-cosolute interactions, as well as the dynamic behaviour of DNA and cationic compacting agents, like lipids, surfactants and polycations. Manipulating DNA in new ways, like DNA networks, allows a better understanding and characterization of DNA-cosolute complexes at the molecular level, and also allows us to follow the assembly structures of these complexes. The use of responsive polymer gels for targeted delivery of toxic and/or labile drugs has, during the past few years, shown to be a promising concept. The features found in the proposed system would find applications in a broader field of gel/drug interaction, for the development of controlled release and targeted delivery devices.

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

    Energy Technology Data Exchange (ETDEWEB)

    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)

  2. DNA interstrand cross-linking by a mycotoxic diepoxide.

    Science.gov (United States)

    Millard, J T; Katz, J L; Goda, J; Frederick, E D; Pierce, S E; Speed, T J; Thamattoor, D M

    2004-06-01

    The diepoxide mycotoxin (2R, 3R, 8R, 9R)-4,6-decadiyne-2,3:8,9-diepoxy-1,10-diol (repandiol) was both isolated from the mushroom Hydnum repandum and synthesized de novo. Repandiol was found to form interstrand cross-links within a restriction fragment of DNA, linking deoxyguanosines on opposite strands primarily within the 5'-GNC and 5'-GNNC sequences preferred by diepoxyoctane. However, repandiol was a significantly less efficient cross-linker than either of the diepoxyalkanes (diepoxyoctane and diepoxybutane) to which it was compared.

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

    Energy Technology Data Exchange (ETDEWEB)

    Phoebe L. Stewart

    2005-06-27

    Exposure to low levels of ionizing radiation causes DNA double-strand breaks (DSBs) that must be repaired for cell survival. Higher eukaryotes respond to DSBs by arresting the cell cycle, presumably to repair the DNA lesions before cell division. In mammalian cells, the nonhomologous end-joining DSB repair pathway is mediated by the 470 kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs) together with the DNA-binding factors Ku70 and Ku80. Mouse knock-out models of these three proteins are all exquisitely sensitive to low doses of ionizing radiation. In the presence of DNA ends, Ku binds to the DNA and then recruits DNA-PKcs. After formation of the complex, the kinase activity associated with DNA-PKcs becomes activated. This kinase activity has been shown to be essential for repairing DNA DSBs in vivo since expression of a kinase-dead form of DNA-PKcs in a mammalian cell line that lacks DNA-PKcs fails to complement the radiosensitive phenotype. The immense size of DNA-PKcs suggests that it may also serve as a docking site for other DNA repair proteins. Since the assembly of the DNA-PK complex onto DNA is a prerequisite for DSB repair, it is critical to obtain structural information on the complex. Cryo-electron microscopy (cryo-EM) and single particle reconstruction methods provide a powerful way to image large macromolecular assemblies at near atomic (10-15 ?) resolution. We have already used cryo-EM methods to examine the structure of the isolated DNA-PKcs protein. This structure reveals numerous cavities throughout the protein that may allow passage of single or double-stranded DNA. Pseudo two-fold symmetry was found for the monomeric protein, suggesting that DNA-PKcs may interact with two DNA ends or two Ku heterodimers simultaneously. Here we propose to study the structure of the cross-linked DNA-PKcs/Ku/DNA complex. Difference imaging with our published DNA-PKcs structure will enable us to elucidate the architecture of the complex. A second

  4. Repair of DNA Double-Strand Breaks

    Science.gov (United States)

    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.

  5. DNA-Protein Cross-Links: Formation, Structural Identities, and Biological Outcomes.

    Science.gov (United States)

    Tretyakova, Natalia Y; Groehler, Arnold; Ji, Shaofei

    2015-06-16

    Noncovalent DNA-protein interactions are at the heart of normal cell function. In eukaryotic cells, genomic DNA is wrapped around histone octamers to allow for chromosomal packaging in the nucleus. Binding of regulatory protein factors to DNA directs replication, controls transcription, and mediates cellular responses to DNA damage. Because of their fundamental significance in all cellular processes involving DNA, dynamic DNA-protein interactions are required for cell survival, and their disruption is likely to have serious biological consequences. DNA-protein cross-links (DPCs) form when cellular proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. DPCs progressively accumulate in the brain and heart tissues as a result of endogenous exposure to reactive oxygen species and lipid peroxidation products, as well as normal cellular metabolism. A range of structurally diverse DPCs are found following treatment with chemotherapeutic drugs, transition metal ions, and metabolically activated carcinogens. Because of their considerable size and their helix-distorting nature, DPCs interfere with the progression of replication and transcription machineries and hence hamper the faithful expression of genetic information, potentially contributing to mutagenesis and carcinogenesis. Mass spectrometry-based studies have identified hundreds of proteins that can become cross-linked to nuclear DNA in the presence of reactive oxygen species, carcinogen metabolites, and antitumor drugs. While many of these proteins including histones, transcription factors, and repair proteins are known DNA binding partners, other gene products with no documented affinity for DNA also participate in DPC formation. Furthermore, multiple sites within DNA can be targeted for cross-linking including the N7 of guanine, the C-5 methyl group of thymine, and the exocyclic amino groups of guanine, cytosine, and adenine. This structural

  6. Efficient DNA interstrand crosslinking by 6-thioguanine and UVA radiation.

    Science.gov (United States)

    Brem, Reto; Daehn, Ilse; Karran, Peter

    2011-08-15

    Patients taking the immunosuppressant and anticancer thiopurines 6-mercaptopurine, azathioprine or 6-thioguanine (6-TG), develop skin cancer at a very high frequency. Their DNA contains 6-TG which absorbs ultraviolet A (UVA) radiation, and their skin is UVA hypersensitive, consistent with the formation of DNA photodamage. Here we demonstrate that UVA irradiation of 6-TG-containing DNA causes DNA interstrand crosslinking. In synthetic duplex oligodeoxynucleotides, the interstrand crosslinks (ICLs) can form between closely opposed 6-TG bases and, in a less favoured reaction, between 6-TG and normal bases on the opposite strand. In vivo, UVA irradiation of cultured cells containing 6-TG-substituted DNA also causes ICL formation and induces the chromosome aberrations that are characteristically associated with this type of DNA lesion. 6-TG/UVA activates the Fanconi anemia (FA) pathway via monoubiquitination of the FANCD2 protein. Cells defective in the FA pathway or other factors involved in ICL processing, such as XPF and DNA Polζ, are all hypersensitive to killing by 6-TG/UVA-consistent with a significant contribution of photochemical ICLs to the cytotoxicity of this treatment. Our findings suggest that sunlight-exposed skin of thiopurine treated patients may experience chronic photochemical DNA damage that requires constant intervention of the FA pathway.

  7. Damage, DNA Repair, Aging, and Neurodegeneration

    Science.gov (United States)

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

    2017-01-01

    Aging in mammals is accompanied by a progressive atrophy of tissues and organs, and stochastic damage accumulation to the macromolecules DNA, RNA, proteins, and lipids. The sequence of the human genome represents our genetic blueprint, and accumulating evidence suggests that loss of genomic maintenance may causally contribute to aging. Distinct evidence for a role of imperfect DNA repair in aging is that several premature aging syndromes have underlying genetic DNA repair defects. Accumulation of DNA damage may be particularly prevalent in the central nervous system owing to the low DNA repair capacity in postmitotic brain tissue. It is generally believed that the cumulative effects of the deleterious changes that occur in aging, mostly after the reproductive phase, contribute to species-specific rates of aging. In addition to nuclear DNA damage contributions to aging, there is also abundant evidence for a causative link between mitochondrial DNA damage and the major phenotypes associated with aging. Understanding the mechanistic basis for the association of DNA damage and DNA repair with aging and age-related diseases, such as neurodegeneration, would give insight into contravening age-related diseases and promoting a healthy life span. PMID:26385091

  8. DNA repair genes in the Megavirales pangenome.

    Science.gov (United States)

    Blanc-Mathieu, Romain; Ogata, Hiroyuki

    2016-06-01

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

  9. Carcinogenesis switched on by DNA cross-link between complementary bases aroused by aflatoxin and N-nitroso compounds

    Institute of Scientific and Technical Information of China (English)

    DAI Qianhuan; LU Ping; PENG Shaohua; ZHANG Qingrong

    2003-01-01

    The di-region theory put forward by Dai Qianhuan, a molecular mechanism of chemical carcinogenesis, concluded that the carcinogenesis induced by most of the environmental carcinogens is switched on by the cross-linking between DNA complementary bases aroused by the bifunctional alkylation of their metabolic intermediates. It was evidenced in this paper with DNA filter elution method that one carcinogenic mycotoxin, aflatoxin G1, four carcinogenic N-nitroso compounds, N-nitrosodiethyl-amine, N-nitrosodibutyl-amine, N-nitrosomorpholine and N-nitrosopyrrolidine, one carcinogenic diazo color, 4-dimethylaminodiazobenzene and one carcinogenic nitrogen-containing heterocyclic compound, quinoline, all induced DNA interstrands cross-linking with dosage correlation after metabolic activation. However, the non-carcinogens in corresponding series for control, aflatoxin B2, N-nitroso-diphenylamine, 4′-bromo-4-dimethylaminodiazobenzene and isoquinoline, cannot induce DNA interstrands cross-linking at all in the same condition. A method for the determination of cross-linking ratio between DNA complementary bases in total DNA interstrands cross-linking, which has no monitoring measure as yet, has been established for the first time based upon a 24 hour repairing experiment. The DNA complementary pair cross-linking ratio induced by a metabolized carcinogen is correlated with its carcinogenic potential. It may be concluded that the mutations including point and frameshift mutagenesis induced by aflatoxin and other carcinogens are switched on by their corresponding cross-linking base pair between complementary bases. Therefore, the di-region theory is a reasonable molecular mechanism for chemical, endogenous and physical carcinogenesis.

  10. Chromatin challenges during DNA replication and repair

    DEFF Research Database (Denmark)

    Groth, Anja; Rocha, Walter; Verreault, Alain

    2007-01-01

    Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge, both in terms of accessibility and maintenance of chromatin organization. To meet...... the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic...

  11. [DNA homologous recombination repair in mammalian cells].

    Science.gov (United States)

    Popławski, Tomasz; Błasiak, Janusz

    2006-01-01

    DNA double-strand breaks (DSBs) are the most serious DNA damage. Due to a great variety of factors causing DSBs, the efficacy of their repair is crucial for the cell's functioning and prevents DNA fragmentation, chromosomal translocation and deletion. In mammalian cells DSBs can be repaired by non-homologous end joining (NHEJ), homologous recombination (HRR) and single strand annealing (SSA). HRR can be divided into the first and second phase. The first phase is initiated by sensor proteins belonging to the MRN complex, that activate the ATM protein which target HRR proteins to obtain the second response phase--repair. HRR is precise because it utilizes a non-damaged homologous DNA fragment as a template. The key players of HRR in mammalian cells are MRN, RPA, Rad51 and its paralogs, Rad52 and Rad54.

  12. DNA repair variants and breast cancer risk.

    Science.gov (United States)

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

    2016-05-01

    A functional DNA repair system has been identified as important in the prevention of tumour development. Previous studies have hypothesized that common polymorphisms in DNA repair genes could play a role in breast cancer risk and also identified the potential for interactions between these polymorphisms and established breast cancer risk factors such as physical activity. Associations with breast cancer risk for 99 single nucleotide polymorphisms (SNPs) from genes in ten DNA repair pathways were examined in a case-control study including both Europeans (644 cases, 809 controls) and East Asians (299 cases, 160 controls). Odds ratios in both additive and dominant genetic models were calculated separately for participants of European and East Asian ancestry using multivariate logistic regression. The impact of multiple comparisons was assessed by correcting for the false discovery rate within each DNA repair pathway. Interactions between several breast cancer risk factors and DNA repair SNPs were also evaluated. One SNP (rs3213282) in the gene XRCC1 was associated with an increased risk of breast cancer in the dominant model of inheritance following adjustment for the false discovery rate (P breast cancer risk or their modification by breast cancer risk factors were observed.

  13. AlkB recognition of a bulky DNA base adduct stabilized by chemical cross-linking

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    E.coli AlkB is a direct DNA/RNA repair protein that oxidatively reverses N1 alkylated purines and N3 alkylated pyrimidines to regular bases.Previous crystal structures have revealed N1-methyl adenine(1-meA) recognition by AlkB and a unique base flipping mechanism,but how the AlkB active site can accommodate bulky base adducts is largely unknown.Employing a previously developed chemical cross-linking technique,we crystallized AlkB with a duplex DNA containing a caged thymine base(cagedT).The structure revealed a flexible hairpin lid and a reorganized substrate recognition loop used by AlkB to accommodate cagedT.These observations demonstrate,at the molecular level,how bulky DNA adducts may be recognized and processed by AlkB.

  14. Genipin-crosslinked fibrin hydrogels as a potential adhesive to augment intervertebral disc annulus repair.

    Science.gov (United States)

    Schek, R M; Michalek, A J; Iatridis, J C

    2011-04-18

    Treatment of damaged intervertebral discs is a significant clinical problem and, despite advances in the repair and replacement of the nucleus pulposus, there are few effective strategies to restore defects in the annulus fibrosus. An annular repair material should meet three specifications: have a modulus similar to the native annulus tissue, support the growth of disc cells, and maintain adhesion to tissue under physiological strain levels. We hypothesized that a genipin crosslinked fibrin gel could meet these requirements. Our mechanical results showed that genipin crosslinked fibrin gels could be created with a modulus in the range of native annular tissue. We also demonstrated that this material is compatible with the in vitro growth of human disc cells, when genipin:fibrin ratios were 0.25:1 or less, although cell proliferation was slower and cell morphology more rounded than for fibrin alone. Finally, lap tests were performed to evaluate adhesion between fibrin gels and pieces of annular tissue. Specimens created without genipin had poor handling properties and readily delaminated, while genipin crosslinked fibrin gels remained adhered to the tissue pieces at strains exceeding physiological levels and failed at 15-30%. This study demonstrated that genipin crosslinked fibrin gels show promise as a gap-filling adhesive biomaterial with tunable material properties, yet the slow cell proliferation suggests this biomaterial may be best suited as a sealant for small annulus fibrosus defects or as an adhesive to augment large annulus repairs. Future studies will evaluate degradation rate, fatigue behaviors, and long-term biocompatibility.

  15. DNA repair: keeping it together

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2004-01-01

    A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest.......A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest....

  16. DNA repair, immunosuppression, and skin cancer.

    Science.gov (United States)

    Yarosh, Daniel B

    2004-11-01

    UV radiation (UVR) produces erythema within the first 24 hours of exposure, suppression of the immune system within the first 10 days, and, for many people, over the course of decades, skin cancer. Although UVR damages many skin targets, DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) is an important mediator of these sequelae. The action spectrum for erythema parallels the action spectrum for CPD formation in skin, and in the absence of repair, as in the genetic disease xeroderma pigmentosum (XP), skin cancer rates are dramatically increased. DNA repair in skin can be enhanced by the delivery of DNA repair enzymes encapsulated in liposomes. Used in this way, photoreactivation of CPDs greatly diminishes erythema and the suppression of contact hypersensitivity (CHS). UV endonucleases delivered by liposomes also prevent UV-induced suppression of delayed-type hypersensitivity. In a clinical study of patients with XP, T4 endonuclease V (T4N5) liposome lotion applied for one year reduced the rates of actinic keratosis (AK) and skin cancer compared with placebo. These results showed that strategies to increase sun protection should include measures to reduce DNA damage and increase the rate of DNA repair.

  17. Bifunctional alkylating agent-mediated MGMT-DNA cross-linking and its proteolytic cleavage in 16HBE cells.

    Science.gov (United States)

    Cheng, Jin; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Wang, Bin; Zhao, Jiqing; Sai, Yan; Zou, Zhongmin

    2016-08-15

    Nitrogen mustard (NM), a bifunctional alkylating agent (BAA), contains two alkyl arms and can act as a cross-linking bridge between DNA and protein to form a DNA-protein cross-link (DPC). O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme for alkyl adducts removal, is found to enhance cell sensitivity to BAAs and to promote damage, possibly due to its stable covalent cross-linking with DNA mediated by BAAs. To investigate MGMT-DNA cross-link (mDPC) formation and its possible dual roles in NM exposure, human bronchial epithelial cell line 16HBE was subjected to different concentrations of HN2, a kind of NM, and we found mDPC was induced by HN2 in a concentration-dependent manner, but the mRNA and total protein of MGMT were suppressed. As early as 1h after HN2 treatment, high mDPC was achieved and the level maintained for up to 24h. Quick total DPC (tDPC) and γ-H2AX accumulation were observed. To evaluate the effect of newly predicted protease DVC1 on DPC cleavage, we applied siRNA of MGMT and DVC1, MG132 (proteasome inhibitor), and NMS-873 (p97 inhibitor) and found that proteolysis plays a role. DVC1 was proven to be more important in the cleavage of mDPC than tDPC in a p97-dependent manner. HN2 exposure induced DVC1 upregulation, which was at least partially contributed to MGMT cleavage by proteolysis because HN2-induced mDPC level and DNA damage was closely related with DVC1 expression. Homologous recombination (HR) was also activated. Our findings demonstrated that MGMT might turn into a DNA damage promoter by forming DPC when exposed to HN2. Proteolysis, especially DVC1, plays a crucial role in mDPC repair.

  18. Assessment of Human DNA Repair (NER) Capacity With DNA Repair Rate (DRR) by Comet Assay

    Institute of Scientific and Technical Information of China (English)

    WEI ZHENG; JI-LIANG HE; LI-FEN JIN; JIAN-LIN LOU; BAO-HONG WANG

    2005-01-01

    Objective Alkaline comet assay was used to evaluate DNA repair (nucleotide excision repair, NER) capacity of human fresh lymphocytes from 12 young healthy non-smokers (6 males and 6 females). Methods Lymphocytes were exposed to UV-C (254 nm) at the dose rate of 1.5 J/m2/sec. Novobiocin (NOV) and aphidicolin (APC), DNA repair inhibitors, were utilized to imitate the deficiency of DNA repair capacity at the incision and ligation steps of NER. Lymphocytes from each donor were divided into three grougs: UVC group, UVC plus NOV group, and UVC plus APC group. DNA single strand breaks were detected in UVC irradiated cells incubated for 0, 30, 60, 90, 120, 180, and 240 min after UVC irradiation. DNA repair rate (DRR) served as an indicator of DNA repair capacity. Results The results indicated that the maximum DNA damage (i.e. maximum tail length) in the UVC group mainly appeared at 90 min. The ranges of DRRs in the UVC group were 62.84%-98.71%. Average DRR value was 81.84%. The DRR difference between males and females was not significant (P<0.05). However, the average DRR value in the UVC plus NOV group and the UVC plus APC group was 52.98% and 39.57% respectively, which were significantly lower than that in the UVC group (P<0.01). Conclusion The comet assay is a rapid, simple and sensitive screening test to assess individual DNA repair (NER) capacity. It is suggested that the time to detect DNA single strand breaks in comet assay should include 0 (before UV irradiation), 90 and 240 min after exposure to 1.5 J·m-2 UVC at least. The DRR, as an indicator, can represent the individual DNA repair capacity in comet assay.

  19. DNA Repair Systems: Guardians of the Genome

    Indian Academy of Sciences (India)

    2016-10-01

    The 2015 Nobel Prize in Chemistry was awarded jointly to Tomas Lindahl, Paul Modrich and Aziz Sancar to honour their accomplishments in the field of DNA repair. Ever since the discovery of DNA structure and their importance in the storage of genetic information, questions about their stability became pertinent. A molecule which is crucial for the development and propagation of an organism must be closely monitored so that the genetic information is not corrupted. Thanks to the pioneering research work of Lindahl, Sancar, Modrich and their colleagues, we now have an holistic awareness of how DNA damage occurs and how the damage is rectified in bacteria as well as in higher organisms including human beings. A comprehensive understanding of DNA repair has proven crucial in the fight against cancer and other debilitating diseases.

  20. DNA melting properties of the dityrosine cross-linked dimer of Ribonuclease A.

    Science.gov (United States)

    Dinda, Amit Kumar; Chattaraj, Saparya; Ghosh, Sudeshna; Tripathy, Debi Ranjan; Dasgupta, Swagata

    2016-09-01

    Several DNA binding proteins exist in dimeric form when bound with DNA to be able to exhibit various biological processes such as DNA repair, DNA replication and gene expression. Various dimeric forms of Ribonuclease A (RNase A) and other members of the ribonuclease A superfamily are endowed with a multitude of biological activities such as antitumor and antiviral activity. In the present study, we have compared the DNA binding properties between the RNase A monomer and the dityrosine (DT) cross-linked RNase A dimer, and checked the inhibitory effect of DNA on the ribonucleolytic activity of the dimeric protein. An agarose gel based assay shows that like the monomer, the dimer also binds with DNA. The number of nucleotides bound per monomer unit of the dimer is higher than the number of nucleotides that bind with the each monomer. From fluorescence measurements, the association constant (Ka) values for complexation of the monomer and the dimer with ct-DNA are (4.95±0.45)×10(4)M(-1) and (1.29±0.05)×10(6)M(-1) respectively. Binding constant (Kb) values for the binding of the monomer and the dimer with ct-DNA were determined using UV-vis spectroscopy and were found to be (4.96±1.67)×10(4)M(-1) and (4.32±0.31)×10(5)M(-1) respectively. Circular dichroism studies shows that the dimer possesses significant effect on DNA conformation. The melting profile for the ct-DNA-dimer indicated that the melting temperature (Tm) for the ct-DNA-dimer complex is lower compared to the ct-DNA-monomer complex. The ribonucleolytic activity of the dimer, like the monomer, diminishes upon binding with DNA.

  1. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Science.gov (United States)

    2010-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a... killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient...

  2. Investigation of DNA repair in human oocytes and preimplantation embryos

    OpenAIRE

    Jaroudi, S.

    2010-01-01

    DNA repair genes are expressed in mammalian embryos and in human germinal vesicles, however, little is known about DNA repair in human preimplantation embryos. This project had three aims: 1) to produce a DNA repair profile of human MII oocytes and blastocysts using expression arrays and identify repair pathways that may be active before and after embryonic genome activation; 2) to design an in vitro functional assay that targeted mismatch repair and which could be applied to human oocytes...

  3. Transcription-coupled DNA repair in prokaryotes.

    Science.gov (United States)

    Ganesan, Ann; Spivak, Graciela; Hanawalt, Philip C

    2012-01-01

    Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair (NER) that acts specifically on lesions in the transcribed strand of expressed genes. First reported in mammalian cells, TCR was then documented in Escherichia coli. In this organism, an RNA polymerase arrested at a lesion is displaced by the transcription repair coupling factor, Mfd. This protein recruits the NER lesion-recognition factor UvrA, and then dissociates from the DNA. UvrA binds UvrB, and the assembled UvrAB* complex initiates repair. In mutants lacking active Mfd, TCR is absent. A gene transcribed by the bacteriophage T7 RNA polymerase in E. coli also requires Mfd for TCR. The CSB protein (missing or defective in cells of patients with Cockayne syndrome, complementation group B) is essential for TCR in humans. CSB and its homologs in higher eukaryotes are likely functional equivalents of Mfd. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Rupp, W Dean

    2013-12-13

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

  5. Relationships between chromatin remodeling and DNA damage repair induced by 8-methoxypsoralen and UVA in yeast Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Lavínia Almeida Cruz

    2012-01-01

    Full Text Available Eukaryotic cells have developed mechanisms to prevent genomic instability, such as DNA damage detection and repair, control of cell cycle progression and cell death induction. The bifunctional compound furocumarin 8-methoxy-psoralen (8-MOP is widely used in the treatment of various inflammatory skin diseases. In this review, we summarize recent data about the role of chromatin remodeling in the repair of DNA damage induced by treatment with 8-methoxypsoralen plus UVA (8-MOP+UVA, focusing on repair proteins in budding yeast Saccharomyces cerevisiae, an established model system for studying DNA repair pathways. The interstrand crosslinks (ICL formed by the 8-MOP+UVA treatment are detrimental lesions that can block transcription and replication, leading to cell death if not repaired. Current data show the involvement of different pathways in ICL processing, such as nucleotide excision repair (NER, base excision repair (BER, translesion repair (TLS and double-strand break repair. 8-MOP+UVA treatment in yeast enhances the expression of genes involved in the DNA damage response, double strand break repair by homologous replication, as well as genes related to cell cycle regulation. Moreover, alterations in the expression of subtelomeric genes and genes related to chromatin remodeling are consistent with structural modifications of chromatin relevant to DNA repair. Taken together, these findings indicate a specific profile in 8-MOP+UVA responses related to chromatin remodeling and DNA repair.

  6. Visible light crosslinking of methacrylated hyaluronan hydrogels for injectable tissue repair.

    Science.gov (United States)

    Fenn, Spencer L; Oldinski, Rachael A

    2016-08-01

    Tissue engineering hydrogels are primarily cured in situ using ultraviolet (UV) radiation which limits the use of hydrogels as drug or cell carriers. Visible green light activated crosslinking systems are presented as a safe alternative to UV photocrosslinked hydrogels, without compromising material properties such as viscosity and stiffness. The objective of this study was to fabricate and characterize photocrosslinked hydrogels with well-regulated gelation kinetics and mechanical properties for the repair or replacement of soft tissue. An anhydrous methacrylation of hyaluronan (HA) was performed to control the degree of modification (DOM) of HA, verified by (1) H-NMR spectroscopy. UV-activated crosslinking was compared to visible green light activated crosslinking. While the different photocrosslinking techniques resulted in varied crosslinking times, comparable mechanical properties of UV and green light activated crosslinked hydrogels were achieved using each photocrosslinking method by adjusting time of light exposure. Methacrylated HA (HA-MA) hydrogels of varying molecular weight, DOM, and concentration exhibited compressive moduli ranging from 1 kPa to 116 kPa, for UV crosslinking, and 3 kPa to 146 kPa, for green light crosslinking. HA-MA molecular weight and concentration were found to significantly influence moduli values. HA-MA hydrogels did not exhibit any significant cytotoxic effects toward human mesenchymal stem cells. Green light activated crosslinking systems are presented as a viable method to form natural-based hydrogels in situ. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1229-1236, 2016.

  7. DNA repair genotypes and phenotypes and cancer susceptibility

    Institute of Scientific and Technical Information of China (English)

    Qingyi Wei

    2008-01-01

    @@ The role of DNA repair in the etiology of cancers has been well illustrated in several hereditary syndromes, in which an inherited defect in DNA repair and related biological processes is associated with extraordinarily high incidence of cancer.

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

    Directory of Open Access Journals (Sweden)

    Kaja Milanowska

    2011-01-01

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

  9. Fragile DNA Repair Mechanism Reduces Ageing in Multicellular Model

    DEFF Research Database (Denmark)

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

    2012-01-01

    DNA damages, as well as mutations, increase with age. It is believed that these result from increased genotoxic stress and decreased capacity for DNA repair. The two causes are not independent, DNA damage can, for example, through mutations, compromise the capacity for DNA repair, which in turn...... to DNA damage can undergo full repair, go apoptotic, or accumulate mutations thus reducing DNA repair capacity. Our model predicts that at the tissue level repair rate does not continuously decline with age, but instead has a characteristic extended period of high and non-declining DNA repair capacity...... of compromised cells, thus freeing the space for healthy peers. This finding might be a first step toward understanding why a mutation in single DNA repair protein (e.g. Wrn or Blm) is not buffered by other repair proteins and therefore, leads to severe ageing disorders...

  10. Epigenetic reduction of DNA repair in progression togastrointestinal cancer

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Deficiencies in DNA repair due to inherited germ-linemutations in DNA repair genes cause increased risk ofgastrointestinal (GI) cancer. In sporadic GI cancers,mutations in DNA repair genes are relatively rare.However, epigenetic alterations that reduce expressionof DNA repair genes are frequent in sporadic GI cancers.These epigenetic reductions are also found in fielddefects that give rise to cancers. Reduced DNA repairlikely allows excessive DNA damages to accumulatein somatic cells. Then either inaccurate translesionsynthesis past the un-repaired DNA damages or errorproneDNA repair can cause mutations. ErroneousDNA repair can also cause epigenetic alterations (i.e. ,epimutations, transmitted through multiple replicationcycles). Some of these mutations and epimutations maycause progression to cancer. Thus, deficient or absentDNA repair is likely an important underlying cause ofcancer. Whole genome sequencing of GI cancers showthat between thousands to hundreds of thousands ofmutations occur in these cancers. Epimutations thatreduce DNA repair gene expression and occur early inprogression to GI cancers are a likely source of this highgenomic instability. Cancer cells deficient in DNA repairare more vulnerable than normal cells to inactivation byDNA damaging agents. Thus, some of the most clinicallyeffective chemotherapeutic agents in cancer treatmentare DNA damaging agents, and their effectivenessoften depends on deficient DNA repair in cancer cells.Recently, at least 18 DNA repair proteins, each activein one of six DNA repair pathways, were found to besubject to epigenetic reduction of expression in GIcancers. Different DNA repair pathways repair differenttypes of DNA damage. Evaluation of which DNA repairpathway(s) are deficient in particular types of GI cancerand/or particular patients may prove useful in guidingchoice of therapeutic agents in cancer therapy.

  11. DNA Repair and Genome Maintenance in Bacillus subtilis

    OpenAIRE

    2012-01-01

    Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutage...

  12. RPA activates the XPF‐ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks

    KAUST Repository

    Abdullah, Ummi B

    2017-06-13

    During replication‐coupled DNA interstrand crosslink (ICL) repair, the XPF‐ERCC1 endonuclease is required for the incisions that release, or “unhook”, ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL. Here, we report that while purified XPF‐ERCC1 incises simple ICL‐containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single‐stranded DNA (ssDNA)‐binding replication protein A (RPA) selectively restores XPF‐ERCC1 endonuclease activity on this structure. The 5′–3′ exonuclease SNM1A can load from the XPF‐ERCC1‐RPA‐induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF‐ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.

  13. Nuclear translocation contributes to regulation of DNA excision repair activities

    DEFF Research Database (Denmark)

    Knudsen, Nina Østergaard; Andersen, Sofie Dabros; Lützen, Anne;

    2009-01-01

    , it is evident that proteins from the different DNA repair pathways interact [Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev. 14 (2000) 927-939; M. Christmann, M......DNA mutations are circumvented by dedicated specialized excision repair systems, such as the base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways. Although the individual repair pathways have distinct roles in suppressing changes in the nuclear DNA.......T. Tomicic, W.P. Roos, B. Kaina, Mechanisms of human DNA repair: an update, Toxicology 193 (2003) 3-34; N.B. Larsen, M. Rasmussen, L.J. Rasmussen, Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 5 (2005) 89-108]. Protein interactions are not only important for function, but also...

  14. Focus on DNA Repair Replication

    NARCIS (Netherlands)

    A.M. Gourdin (Audrey)

    2010-01-01

    markdownabstract__Abstract__ The crucial factor for the survival of an organism resides in genetic stability. In fact the integrity of the DNA sequence, that carries out and regulates genetic information, can be impaired by inaccurate maintenance processes, endogenous metabolites or

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Cisplatin sensitivity of testis tumour cells is due to deficiency in interstrand-crosslink repair and low ERCC1-XPF expression

    Directory of Open Access Journals (Sweden)

    Kaina Bernd

    2010-09-01

    Full Text Available Abstract Background Cisplatin based chemotherapy cures over 80% of metastatic testicular germ cell tumours (TGCT. In contrast, almost all other solid cancers in adults are incurable once they have spread beyond the primary site. Cell lines derived from TGCTs are hypersensitive to cisplatin reflecting the clinical response. Earlier findings suggested that a reduced repair capacity might contribute to the cisplatin hypersensitivity of testis tumour cells (TTC, but the critical DNA damage has not been defined. This study was aimed at investigating the formation and repair of intrastrand and interstrand crosslinks (ICLs induced by cisplatin in TTC and their contribution to TTC hypersensitivity. Results We observed that repair of intrastrand crosslinks is similar in cisplatin sensitive TTC and resistant bladder cancer cells, whereas repair of ICLs was significantly reduced in TTC. γH2AX formation, which serves as a marker of DNA breaks formed in response to ICLs, persisted in cisplatin-treated TTC and correlated with sustained phosphorylation of Chk2 and enhanced PARP-1 cleavage. Expression of the nucleotide excision repair factor ERCC1-XPF, which is implicated in the processing of ICLs, is reduced in TTC. To analyse the causal role of ERCC1-XPF for ICL repair and cisplatin sensitivity, we over-expressed ERCC1-XPF in TTC by transient transfection. Over-expression increased ICL repair and rendered TTC more resistant to cisplatin, which suggests that ERCC1-XPF is rate-limiting for repair of ICLs resulting in the observed cisplatin hypersensitivity of TTC. Conclusion Our data indicate for the first time that the exceptional sensitivity of TTC and, therefore, very likely the curability of TGCT rests on their limited ICL repair due to low level of expression of ERCC1-XPF.

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

    Science.gov (United States)

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

    1999-02-01

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

  18. Relative toxicities of DNA cross-links and monoadducts: new insights from studies of decarbamoyl mitomycin C and mitomycin C.

    Science.gov (United States)

    Palom, Yolanda; Suresh Kumar, Gopinatha; Tang, Li-Qian; Paz, Manuel M; Musser, Steven M; Rockwell, Sara; Tomasz, Maria

    2002-11-01

    Mitomycin C (MC), a cytotoxic anticancer drug and bifunctional DNA DNA alkylating agent, induces cross-linking of the complementary strands of DNA. The DNA interstrand cross-links (ICLs) are thought to be the critical cytotoxic lesions produced by MC. Decarbamoyl mitomycin C (DMC) has been regarded as a monofunctional mitomycin, incapable of causing ICLs. Paradoxically, DMC is slightly more toxic than MC to hypoxic EMT6 mouse mammary tumor cells as well as to CHO cells. To resolve this paradox, EMT6 cells were treated with MC or DMC under hypoxia at equimolar concentrations and the resulting DNA adducts were analyzed using HPLC and UV detection. MC treatment generated both intrastrand and interstrand cross-link adducts and four monoadducts, as shown previously. DMC generated two stereoisomeric monoadducts and two stereoisomeric ICL adducts, all of which were structurally characterized; one was identical with that formed with MC, the other was new and unique to DMC. Overall, adduct frequencies were strikingly higher (20-30-fold) with DMC than with MC. Although DMC monoadducts greatly exceeded DMC cross-link adducts ( approximately 10:1 ratio), the latter were equal or higher in number than the cross-link adducts from MC. DMC displayed a much higher monoadduct:cross-link ratio than MC. The similar cytotoxicities of the two drug show a correlation with their similar DNA cross-link adduct frequencies, but not with their total adduct or monoadduct frequencies. This provides specific experimental evidence that the ICLs rather than the monoadducts are critical factors in the cell death induced by MC. In vitro, overall alkylation of calf thymus DNA by DMC was much less efficient than by MC. Nevertheless, ICLs formed with DMC were clearly detectable. The chemical pathway of the cross-linking was shown to be analogous to that occurring with MC. These results also suggest that the differential sensitivity of Fanconi's Anemia cells to MC and DMC is related to factors other

  19. Genipin-crosslinked fibrin hydrogels as a potential adhesive to augment intervertebral disc annulus repair

    Directory of Open Access Journals (Sweden)

    RM Schek

    2011-04-01

    Full Text Available Treatment of damaged intervertebral discs is a significant clinical problem and, despite advances in the repair and replacement of the nucleus pulposus, there are few effective strategies to restore defects in the annulus fibrosus. An annular repair material should meet three specifications: have a modulus similar to the native annulus tissue, support the growth of disc cells, and maintain adhesion to tissue under physiological strain levels. We hypothesized that a genipin crosslinked fibrin gel could meet these requirements. Our mechanical results showed that genipin crosslinked fibrin gels could be created with a modulus in the range of native annular tissue. We also demonstrated that this material is compatible with the in vitro growth of human disc cells, when genipin:fibrin ratios were 0.25:1 or less, although cell proliferation was slower and cell morphology more rounded than for fibrin alone. Finally, lap tests were performed to evaluate adhesion between fibrin gels and pieces of annular tissue. Specimens created without genipin had poor handling properties and readily delaminated, while genipin crosslinked fibrin gels remained adhered to the tissue pieces at strains exceeding physiological levels and failed at 15-30%. This study demonstrated that genipin crosslinked fibrin gels show promise as a gap-filling adhesive biomaterial with tunable material properties, yet the slow cell proliferation suggests this biomaterial may be best suited as a sealant for small annulus fibrosus defects or as an adhesive to augment large annulus repairs. Future studies will evaluate degradation rate, fatigue behaviors, and long-term biocompatibility.

  20. Lack of increase in DNA crosslinking in Drosophila melanogaster with age.

    Science.gov (United States)

    Massie, H R; Baird, M B; Williams, T R

    1975-01-01

    Adult Drosophila melanogaster fruit flies ranging in age from 2 to 7.5 weeks with a median colony survival time of 6.4 weeks at 25 degrees C showed no increase in DNA crosslinking with age. The purified denatured DNA used for crosslink determinations varied in molecular weight from 2.02 to 3.84 times 10(5) daltons and was crosslinked to the extent of 6.2-8.8% with no age-related trend.

  1. Mechanisms and functions of DNA mismatch repair

    Institute of Scientific and Technical Information of China (English)

    Guo MinLi

    2008-01-01

    DNA mismatch repair (MMR) is a highly conserved biological pathway that plays a key role in maintaining genomic stability. The specificity of MMR is primarily for base-base mismatches and insertion/deletion mispairs generated dur-ing DNA replication and recombination. MMR also suppresses homeologous recombination and was recently shown to play a role in DNA damage signaling in eukaryotic cells. Escherichia coli MutS and MutL and their eukaryotic homo-logs, MutSα and MutLα, respectively, are key players in MMR-associated genome maintenance. Many other protein components that participate in various DNA metabolic pathways, such as PCNA and RPA, are also essential for MMR. Defects in MMR are associated with genome-wide instability, predisposition to certain types of cancer including he-reditary non-polyposis colorectal cancer, resistance to certain chemotherapeutic agents, and abnormalities in meiosis and sterility in mammalian systems.

  2. Human XPC-hHR23B interacts with XPA-RPA in the recognition of triplex-directed psoralen DNA interstrand crosslinks

    DEFF Research Database (Denmark)

    Thoma, Brian S; Wakasugi, Mitsuo; Christensen, Jesper;

    2005-01-01

    DNA interstrand crosslinks (ICLs) represent a severe form of damage that blocks DNA metabolic processes and can lead to cell death or carcinogenesis. The repair of DNA ICLs in mammals is not well characterized. We have reported previously that a key protein complex of nucleotide excision repair...... recognizes psoralen ICLs, which have a structure fundamentally different from other lesions that XPC-hHR23B is known to bind, with high affinity and specificity. XPC-hHR23B and XPA-RPA protein complexes were also observed to bind psoralen ICLs simultaneously, demonstrating not only that psoralen ICLs...

  3. Epigenetic changes of DNA repair genes in cancer

    Institute of Scientific and Technical Information of China (English)

    Christoph Lahtz; Gerd P. Pfeifer

    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.

  4. DNA Repair Defects and Chromosomal Aberrations

    Science.gov (United States)

    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

  5. DNA Repair Defects and Chromosomal Aberrations

    Science.gov (United States)

    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

  6. Characterization of a novel DNA glycosylase from S. sahachiroi involved in the reduction and repair of azinomycin B induced DNA damage.

    Science.gov (United States)

    Wang, Shan; Liu, Kai; Xiao, Le; Yang, LiYuan; Li, Hong; Zhang, FeiXue; Lei, Lei; Li, ShengQing; Feng, Xu; Li, AiYing; He, Jing

    2016-01-01

    Azinomycin B is a hybrid polyketide/nonribosomal peptide natural product and possesses antitumor activity by interacting covalently with duplex DNA and inducing interstrand crosslinks. In the biosynthetic study of azinomycin B, a gene (orf1) adjacent to the azinomycin B gene cluster was found to be essential for the survival of the producer, Streptomyces sahachiroi ATCC33158. Sequence analyses revealed that Orf1 belongs to the HTH_42 superfamily of conserved bacterial proteins which are widely distributed in pathogenic and antibiotic-producing bacteria with unknown functions. The protein exhibits a protective effect against azinomycin B when heterologously expressed in azinomycin-sensitive strains. EMSA assays showed its sequence nonspecific binding to DNA and structure-specific binding to azinomycin B-adducted sites, and ChIP assays revealed extensive association of Orf1 with chromatin in vivo. Interestingly, Orf1 not only protects target sites by protein-DNA interaction but is also capable of repairing azinomycin B-mediated DNA cross-linking. It possesses the DNA glycosylase-like activity and specifically repairs DNA damage induced by azinomycin B through removal of both adducted nitrogenous bases in the cross-link. This bifunctional protein massively binds to genomic DNA to reduce drug attack risk as a novel DNA binding protein and triggers the base excision repair system as a novel DNA glycosylase.

  7. Dual roles for DNA polymerase theta in alternative end-joining repair of double-strand breaks in Drosophila.

    Directory of Open Access Journals (Sweden)

    Sze Ham Chan

    2010-07-01

    Full Text Available DNA double-strand breaks are repaired by multiple mechanisms that are roughly grouped into the categories of homology-directed repair and non-homologous end joining. End-joining repair can be further classified as either classical non-homologous end joining, which requires DNA ligase 4, or "alternative" end joining, which does not. Alternative end joining has been associated with genomic deletions and translocations, but its molecular mechanism(s are largely uncharacterized. Here, we report that Drosophila melanogaster DNA polymerase theta (pol theta, encoded by the mus308 gene and previously implicated in DNA interstrand crosslink repair, plays a crucial role in DNA ligase 4-independent alternative end joining. In the absence of pol theta, end joining is impaired and residual repair often creates large deletions flanking the break site. Analysis of break repair junctions from flies with mus308 separation-of-function alleles suggests that pol theta promotes the use of long microhomologies during alternative end joining and increases the likelihood of complex insertion events. Our results establish pol theta as a key protein in alternative end joining in Drosophila and suggest a potential mechanistic link between alternative end joining and interstrand crosslink repair.

  8. Detection of DNA cross-links in tumor cells with the ethidium bromide fluorescence assay

    NARCIS (Netherlands)

    de Jong, Steven; Zijlstra, J G; Timmer-Bosscha, H; Mulder, N H; de Vries, Liesbeth

    1986-01-01

    Until now the fluorescence assay with ethidium bromide has only been used on pure DNA. This assay depends on the difference in fluorescence between single- and double-stranded DNA (dsDNA). Cross-links in DNA are measured by the return of fluorescence of dsDNA after heat denaturation at pH 12. Under

  9. DNA repair in species with extreme lifespan differences

    Science.gov (United States)

    MacRae, Sheila L.; Croken, Matthew McKnight; Calder, R.B.; Aliper, Alexander; Milholland, Brandon; White, Ryan R.; Zhavoronkov, Alexander; Gladyshev, Vadim N.; Seluanov, Andrei; Gorbunova, Vera; Zhang, Zhengdong D.; Vijg, Jan

    2015-01-01

    Differences in DNA repair capacity have been hypothesized to underlie the great range of maximum lifespans among mammals. However, measurements of individual DNA repair activities in cells and animals have not substantiated such a relationship because utilization of repair pathways among animals—depending on habitats, anatomical characteristics, and life styles—varies greatly between mammalian species. Recent advances in high-throughput genomics, in combination with increased knowledge of the genetic pathways involved in genome maintenance, now enable a comprehensive comparison of DNA repair transcriptomes in animal species with extreme lifespan differences. Here we compare transcriptomes of liver, an organ with high oxidative metabolism and abundant spontaneous DNA damage, from humans, naked mole rats, and mice, with maximum lifespans of ∼120, 30, and 3 years, respectively, with a focus on genes involved in DNA repair. The results show that the longer-lived species, human and naked mole rat, share higher expression of DNA repair genes, including core genes in several DNA repair pathways. A more systematic approach of signaling pathway analysis indicates statistically significant upregulation of several DNA repair signaling pathways in human and naked mole rat compared with mouse. The results of this present work indicate, for the first time, that DNA repair is upregulated in a major metabolic organ in long-lived humans and naked mole rats compared with short-lived mice. These results strongly suggest that DNA repair can be considered a genuine longevity assurance system. PMID:26729707

  10. DNA repair and mutagenesis of singlestranded bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Doubleday, O.P.; Brandenburger, A.; Wagner, R. Jr.; Radman, M. (Brussels Univ. (Belgium)); Godson, G.N.

    1981-01-01

    Virtually all radiation-induced mutagenesis is believed to result from an error-prone repair activity (SOS repair) and to involve mutations occurring both at the site of radiation-induced lesions (targeted mutations) and in undamaged DNA (untargeted mutations). To examine the relative contributions of targeted and untargeted mutations to ..gamma.. and ultraviolet (UV) radiation mutagenesis we have determined the DNA sequences of 174 M13 revertant phages isolated from stocks of irradiated or unirradiated amber mutants grown in irradiated or unirradiated host bacteria. We have detected no obvious specificity of mutagenesis and find no evidence of a predominance of targeted mutations associated with either UV- or ..gamma..-irradiation of the phages or with the induction of the host SOS repair system. In particular, pyrimidine dimers do not appear to be the principal sites of UV-induced bare substitution mutagenesis, suggesting that such UV-induced mutagenesis may be untargeted or occur at sites of lesions other than pyrimidine dimers.

  11. A brief history of the DNA repair field.

    Science.gov (United States)

    Friedberg, Errol C

    2008-01-01

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

  12. A brief history of the DNA repair field

    Institute of Scientific and Technical Information of China (English)

    Errol C Friedberg

    2008-01-01

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

  13. Making the cut : How XPF-ERCC1 unhooks DNA interstrand crosslinks

    NARCIS (Netherlands)

    Klein Douwel, Daisy

    2017-01-01

    DNA interstrand crosslinks (ICLs) are highly toxic lesions that bind both strands of the DNA helix together, which prevents the DNA from unwinding. This blocks important cellular processes such as DNA replication and transcription. ICL inducing agents were among the first chemotherapeutic drugs, mak

  14. DNA repair and radiation sensitivity in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

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

    1993-02-01

    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.

  15. DNA repair and radiation sensitivity in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

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

    1993-01-01

    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.

  16. Ancient bacteria show evidence of DNA repair

    DEFF Research Database (Denmark)

    Johnson, Sarah Stewart; Hebsgaard, Martin B; Christensen, Torben R

    2007-01-01

    Recent claims of cultivable ancient bacteria within sealed environments highlight our limited understanding of the mechanisms behind long-term cell survival. It remains unclear how dormancy, a favored explanation for extended cellular persistence, can cope with spontaneous genomic decay over......-term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence of bacterial survival in samples up to half a million years in age, making this the oldest independently authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence...... that this long-term survival is closely tied to cellular metabolic activity and DNA repair that over time proves to be superior to dormancy as a mechanism in sustaining bacteria viability....

  17. A microdosing approach for characterizing formation and repair of carboplatin–DNA monoadducts and chemoresistance

    Science.gov (United States)

    Henderson, Paul T.; Li, Tao; He, Miaoling; Zhang, Hongyong; Malfatti, Michael; Gandara, David; Grimminger, Peter P.; Danenberg, Kathleen D.; Beckett, Laurel; de Vere White, Ralph W.; Turteltaub, Kenneth W.; Pan, Chong-Xian

    2011-01-01

    Formation and repair of platinum (Pt)-induced DNA adducts is a critical step in Pt drug-mediated cytotoxicity. Measurement of Pt–DNA adduct kinetics in tumors may be useful for better understanding chemoresistance and therapeutic response. However, this concept has yet to be rigorously tested because of technical challenges in measuring the adducts at low concentrations and consistent access to sufficient tumor biopsy material. Ultrasensitive accelerator mass spectrometry was used to detect [14C]carboplatin–DNA monoadducts at the attomole level, which are the precursors to Pt–DNA crosslink formation, in six cancer cell lines as a proof-of-concept. The most resistant cells had the lowest monoadduct levels at all time points over 24 hr. [14C]Carboplatin “microdoses" (1/100th the pharmacologically effective concentration) had nearly identical adduct formation and repair kinetics compared to therapeutically relevant doses, suggesting that the microdosing approach can potentially be used to determine the pharmacological effects of therapeutic treatment. Some of the possible chemoresistance mechanisms were also studied, such as drug uptake/efflux, intracellular inactivation and DNA repair in selected cell lines. Intracellular inactivation and efficient DNA repair each contributed significantly to the suppression of DNA monoadduct formation in the most resistant cell line compared to the most sensitive cell line studied (p < 0.001). Nucleotide excision repair (NER)-deficient and - proficient cells showed substantial differences in carboplatin monoadduct concentrations over 24 hr that likely contributed to chemoresistance. The data support the utility of carboplatin microdosing as a translatable approach for defining carboplatin–DNA monoadduct formation and repair, possibly by NER, which may be useful for characterizing chemoresistance in vivo. PMID:21128223

  18. Recombinational DNA repair and human disease

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Larry H.; Schild, David

    2002-11-30

    We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities.

  19. Protein found to promote DNA repair, prevent cancer

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    @@ An abundant chromosomal protein that binds to damaged DNA prevents cancer development by enhancing DNA repair, researchers at University of Texas reported on-line in the Proceedings of the National Academies of Science.

  20. Princess takamatsu symposium on DNA repair and human cancers.

    Science.gov (United States)

    Loeb, Lawrence A; Nishimura, Susumu

    2010-06-01

    The 40th International Symposium of the Princess Takamatsu Cancer Research Fund, entitled "DNA Repair and Human Cancers," was held on November 10-12, 2009 at Hotel Grand Palace, Tokyo, Japan. The meeting focused on the role of DNA repair in preventing mutations by endogenous and exogenous DNA damage and increasing the efficacy of chemotherapeutic agents by interfering with DNA repair. The 14 presentations by the speakers from the United States, four from the United Kingdom, one each from Italy, The Netherlands, and France, and 13 from Japan, covered most aspects of DNA repair, spanning DNA damage, molecular structures of repair enzymes, and clinical studies on inhibition of DNA repair processes. Extensive time was reserved for discussions with the active participation of the 150 invited Japanese scientists. The choice of a symposium on DNA repair in human cancers resulted in part from the excellent basic and clinical studies that have been carried out for many years in Japan, and the general lack of recognition versus the importance of DNA repair in understanding carcinogenesis. Copyright 2010 AACR.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A., E-mail: sarah.martin@qmul.ac.uk [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ (United Kingdom)

    2014-08-05

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

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

    Science.gov (United States)

    Keskin, Havva; Meers, Chance; Storici, Francesca

    2016-01-01

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

  4. Rad52 SUMOylation affects the efficiency of the DNA repair

    DEFF Research Database (Denmark)

    Altmannova, Veronika; Eckert-Boulet, Nadine; Arneric, Milica

    2010-01-01

    Homologous recombination (HR) plays a vital role in DNA metabolic processes including meiosis, DNA repair, DNA replication and rDNA homeostasis. HR defects can lead to pathological outcomes, including genetic diseases and cancer. Recent studies suggest that the post-translational modification...... recombination mediator protein Rad52. Interestingly, Rad52 SUMOylation is enhanced by single-stranded DNA, and we show that SUMOylation of Rad52 also inhibits its DNA binding and annealing activities. The biochemical effects of SUMO modification in vitro are accompanied by a shorter duration of spontaneous Rad...... of recombination and DNA repair....

  5. Targeting DNA-repair systems brings hopes to cancer patients

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ CAS researchers have recently raised a hypothesis to circumvent tumor resistance to radio- and chemo-therapy and to enhance the efficacy of DNAdamaging agents by interfering with DNA repair. "There are emerging anticancer therapeutic opportunities in targeting DNA-repair systems," they asserted.

  6. Destabilizing DNA during Rejoining Enhances Fidelity of Repair.

    Directory of Open Access Journals (Sweden)

    Richard Robinson

    2015-08-01

    Full Text Available A new study shows that during repair of DNA, the effect of a single-strand annealing protein is to destabilize DNA duplex formation so that annealing only occurs between perfectly matched strands; the protein then clamps the strands together for repair. Read the Research Article.

  7. An Overview of DNA Repair in Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Fabio Coppedè

    2011-01-01

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

  8. Differential recruitment of DNA Ligase I and III to DNA repair sites

    OpenAIRE

    Mortusewicz, O; Rothbauer, U.; Cardoso, M C; Leonhardt, H.

    2006-01-01

    DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use the same catalytic reaction mechanism. To compare the recruitment of the three eukaryotic DNA Ligases to repair sites in vivo we introduced DNA lesions in human cells by laser microirradiation. Time lapse microscopy of fluorescently tagged proteins showed that DNA Ligase III accumulated at microirradiated sites before DNA Liga...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-08-29

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    DEFF Research Database (Denmark)

    Forsberg, Kristin; Aalling, Nadia; Wörtwein, Gitta

    2015-01-01

    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...... 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...

  12. SIRT1 promotes DNA repair activity in response to radiation

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Jae-Min; Lee, Kee-Ho [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2006-07-01

    Human SIRT1 controls various physiological responses including cell fate, stress, and aging, through deacetylation of its specific substrate protein. In processing DNA damage signaling, SIRT1 attenuates a cellular apoptotic response by deacetylation of p53 tumor suppressor. Ectopically over-expressed SIRT1 resulted in the increase of repair of DNA strand breakages produced by radiation. On the other hand, repression of endogenous SIRT1 expression by SIRT1 siRNA led to the decrease of this repair activity, indicating that SIRT1 can regulate DNA repair capacity of cells with DNA strand breaks.

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

    Science.gov (United States)

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

    2008-01-01

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

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

    Science.gov (United States)

    Sokhansanj, B A

    2005-01-01

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

  15. DNA repair: Dynamic defenders against cancer and aging

    Energy Technology Data Exchange (ETDEWEB)

    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

  16. Modulation of DNA base excision repair during neuronal differentiation

    DEFF Research Database (Denmark)

    Sykora, Peter; Yang, Jenq-Lin; Ferrarelli, Leslie K

    2013-01-01

    Neurons are terminally differentiated cells with a high rate of metabolism and multiple biological properties distinct from their undifferentiated precursors. Previous studies showed that nucleotide excision DNA repair is downregulated in postmitotic muscle cells and neurons. Here, we characterize...... DNA damage susceptibility and base excision DNA repair (BER) capacity in undifferentiated and differentiated human neural cells. The results show that undifferentiated human SH-SY5Y neuroblastoma cells are less sensitive to oxidative damage than their differentiated counterparts, in part because...

  17. Chloroacetamide-Linked Nucleotides and DNA for Cross-Linking with Peptides and Proteins.

    Science.gov (United States)

    Olszewska, Agata; Pohl, Radek; Brázdová, Marie; Fojta, Miroslav; Hocek, Michal

    2016-09-21

    Nucleotides, 2'-deoxyribonucleoside triphosphates (dNTPs), and DNA probes bearing reactive chloroacetamido group linked to nucleobase (cytosine or 7-deazadaenine) through a propargyl tether were prepared and tested in cross-linking with cysteine- or histidine-containing peptides and proteins. The chloroacetamide-modifed dNTPs proved to be good substrates for DNA polymerases in the enzymatic synthesis of modified DNA probes. Modified nucleotides and DNA reacted efficiently with cysteine and cysteine-containing peptides, whereas the reaction with histidine was sluggish and low yielding. The modified DNA efficiently cross-linked with p53 protein through alkylation of cysteine and showed potential for cross-linking with histidine (in C277H mutant of p53).

  18. Human longevity and variation in DNA damage response and repair

    DEFF Research Database (Denmark)

    Debrabant, Birgit; Soerensen, Mette; Flachsbart, Friederike

    2014-01-01

    others. Data were applied on 592 SNPs from 77 genes involved in nine sub-processes: DNA-damage response, base excision repair (BER), nucleotide excision repair, mismatch repair, non-homologous end-joining, homologous recombinational repair (HRR), RecQ helicase activities (RECQ), telomere functioning...... and mitochondrial DNA processes. The study population was 1089 long-lived and 736 middle-aged Danes. A self-contained set-based test of all SNPs displayed association with longevity (P-value=9.9 × 10-5), supporting that the overall pathway could affect longevity. Investigation of the nine sub-processes using...

  19. Automated Image Processing for the Analysis of DNA Repair Dynamics

    CERN Document Server

    Riess, Thorsten; Tomas, Martin; Ferrando-May, Elisa; Merhof, Dorit

    2011-01-01

    The efficient repair of cellular DNA is essential for the maintenance and inheritance of genomic information. In order to cope with the high frequency of spontaneous and induced DNA damage, a multitude of repair mechanisms have evolved. These are enabled by a wide range of protein factors specifically recognizing different types of lesions and finally restoring the normal DNA sequence. This work focuses on the repair factor XPC (xeroderma pigmentosum complementation group C), which identifies bulky DNA lesions and initiates their removal via the nucleotide excision repair pathway. The binding of XPC to damaged DNA can be visualized in living cells by following the accumulation of a fluorescent XPC fusion at lesions induced by laser microirradiation in a fluorescence microscope. In this work, an automated image processing pipeline is presented which allows to identify and quantify the accumulation reaction without any user interaction. The image processing pipeline comprises a preprocessing stage where the ima...

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

    Directory of Open Access Journals (Sweden)

    Devita Surjana

    2010-01-01

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

  1. Crosslinks rather than strand breaks determine access to ancient DNA sequences from frozen sediments

    DEFF Research Database (Denmark)

    Hansen, Anders Johannes; Mitchell, D.L.; Wiuf, C.

    2006-01-01

    Diagenesis was studied in DNA obtained from Siberian permafrost (permanently frozen soil) ranging from 10 to 400 thousand years in age. Despite optimal preservation conditions, we found the sedimentary DNA to be severely modified by interstrand crosslinks, single and double stranded breaks, and f...

  2. Error-prone translesion synthesis past DNA-peptide cross-links conjugated to the major groove of DNA via C5 of thymidine.

    Science.gov (United States)

    Wickramaratne, Susith; Boldry, Emily J; Buehler, Charles; Wang, Yen-Chih; Distefano, Mark D; Tretyakova, Natalia Y

    2015-01-01

    DNA-protein cross-links (DPCs) are exceptionally bulky, structurally diverse DNA adducts formed in cells upon exposure to endogenous and exogenous bis-electrophiles, reactive oxygen species, and ionizing radiation. If not repaired, DPCs can induce toxicity and mutations. It has been proposed that the protein component of a DPC is proteolytically degraded, giving rise to smaller DNA-peptide conjugates, which can be subject to nucleotide excision repair and replication bypass. In this study, polymerase bypass of model DNA-peptide conjugates structurally analogous to the lesions induced by reactive oxygen species and DNA methyltransferase inhibitors was examined. DNA oligomers containing site-specific DNA-peptide conjugates were generated by copper-catalyzed [3 + 2] Huisgen cyclo-addition between an alkyne-functionalized C5-thymidine in DNA and an azide-containing 10-mer peptide. The resulting DNA-peptide conjugates were subjected to steady-state kinetic experiments in the presence of recombinant human lesion bypass polymerases κ and η, followed by PAGE-based assays to determine the catalytic efficiency and the misinsertion frequency opposite the lesion. We found that human polymerase κ and η can incorporate A, G, C, or T opposite the C5-dT-conjugated DNA-peptide conjugates, whereas human polymerase η preferentially inserts G opposite the lesion. Furthermore, HPLC-ESI(-)-MS/MS sequencing of the extension products has revealed that post-lesion synthesis was highly error-prone, resulting in mutations opposite the adducted site or at the +1 position from the adduct and multiple deletions. Collectively, our results indicate that replication bypass of peptides conjugated to the C5 position of thymine by human translesion synthesis polymerases leads to large numbers of base substitution and frameshift mutations.

  3. DNA interstrand cross-link induced by estrogens as well as their complete and synergic carcinogenesis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The estrogens show negative activity in Ames test, but estrodiol and diethylstilbestrol in estrogens both are carcinogens based upon animal experiments and epidemiological investigation. It is concluded from the di-region theory, a mechanism conception put forward by one of the present authors, that the carcinogenesis of estrogens is switched on by the covalent cross-link between complementary DNA bases induced by them. We verified for the first time by the DNA alkaline elution method that both estrodiol and diethylstilbestrol cause covalent cross-link between DNA-protein and DNA interstrands after metabolic activation with dosage correlation, but neither the non-carcinogens cholesterol nor pyrene can lead to these sorts of cross-link in the same condition. It has been known that there is a synergetic effect between estrogen and pollution of polycyclic aromatic hydrocarbons. Although non-carcinogenic pyrene alone cannot induce cross-link, its addition with equal molar quantity to estrodiol culture causes synergically the total and DNA interstrand cross-link ratios to be respectively four and three times more than the ones in the cultivation with estrodiol only. It is shown that not only the estrodiol set off the formation of pyrene bi-radicals, but also the pyrene radicals arouse conversely the production of estrodiol bi-radicals.

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

    Science.gov (United States)

    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. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. D-ribose inhibits DNA repair synthesis in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-07-31

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

  6. Molecular mechanisms of DNA repair inhibition by caffeine

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

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

    Science.gov (United States)

    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 β, 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.

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

    Science.gov (United States)

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

    2014-11-20

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

  9. Platinum drugs and DNA repair mechanisms in lung cancer.

    Science.gov (United States)

    Bonanno, Laura; Favaretto, Adolfo; Rosell, Rafael

    2014-01-01

    The standard first-line treatment for around 80% of newly-diagnosed advanced non-small cell lung cancer (NSCLC) is chemotherapy. Currently, patients are allocated to chemotherapy on the basis of clinical conditions, comorbidities and histology. If feasible, platinum-based chemotherapy is considered as the most efficacious option. Due to the heterogeneity in terms of platinum-sensitivity among patients with NSCLC, great efforts have been made in order to identify molecular predictive markers of platinum resistance. Based on the mechanism of action of platinum, several components of DNA repair pathways have been investigated as potential predictive markers. The main DNA repair pathways involved in the repair of platinum-induced DNA damage are nucleotide excision repair and homologous recombination. The most studied potential predictive markers of platinum-sensitivity are Excision Repair Cross Complementing-1 (ERCC1) and Brest Cancer Type-I Susceptibility protein (BRCA1); however, increasing biological knowledge about DNA repair pathways suggests the potential clinical usefulness of integrated analysis of multiple DNA repair components.

  10. 4. DNA REPAIR CAPACITY IN LUNG CANCER PATIENTS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@The ability for DNA repair is an important host factor which influences the individual susceptibility to genotoxic carcinogen exposures. It has been shown in different case-control studies that DNA repair capacity (DRC) can be reduced in lung cancer patients.We have used an alkaline comet assay to measure the cellular DRC in peri-pheral blood lymphocytes of lung cancer patients and tumor-free control

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

    Science.gov (United States)

    Marín-García, José

    2016-09-01

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

  12. Biomarkers of oxidative damage to DNA and repair

    DEFF Research Database (Denmark)

    Loft, Steffen; Høgh Danielsen, Pernille; Mikkelsen, Lone

    2008-01-01

    Oxidative-stress-induced damage to DNA includes a multitude of lesions, many of which are mutagenic and have multiple roles in cancer and aging. Many lesions have been characterized by MS-based methods after extraction and digestion of DNA. These preparation steps may cause spurious base oxidation...... DNA glycosylase 1), responsible for repair of 8-oxodG, by genotyping. Products of repair in DNA or the nucleotide pool, such as 8-oxodG, excreted into the urine can be assessed by MS-based methods and generally reflects the rate of damage. Experimental and population-based studies indicate that many...

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

    Directory of Open Access Journals (Sweden)

    Maria E Morales

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

  14. Purification of mammalian DNA repair protein XRCC1

    Energy Technology Data Exchange (ETDEWEB)

    Chen, I. [Univ. of California, Berkeley, CA (United States)

    1995-11-01

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

  15. Preferential repair in Saccharomyces cerevisiae rad mutants after induction of interstrand cross-links by 8-methoxypsoralen plus UVA.

    Science.gov (United States)

    Meniel, V; Magaña-Schwencke, N; Averbeck, D

    1995-11-01

    The gene specific induction and the incision step of the removal of 8-methoxypsoralen (8-MOP) plus UVA-induced interstrand cross-links (ICL) was measured in repair mutants of Saccharomyces cerevisiae. Events were examined at the MAT alpha and HML alpha loci in mutants deficient in the repair of ICL, namely rad1, rad2 delta, rad52, pso2 and the rad16 mutant which is impaired in the removal of UV-induced pyrimidine dimers from the silent HML alpha locus. Previously, we observed in a wild-type strain (K107) preferential repair concerning the incision of 8-MOP photo-induced ICL. The present study indicates that the two mutants rad1 and rad2 delta show no repair in either locus, due presumably to their deficiency in the incision step of ICL repair. The rad52 mutant which is defective in recombination, is proficient in the preferential incision of ICL at the MAT alpha locus versus the HML alpha locus. The same is true for the pso2 mutant which also lacks the ability to perform complete repair of ICL. The rad16 mutant is unable to repair ICL in the silent locus HML alpha but is proficient in repair (i.e. the incision of ICL) in the transcriptionally active MAT alpha locus.

  16. Dehydration of an ethanol/water azeotrope through alginate-DNA membranes cross-linked with metal ions by pervaporation.

    Science.gov (United States)

    Uragami, Tadashi; Banno, Masashi; Miyata, Takashi

    2015-12-10

    To obtain high dehydration membranes for an ethanol/water azeotrope, dried blend membranes prepared from mixtures of sodium alginate (Alg-Na) and sodium deoxyribonucleate (DNA-Na) were cross-linked by immersing in a methanol solution of CaCl2 or MaCl2. In the dehydration of an ethanol/water azeotropic mixture by pervaporation, the effects of immersion time in methanol solution of CaCl2 or MaCl2 on the permeation rate and water/ethanol selectivity through Alg-DNA/Ca(2+) and Alg-DNA/Mg(2+) cross-linked membranes were investigated. Alg-DNA/Mg(2+) cross-linked membrane immersed for 12h in methanol solution of MaCl2 exhibited the highest water/ethanol selectivity. This results from depressed swelling of the membranes by formation of a cross-linked structure. However, excess immersion in solution containing cross-linker led to an increase in the hydrophobicity of cross-linked membrane. Therefore, the water/ethanol selectivity of Alg-DNA/Mg(2+) cross-linked membranes with an excess immersion in cross-linking solution was lowered. The relationship between the structure of Alg-DNA/Ca(2+) and Alg-DNA/Mg(2+) cross-linked membranes and their permeation and separation characteristics during pervaporation of an ethanol/water azeotropic mixture is discussed in detail.

  17. Role of poly(ADP-ribosepolymerase 2 in DNA repair

    Directory of Open Access Journals (Sweden)

    Lavrik O. I.

    2012-06-01

    Full Text Available Poly(ADP-ribosylation is a posttranslational protein modification significant for the genomic stability and cell survival in response to DNA damage. Poly(ADP-ribosylation is catalyzed by poly(ADP-ribosepolymerases (PARPs, which use NAD+ as a substrate, synthesize polymer of (ADP-ribose (PAR covalently attached to nuclear proteins including PARP themselves. PARPs constitute a large family of proteins, in which PARP1 is the most abundant and best-characterized member. In spite of growing body of PARPs’ role in cellular processes, PARP2, the closest homolog of PARP1, still remains poorly characterized at the level of its contribution to different pathways of DNA repair. An overview summarizes in vivo and in vitro data on PARP2 implication in specialized DNA repair processes, base excision repair and double strand break repair.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2013-07-01

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

  20. Riboflavin crosslinked high-density collagen gel for the repair of annular defects in intervertebral discs: An in vivo study.

    Science.gov (United States)

    Grunert, Peter; Borde, Brandon H; Towne, Sara B; Moriguchi, Yu; Hudson, Katherine D; Bonassar, Lawrence J; Härtl, Roger

    2015-10-01

    Open annular defects compromise the ability of the annulus fibrosus to contain nuclear tissue in the disc space, and therefore lead to disc herniation with subsequent degenerative changes to the entire intervertebral disc. This study reports the use of riboflavin crosslinked high-density collagen gel for the repair of annular defects in a needle-punctured rat-tail model. High-density collagen has increased stiffness and greater hydraulic permeability than conventional low-density gels; riboflavin crosslinking further increases these properties. This study found that treating annular defects with crosslinked high-density collagen inhibited the progression of disc degeneration over 18 weeks compared to untreated control discs. Histological sections of FITC-labeled collagen gel revealed an early tight attachment to host annular tissue. The gel was subsequently infiltrated by host fibroblasts which remodeled it into a fibrous cap that bridged the outer disrupted annular fibers and partially repaired the defect. This repair tissue enhanced retention of nucleus pulposus tissue, maintained physiological disc hydration, and preserved hydraulic permeability, according to MRI, histological, and mechanical assessments. Degenerative changes were partially reversed in treated discs, as indicated by an increase in nucleus pulposus size and hydration between weeks 5 and 18. The collagen gel appeared to work as an instant sealant and by enhancing the intrinsic healing capabilities of the host tissue.

  1. Chromatin Dynamics in Genome Stability: Roles in Suppressing Endogenous DNA Damage and Facilitating DNA Repair

    Directory of Open Access Journals (Sweden)

    Nidhi Nair

    2017-07-01

    Full Text Available Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome stability. This is exemplified in the response to DNA damage where chromatin relaxation near genomic lesions serves to promote access of relevant enzymes to specific DNA regions for signaling and repair. Furthermore, recent data highlight genome maintenance roles of chromatin through the regulation of endogenous DNA-templated processes including transcription and replication. Here, we review research that shows the importance of chromatin structure regulation in maintaining genome integrity by multiple mechanisms including facilitating DNA repair and directly suppressing endogenous DNA damage.

  2. Ouabain, a cardiac glycoside, inhibits the Fanconi anemia/BRCA pathway activated by DNA interstrand cross-linking agents.

    Directory of Open Access Journals (Sweden)

    Dong Wha Jun

    Full Text Available Modulation of the DNA repair pathway is an emerging target for the development of anticancer drugs. DNA interstrand cross-links (ICLs, one of the most severe forms of DNA damage caused by anticancer drugs such as cisplatin and mitomycin C (MMC, activates the Fanconi anemia (FA/BRCA DNA repair pathway. Inhibition of the FA/BRCA pathway can enhance the cytotoxic effects of ICL-inducing anticancer drugs and can reduce anticancer drug resistance. To find FA/BRCA pathway inhibitory small molecules, we established a cell-based high-content screening method for quantitating the activation of the FA/BRCA pathway by measuring FANCD2 foci on DNA lesions and then applied our method to chemical screening. Using commercial LOPAC1280 chemical library screening, ouabain was identified as a competent FA/BRCA pathway inhibitory compound. Ouabain, a member of the cardiac glycoside family, binds to and inhibits Na(+/K(+-ATPase and has been used to treat heart disease for many years. We observed that ouabain, as well as other cardiac glycoside family members--digitoxin and digoxin--down-regulated FANCD2 and FANCI mRNA levels, reduced monoubiquitination of FANCD2, inhibited FANCD2 foci formation on DNA lesions, and abrogated cell cycle arrest induced by MMC treatment. These inhibitory activities of ouabain required p38 MAPK and were independent of cellular Ca(2+ ion increase or the drug uptake-inhibition effect of ouabain. Furthermore, we found that ouabain potentiated the cytotoxic effects of MMC in tumor cells. Taken together, we identified an additional effect of ouabain as a FA/BRCA pathway-inhibiting chemosensitization compound. The results of this study suggest that ouabain may serve as a chemosensitizer to ICL-inducing anticancer drugs.

  3. DNA mismatch repair: Dr. Jekyll and Mr. Hyde?

    Science.gov (United States)

    Hsieh, Peggy

    2012-09-14

    In this issue, Peña-Diaz et al. (2012) describe a pathway for somatic mutation in nonlymphoid cells termed noncanonical DNA mismatch repair, whereby the error-prone translesion polymerase Pol-η substitutes for high-fidelity replicative polymerases to resynthesize excised regions opposite DNA damage. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    DEFF Research Database (Denmark)

    Croteau, Deborah L; de Souza-Pinto, Nadja C; Harboe, Charlotte

    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......-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....

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

    Science.gov (United States)

    Demple, B; Halbrook, J

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

  6. Hsp90: A New Player in DNA Repair?

    Directory of Open Access Journals (Sweden)

    Rosa Pennisi

    2015-10-01

    Full Text Available 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.

  7. Ionizing radiation-induced DNA damage and its repair in human cells. Final performance report, July 1992--June 1995

    Energy Technology Data Exchange (ETDEWEB)

    Dizdaroglu, M.

    1995-12-31

    The studies of DNA damage in living cells in vitro and in vivo were continued. A variety of systems including cultured mammalian cells, animals, and human tissues were used to conduct these studies. In addition, enzymatic repair of DNA base damage was studied using several DNA glycosylases. To this end, substrate specificities of these enzymes were examined in terms of a large number of base lesions in DNA. In the first phase of the studies, the author sought to introduce improvements to his methodologies for measurement of DNA damage using the technique of gas chromatography/mass spectrometry (GC/MS). In particular, the quantitative measurement of DNA base damage and DNA-protein crosslinks was improved by incorporation of isotope-dilution mass spectrometry into the methodologies. This is one of the most accurate techniques for quantification of organic compounds. Having improved the measurement technique, studies of DNA damage in living cells and DNA repair by repair enzymes were pursued. This report provides a summary of these studies with references to the original work.

  8. Regulation of DNA double-strand break repair pathway choice

    Institute of Scientific and Technical Information of China (English)

    Meena Shrivastav; Leyma P De Haro; Jac A Nickoloff

    2008-01-01

    DNA double-strand breaks (DSBs) are critical lesions that can result in cell death or a wide variety of genetic alterations including large- or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources includ-ing reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1 (XRS2) complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.

  9. Repair of mismatched basepairs in mammalian DNA

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-08-01

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

  10. Effect of varying the exposure and /sup 3/H-thymidine labeling period upon the outcome of the primary hepatocyte DNA repair assay

    Energy Technology Data Exchange (ETDEWEB)

    Barfknecht, T.R.; Mecca, D.J.; Naismith, R.W.

    1988-06-01

    The results presented in this report demonstrate that an 18-20 hour exposure//sup 3/H-thymidine DNA labeling period is superior to a 4 hour incubation interval for general genotoxicity screening studies in the rat primary hepatocyte DNA repair assay. When DNA damaging agents which give rise to bulky-type DNA base adducts such as 2-acetylaminofluorene, aflatoxin B1 and benzidine were evaluated, little or no difference was observed between the 4 hour or an 18-20-hour exposure/labeling period. Similar results were also noted for the DNA ethylating agent diethylnitrosamine. However, when DNA damaging chemicals which produce a broader spectrum of DNA lesions were studied, differences in the amount of DNA repair as determined by autoradiographic analysis did occur. Methyl methanesulfonate and dimethylnitrosamine induced repairable DNA damage that was detected at lower dose levels with the 18-20 hour exposure/labeling period. Similar results were also observed for the DNA cross-linking agents, mitomycin C and nitrogen mustard. Ethyl methanesulfonate produced only a marginal amount of DNA repair in primary hepatocytes up to a dose level of 10(-3) M during the 4 hour incubation period, whereas a substantial amount of DNA repair was detectable at a dose level of 2.5 X 10(-4) M when the 18-20 hour exposure/labeling period was employed. The DNA alkylating agent 4-nitroquinoline-1-oxide, which creates DNA base adducts that are slowly removed from mammalian cell DNA, induced no detectable DNA repair in hepatocytes up to a toxic dose level of 2 X 10(-5) M with the 4 hour exposure period, whereas a marked DNA repair response was observed at 10(-5) M when the 18-20 hour exposure/labeling period was used.

  11. Electronic Pathways in Photoactivated Repair of UV Mutated DNA

    Science.gov (United States)

    Bohr, Henrik; Jalkanen, K. J.; Bary Malik, F.

    An investigation of the physics, underlying the damage caused to DNA by UV radiation and its subsequent repair via a photoreactivation mechanism, is presented in this study. Electronic pathways, starting from the initial damage to the final repair process, are presented. UV radiation is absorbed to create a hole-excited thymine or other pyrimidine that subsequently is responsible for the formation of a dimer. The negative-ion of the cofactor riboflavin, FADH-, formed by the exposure of the photolyase protein to visible light, interacts with the hole-excited electronic orbital of the thymine dimer inducing a photon-less Auger transition, which restores the two thymines to the ground state, thereby detaching the lesion and repairing the DNA. Density functional theoretical calculations supporting the theory are presented. The mechanism involves the least amount of energy dissipation and is charge neutral. It also avoids radiation damage in the repair process. Recent experimental data are compatible with this theory.

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2017-04-03

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

  14. Identification of small molecule inhibitors of ERCC1-XPF that inhibit DNA repair and potentiate cisplatin efficacy in cancer cells.

    Science.gov (United States)

    Arora, Sanjeevani; Heyza, Joshua; Zhang, Hao; Kalman-Maltese, Vivian; Tillison, Kristin; Floyd, Ashley M; Chalfin, Elaine M; Bepler, Gerold; Patrick, Steve M

    2016-11-15

    ERCC1-XPF heterodimer is a 5'-3' structure-specific endonuclease which is essential in multiple DNA repair pathways in mammalian cells. ERCC1-XPF (ERCC1-ERCC4) repairs cisplatin-DNA intrastrand adducts and interstrand crosslinks and its specific inhibition has been shown to enhance cisplatin cytotoxicity in cancer cells. In this study, we describe a high throughput screen (HTS) used to identify small molecules that inhibit the endonuclease activity of ERCC1-XPF. Primary screens identified two compounds that inhibit ERCC1-XPF activity in the nanomolar range. These compounds were validated in secondary screens against two other non-related endonucleases to ensure specificity. Results from these screens were validated using an in vitro gel-based nuclease assay. Electrophoretic mobility shift assays (EMSAs) further show that these compounds do not inhibit the binding of purified ERCC1-XPF to DNA. Next, in lung cancer cells these compounds potentiated cisplatin cytotoxicity and inhibited DNA repair. Structure activity relationship (SAR) studies identified related compounds for one of the original Hits, which also potentiated cisplatin cytotoxicity in cancer cells. Excitingly, dosing with NSC16168 compound potentiated cisplatin antitumor activity in a lung cancer xenograft model. Further development of ERCC1-XPF DNA repair inhibitors is expected to sensitize cancer cells to DNA damage-based chemotherapy.

  15. Active DNA demethylation by oxidation and repair

    Institute of Scientific and Technical Information of China (English)

    Zhizhong Gong; Jian-Kang Zhu

    2011-01-01

    DNA methylation and demethylation are increasingly recognized as important epigenetic factors in both plants and animals.DNA methylation,which is catalyzed by DNA methyltransferases (DNMTs),is a relatively stable and heritable modification that controls gene expression,cellular differentiation,genomic imprinting,paramutation,transposon movement,X-inactivation,and embryogenesis [1].The methylation of cytosine to 5-methylcytosine (5mC) is an important example of DNA modification in animals and plants.This highlight concerns DNA demethylation mechanisms in mammals and whether they are similar to that in plants.

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

    DEFF Research Database (Denmark)

    Géli, Vincent; Lisby, Michael

    2015-01-01

    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....

  17. Damage and repair of ancient DNA

    DEFF Research Database (Denmark)

    Mitchell, David; Willerslev, Eske; Hansen, Anders

    2005-01-01

    , and extensive degradation. In the course of this review, we will discuss the current aDNA literature describing the importance of aDNA studies as they relate to important biological questions and the difficulties associated with extracting useful information from highly degraded and damaged substrates derived......Under certain conditions small amounts of DNA can survive for long periods of time and can be used as polymerase chain reaction (PCR) substrates for the study of phylogenetic relationships and population genetics of extinct plants and animals, including hominids. Because of extensive DNA...... degradation, these studies are limited to species that lived within the past 10(4)-10(5) years (Late Pleistocene), although DNA sequences from 10(6) years have been reported. Ancient DNA (aDNA) has been used to study phylogenetic relationships of protists, fungi, algae, plants, and higher eukaryotes...

  18. 1,2,3,4-Diepoxybutane-Induced DNA-Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells

    OpenAIRE

    2013-01-01

    1,2,3,4-diepoxybutane (DEB) is the key carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is a genotoxic bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs). In the present work, mass spectrometry-based proteomics was employed to characterize DEB-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. Over 150 proteins ...

  19. Microhomology directs diverse DNA break repair pathways and chromosomal translocations.

    Directory of Open Access Journals (Sweden)

    Diana D Villarreal

    Full Text Available Chromosomal structural change triggers carcinogenesis and the formation of other genetic diseases. The breakpoint junctions of these rearrangements often contain small overlapping sequences called "microhomology," yet the genetic pathway(s responsible have yet to be defined. We report a simple genetic system to detect microhomology-mediated repair (MHMR events after a DNA double-strand break (DSB in budding yeast cells. MHMR using >15 bp operates as a single-strand annealing variant, requiring the non-essential DNA polymerase subunit Pol32. MHMR is inhibited by sequence mismatches, but independent of extensive DNA synthesis like break-induced replication. However, MHMR using less than 14 bp is genetically distinct from that using longer microhomology and far less efficient for the repair of distant DSBs. MHMR catalyzes chromosomal translocation almost as efficiently as intra-chromosomal repair. The results suggest that the intrinsic annealing propensity between microhomology sequences efficiently leads to chromosomal rearrangements.

  20. Exonuclease 1 and its versatile roles in DNA repair

    DEFF Research Database (Denmark)

    Keijzers, Guido; Liu, Dekang; Rasmussen, Lene Juel

    2016-01-01

    Exonuclease 1 (EXO1) is a multifunctional 5' → 3' exonuclease and a DNA structure-specific DNA endonuclease. EXO1 plays roles in DNA replication, DNA mismatch repair (MMR) and DNA double-stranded break repair (DSBR) in lower and higher eukaryotes and contributes to meiosis, immunoglobulin...... maturation, and micro-mediated end-joining in higher eukaryotes. In human cells, EXO1 is also thought to play a role in telomere maintenance. Mutations in the human EXO1 gene correlate with increased susceptibility to some cancers. This review summarizes recent studies on the enzymatic functions...... and biological roles of EXO1, its possible protective role against cancer and aging, and regulation of EXO1 by posttranslational modification....

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

    Directory of Open Access Journals (Sweden)

    Michel Lebel

    2011-01-01

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

  2. Nampt is involved in DNA double-strand break repair

    Institute of Scientific and Technical Information of China (English)

    Bingtao Zhu; Xiaoli Deng; Yifan Sun; Lin Bai; Zhikai Xiahou; Yusheng Cong; Xingzhi Xu

    2012-01-01

    DNA double-strand break (DSB) is the most severe form of DNA damage,which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ).Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer.Nicotinamide phosphoribosyltransferase (Nampt),which is involved in nicotinamide adenine dinucleotide metabolism,is overexpressed in a variety of tumors.In this report,we found that Nampt physically associated with CtlP and DNA-PKcs/Ku80,which are key factors in HR and NHEJ,respectively.Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair.Furthermore,the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining,indicating a delay in the onset of cellular senescence in normal human fibroblasts.Taken together,our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair,contributing to the acceleration of cellular senescence.

  3. Intrinsic mitochondrial DNA repair defects in Ataxia Telangiectasia.

    Science.gov (United States)

    Sharma, Nilesh K; Lebedeva, Maria; Thomas, Terace; Kovalenko, Olga A; Stumpf, Jeffrey D; Shadel, Gerald S; Santos, Janine H

    2014-01-01

    Ataxia Telangiectasia (A-T) is a progressive childhood disorder characterized most notably by cerebellar degeneration and predisposition to cancer. A-T is caused by mutations in the kinase ATM, a master regulator of the DNA double-strand break response. In addition to DNA-damage signaling defects, A-T cells display mitochondrial dysfunction that is thought to contribute to A-T pathogenesis. However, the molecular mechanism leading to mitochondrial dysfunction in A-T remains unclear. Here, we show that lack of ATM leads to reduced mitochondrial DNA (mtDNA) integrity and mitochondrial dysfunction, which are associated to defective mtDNA repair. While protein levels of mtDNA repair proteins are essentially normal, in the absence of ATM levels specifically of DNA ligase III (Lig3), the only DNA ligase working in mitochondria is reduced. The reduction of Lig3 is observed in different A-T patient cells, in brain and pre-B cells derived from ATM knockout mice as well as upon transient or stable knockdown of ATM. Furthermore, pharmacological inhibition of Lig3 in wild type cells phenocopies the mtDNA repair defects observed in A-T patient cells. As targeted deletion of LIG3 in the central nervous system causes debilitating ataxia in mice, reduced Lig3 protein levels and the consequent mtDNA repair defect may contribute to A-T neurodegeneration. A-T is thus the first disease characterized by diminished Lig3. Published by Elsevier B.V.

  4. Repairing split ends: SIRT6, mono-ADP ribosylation and DNA repair

    Science.gov (United States)

    Van Meter, Michael; Mao, Zhiyong; Gorbunova, Vera; Seluanov, Andrei

    2011-01-01

    The sirtuin gene family comprises an evolutionarily ancient set of NAD+ dependent protein deacetylase and mono-ADP ribosyltransferase enzymes. Found in all domains of life, sirtuins regulate a diverse array of biological processes, including DNA repair, gene silencing, apoptosis and metabolism. Studies in multiple model organisms have indicated that sirtuins may also function to extend lifespan and attenuate age-related pathologies. To date, most of these studies have focused on the deacetylase activity of sirtuins, and relatively little is known about the other biochemical activity of sirtuins, mono-ADP ribosylation. We recently reported that the mammalian sirtuin, SIRT6, mono-ADP ribosylates PARP1 to promote DNA repair in response to oxidative stress. In this research perspective we review the role of SIRT6 in DNA repair and discuss the emerging implications for sirtuin directed mono-ADP ribosylation in aging and age-related diseases. PMID:21946623

  5. Approaches to diagnose DNA mismatch repair gene defects in cancer

    DEFF Research Database (Denmark)

    Peña-Diaz, Javier; Rasmussen, Lene Juel

    2016-01-01

    The DNA repair pathway mismatch repair (MMR) is responsible for the recognition and correction of DNA biosynthetic errors caused by inaccurate nucleotide incorporation during replication. Faulty MMR leads to failure to address the mispairs or insertion deletion loops (IDLs) left behind...... already been well defined and their pathogenicity assessed. Despite this substantial wealth of knowledge, the effects of a large number of alterations remain uncharacterized (variants of uncertain significance, VUSs). The advent of personalized genomics is likely to increase the list of VUSs found in MMR...

  6. Decreased repair of gamma damaged DNA in progeria

    Energy Technology Data Exchange (ETDEWEB)

    Rainbow, A.J.; Howes, M.

    1977-01-01

    A sensitive host-cell reactivation technique was used to examine the DNA repair ability of fibroblasts from two patients with classical progeria. Fibroblasts were infected with either non-irradiated or gamma-irradiated adenovirus type 2 and at 48 hrs after infection cells were examined for the presence of viral structural antigens using immunofluorescent staining. The production of viral structural antigens was considerably reduced in the progeria lines as compared to normal fibroblasts when gamma-irradiated virus was used, indicating a defect in the repair of gamma ray damaged DNA in the progeria cells.

  7. Naturally occurring branched-chain polyamines induce a crosslinked meshwork structure in a giant DNA

    Science.gov (United States)

    Muramatsu, Akira; Shimizu, Yuta; Yoshikawa, Yuko; Fukuda, Wakao; Umezawa, Naoki; Horai, Yuhei; Higuchi, Tsunehiko; Fujiwara, Shinsuke; Imanaka, Tadayuki; Yoshikawa, Kenichi

    2016-12-01

    We studied the effect of branched-chain polyamines on the folding transition of genome-sized DNA molecules in aqueous solution by the use of single-molecule observation with fluorescence microcopy. Detailed morphological features of polyamine/DNA complexes were characterized by atomic force microscopy (AFM). The AFM observations indicated that branched-chain polyamines tend to induce a characteristic change in the higher-order structure of DNA by forming bridges or crosslinks between the segments of a DNA molecule. In contrast, natural linear-chain polyamines cause a parallel alignment between DNA segments. Circular dichroism measurements revealed that branched-chain polyamines induce the A-form in the secondary structure of DNA, while linear-chain polyamines have only a minimum effect. This large difference in the effects of branched- and linear-chain polyamines is discussed in relation to the difference in the manner of binding of these polyamines to negatively charged double-stranded DNA.

  8. Site-specific uv crosslinking of minihelix DNA and TrpRS from Bacillus subtilis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to investigate the recognition mechanism and the relationship between structure and function of minihelix DNA with Tryptophanyl-tRNA Synthetase (TrpRS), TrpRS from Bacillus Subtilis was purified. Four minihelix DNAs were chemically synthesized and the photoreactive reagent s4T was incorporated into three of them at the positions of G73, T72 and T55 corresponding to tRNATrp.The apparatus for uv crossiinking was devised and the parameters for uv crosslinking were optimized. The results indicated that the G73 and T72 base of minihelix DNA interacted with TrpRS directly. The uv crosslinking reaction was improved by the dose of uv irradiation and the concentration of both TrpRS and minihelix DNA.``

  9. Site specificity of psoralen-DNA interstrand cross-linking determined by nuclease Bal31 digestion

    DEFF Research Database (Denmark)

    Zhen, W P; Buchardt, O; Nielsen, Henrik

    1986-01-01

    A novel method for determination of psoralen photo-cross-linking sites in double-stranded DNA is described, which is based on a pronounced inhibition of Bal31 exonuclease activity by psoralen-DNA interstrand cross-links. The results using a 51 base pair fragment of plasmid pUC19 and a 346 base pair...... fragment of pBR322 show that 5'-TA sequences are preferred cross-linking sites compared to 3'-TA sequences. They also indicate that sequences flanking the 5'-TA site influence the cross-linking efficiency at the site. The DNA photo-cross-linking by 4,5',8-trimethylpsoralen and 8-methoxypsoralen...... was analyzed, and these two psoralens showed identical site specificity. The 5'-TA preference is rationalized on the basis of the local DNA structure in terms of the pi-pi electronic interaction between the thymines and the intercalated psoralens, as well as on the base tilt angles of the DNA....

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

    Directory of Open Access Journals (Sweden)

    Elisa Mentegari

    2016-08-01

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

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

    Science.gov (United States)

    Gorbunova, Vera; Seluanov, Andrei

    2016-06-01

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

  12. Diaphragm Repair with a Novel Cross-Linked Collagen Biomaterial in a Growing Rabbit Model.

    Directory of Open Access Journals (Sweden)

    Steffi Mayer

    Full Text Available Neonates with congenital diaphragmatic hernia and large defects often require patch closure. Acellular collagen matrices (ACM have been suggested as an alternative to synthetic durable patches as they are remodeled by the host or could also be used for tissue engineering purposes.2.0x1.0 cm diaphragmatic defects were created in 6-weeks old New-Zealand white rabbits. We compared reconstruction with a purpose-designed cross-linked ACM (Matricel to 4-layer non-cross-linked small intestinal submucosa (SIS and a 1-layer synthetic Dual Mesh (Gore-Tex. Unoperated animals or animals undergoing primary closure (4/0 polyglecaprone served as age-matched controls. 60 (n = 25 resp. 90 (n = 17 days later, animals underwent chest x-ray and obduction for gross examination of explants, scoring of adhesion and inflammatory response. Also, uniaxial tensiometry was done, comparing explants to contralateral native diaphragmatic tissue.Overall weight nearly doubled from 1,554±242 g at surgery to 2,837±265 g at obduction (+84%. X-rays did show rare elevation of the left diaphragm (SIS = 1, Gore-Tex = 1, unoperated control = 1, but no herniation of abdominal organs. 56% of SIS and 10% of Matricel patches degraded with visceral bulging in four (SIS = 3, Matricel = 1. Adhesion scores were limited: 0.5 (Matricel to 1 (SIS, Gore-Tex to the left lung (p = 0.008 and 2.5 (Gore-Tex, 3 (SIS and 4 (Matricel to the liver (p<0.0001. Tensiometry revealed a reduced bursting strength but normal compliance for SIS. Compliance was reduced in Matricel and Gore-Tex (p<0.01. Inflammatory response was characterized by a more polymorphonuclear cell (SIS resp. macrophage (Matricel type of infiltrate (p<0.05. Fibrosis was similar for all groups, except there was less mature collagen deposited to Gore-Tex implants (p<0.05.Matricel induced a macrophage-dominated inflammatory response, more adhesions, had appropriate strength but a lesser compliance compared to native tissue. The herein

  13. Integral parametrization of the Kinetics of Crosslink production in plasmid DNA as a function of 8-methoxypsoralen concentration

    Energy Technology Data Exchange (ETDEWEB)

    Vidania, R. de; Paramio, J. M.; Bauluz, C.

    1986-07-01

    In this paper we present results of crosslink production in pBR322 DNA along a wide range of 8-methoxypsoralen (8-MOP) concentration. Experimental data were obtained as DNA renaturation percentages, from the shift in hyperchromicity after a temperature-dependent denaturation-renaturation process. the experimental results showed a three-stage profile when represented as a function of the natural logarithms of 8-MOP concentration. an integral parametrization which allows a simultaneous fit of the three observed stages is presented here. the theoretical values of crosslink production determined from the fit are useful to asses the genotoxicity of psoralen-induced crosslinks in plasmid DNA. (Author) 24 refs.

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

    Science.gov (United States)

    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.

  15. Principles of ubiquitin and SUMO modifications in DNA repair

    NARCIS (Netherlands)

    Bergink, Steven; Jentsch, Stefan

    2009-01-01

    With the discovery in the late 1980s that the DNA-repair gene RAD6 encodes a ubiquitin-conjugating enzyme, it became clear that protein modification by ubiquitin conjugation has a much broader significance than had previously been assumed. Now, two decades later, ubiquitin and its cousin SUMO are im

  16. Selective targeting of homologous DNA recombination repair by gemcitabine

    NARCIS (Netherlands)

    Wachters, FM; van Putten, JWG; Maring, JG; Zdzienicka, MZ; Groen, HJM; Kampinga, HH

    2003-01-01

    Purpose: Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) is a potent radiosensitizer. The mechanism of dFdC-mediated radiosensitization is yet poorly understood. We recently excluded inhibition of DNA double-strand break (DSB) repair by nonhomologous end-joining (NHEJ) as a means of

  17. DNA Repair-Protein Relocalization After Heavy Ion Exposure

    Science.gov (United States)

    Metting, N. F.

    1999-01-01

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

  18. UV Radiation Damage and Bacterial DNA Repair Systems

    Science.gov (United States)

    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…

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

    Directory of Open Access Journals (Sweden)

    De Felice F

    2017-03-01

    Full Text Available Francesca De Felice,1 Vincenzo Tombolini,1 Francesco Marampon,2 Angela Musella,3 Claudia Marchetti3 1Department of Radiotherapy, Policlinico Umberto I, “Sapienza” University of Rome, Rome, 2Department of Biotechnological and Applied Clinical Sciences, Laboratory of Radiobiology, University of L’Aquila, L’Aquila, 3Department of Gynecological and Obstetrical Sciences and Urological Sciences, “Sapienza” University of Rome, Rome, Italy Abstract: The field of prostate oncology has continued to change dramatically. It has truly become a field that is intensely linked to molecular genetic alterations, especially DNA-repair defects. Germline breast cancer 1 gene (BRCA1 and breast cancer 2 gene (BRCA2 mutations are implicated in the highest risk of prostate cancer (PC predisposition and aggressiveness. Poly adenosine diphosphate ribose polymerase (PARP proteins play a key role in DNA repair mechanisms and represent a valid target for new therapies. Olaparib is an oral PARP inhibitor that blocks DNA repair pathway and coupled with BRCA mutated-disease results in tumor cell death. In phase II clinical trials, including patients with advanced castration-resistant PC, olaparib seems to be efficacious and well tolerated. Waiting for randomized phase III trials, olaparib should be considered as a promising treatment option for PC. Keywords: prostate cancer, metastatic disease, castration resistant, BRCA, DNA-repair, PARP, olaparib

  20. Distribution of DNA repair-related ESTs in sugarcane

    Directory of Open Access Journals (Sweden)

    W.C. Lima

    2001-12-01

    Full Text Available DNA repair pathways are necessary to maintain the proper genomic stability and ensure the survival of the organism, protecting it against the damaging effects of endogenous and exogenous agents. In this work, we made an analysis of the expression patterns of DNA repair-related genes in sugarcane, by determining the EST (expressed sequence tags distribution in the different cDNA libraries of the SUCEST transcriptome project. Three different pathways - photoreactivation, base excision repair and nucleotide excision repair - were investigated by employing known DNA repair proteins as probes to identify homologous ESTs in sugarcane, by means of computer similarity search. The results showed that DNA repair genes may have differential expressions in tissues, depending on the pathway studied. These in silico data provide important clues on the potential variation of gene expression, to be confirmed by direct biochemical analysis.As vias de reparo de DNA são requeridas para manter a necessária estabilidade genômica e garantir a sobrevivência do organismo, frente aos efeitos deletérios causados por fatores endógenos e exógenos. Neste trabalho, realizamos a análise dos padrões de expressão dos genes de reparo de DNA encontrados na cana-de-açúcar, pela determinação da distribuição de ESTs nas diferentes bibliotecas de cDNA no projeto de transcriptoma SUCEST. Três vias de reparo - fotorreativação, reparo por excisão de bases e reparo por excisão de nucleotídeos - foram estudadas através do uso de proteínas de reparo como sondas para identificação de ESTs homólogos em cana-de-açúcar, com base na procura computacional de similaridade. Os resultados indicam que os genes de reparo de DNA possuem uma expressão diferencial nos tecidos, dependendo da via de reparo analisada. Esses dados in silico fornecem importantes indícios da expressão diferencial, a qual deve ser confirmada por análises bioquímicas diretas.

  1. DNA repair and transcription deficiency syndromes

    NARCIS (Netherlands)

    W. Vermeulen (Wim)

    1995-01-01

    textabstractThe genetic information of all living organisms is stored in DNA, a long macromolecule composed of four different nucleotides. Preservation of the sequence of nucleotides, defining the genetic code, is a prerequisite for a faithful transmission of the genetic information to subsequent ge

  2. Generation of Guanine – Thymidine Cross-links in DNA by Peroxynitrite/Carbon Dioxide

    OpenAIRE

    Yun, Byeong Hwa; Geacintov, Nicholas E.; Shafirovich, Vladimir

    2011-01-01

    Nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, is an important chemical mediator of inflammation. In aqueous solutions, it rapidly decomposes to the reactive species CO3•− and •NO2 radicals that are known to initiate the selective oxidation and nitration of guanine in DNA. We have previously demonstrated that the reactions of carbonate radical anions with guanine in 2′-deoxyoligoribonucleotides generate a previously unknown intrastrand cross-linked gu...

  3. Cycling with BRCA2 from DNA repair to mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hyunsook, E-mail: HL212@snu.ac.kr

    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.

  4. Eukaryotic Mismatch Repair in Relation to DNA Replication.

    Science.gov (United States)

    Kunkel, Thomas A; Erie, Dorothy A

    2015-01-01

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

  5. Molecular Understanding of Efficient DNA Repair Machinery of Photolyase

    Science.gov (United States)

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

    2012-06-01

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

  6. DNA ligase I selectively affects DNA synthesis by DNA polymerases delta and epsilon suggesting differential functions in DNA replication and repair.

    OpenAIRE

    Mossi, R; Ferrari, E; Hübscher, U

    1998-01-01

    The joining of single-stranded breaks in double-stranded DNA is an essential step in many important processes such as DNA replication, DNA repair, and genetic recombination. Several data implicate a role for DNA ligase I in DNA replication, probably coordinated by the action of other enzymes and proteins. Since both DNA polymerases delta and epsilon show multiple functions in different DNA transactions, we investigated the effect of DNA ligase I on various DNA synthesis events catalyzed by th...

  7. Aspects of DNA repair and nucleotide pool imbalance

    Energy Technology Data Exchange (ETDEWEB)

    Holliday, R.

    1985-01-01

    Evidence that optimum repair depends on adequate pools of deoxynucleotide triphosphates (dNTPs) comes from the study of pyrimidine auxotrophs of Ustilago maydis. These strains are sensitive to UV light and X-rays, and for pyr1-1 it has been shown that the intracellular concentration of dTTP is reduced about 7-fold. The survival curve of pyr1-1 after UV-treatment, and split dose experiments with wild-type cells, provide evidence for an inducible repair mechanism, which probably depends on genetic recombination. Although inducible repair saves cellular resources, it has the disadvantage of becoming ineffective at doses which are high enough to inactivate the repressed structural gene(s) for repair enzymes. It is clear that a wide variety of repair mechanisms have evolved to remove lesions which arise either spontaneously or as a result of damage from external agents. Nevertheless, it would be incorrect to assume that all species require all possible pathways of repair. It is now well established that the accuracy of DNA and protein synthesis depends on proof-reading or editing mechanisms. Optimum accuracy levels will evolve from the balance between error avoidance in macromolecular synthesis and physiological efficiency in growth and propagation.

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

    Science.gov (United States)

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

    2016-05-08

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

  9. DNA repair in neurons: So if they don't divide what's to repair?

    Energy Technology Data Exchange (ETDEWEB)

    Fishel, Melissa L. [Department of Pediatrics (Section of Hematology/Oncology), Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 W. Walnut, Room 302C, Indianapolis, IN 46202 (United States); Vasko, Michael R. [Department of Pharmacology and Toxicology, Indiana University School of Medicine, 1044 W. Walnut St., Indianapolis, IN 46202 (United States); Kelley, Mark R. [Department of Pediatrics (Section of Hematology/Oncology), Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 W. Walnut, Room 302C, Indianapolis, IN 46202 (United States) and Department of Pharmacology and Toxicology, Indiana University School of Medicine, 1044 W. Walnut St., Indianapolis, IN 46202 (United States) and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 1044 W. Walnut, Room 302C, Indianapolis, IN 46202 (United States)]. E-mail: mkelley@iupui.edu

    2007-01-03

    Neuronal DNA repair remains one of the most exciting areas for investigation, particularly as a means to compare the DNA repair response in mitotic (cancer) vs. post-mitotic (neuronal) cells. In addition, the role of DNA repair in neuronal cell survival and response to aging and environmental insults is of particular interest. DNA damage caused by reactive oxygen species (ROS) such as generated by mitochondrial respiration includes altered bases, abasic sites, and single- and double-strand breaks which can be prevented by the DNA base excision repair (BER) pathway. Oxidative stress accumulates in the DNA of the human brain over time especially in the mitochondrial DNA (mtDNA) and is proposed to play a critical role in aging and in the pathogenesis of several neurological disorders including Parkinson's disease, ALS, and Alzheimer's diseases. Because DNA damage accumulates in the mtDNA more than nuclear DNA, there is increased interest in DNA repair pathways and the consequence of DNA damage in the mitochondria of neurons. The type of damage that is most likely to occur in neuronal cells is oxidative DNA damage which is primarily removed by the BER pathway. Following the notion that the bulk of neuronal DNA damage is acquired by oxidative DNA damage and ROS, the BER pathway is a likely area of focus for neuronal studies of DNA repair. BER variations in brain aging and pathology in various brain regions and tissues are presented. Therefore, the BER pathway is discussed in greater detail in this review than other repair pathways. Other repair pathways including direct reversal, nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination and non-homologous end joining are also discussed. Finally, there is a growing interest in the role that DNA repair pathways play in the clinical arena as they relate to the neurotoxicity and neuropathy associated with cancer treatments. Among the numerous side effects of cancer treatments, major

  10. Structural Elucidation of DNA-Protein Crosslinks Using Reductive Desulfurization and Liquid Chromatography-Tandem Mass Spectrometry

    OpenAIRE

    Wickramaratne, Susith; Tretyakova, Natalia Y.

    2014-01-01

    Structural characterization of DNA-protein crosslinks involving cysteine using reductive desulfurization in combination with liquid chromatography-tandem mass spectrometry is highlighted. The novel approach was used to identify hydrolytically stable DNA-protein lesions involving alkylguanine DNA alkyltransferase (AGT).

  11. DNA interstrand cross-links of an antitumor trinuclear platinum(II) complex: thermodynamic analysis and chemical probing.

    Science.gov (United States)

    Malina, Jaroslav; Farrell, Nicholas P; Brabec, Viktor

    2011-06-06

    The trinuclear platinum compound [{trans-PtCl(NH(3))(2)}(2)(μ-trans-Pt(NH(3))(2){NH(2)(CH(2))(6)NH(2)}(2))](4+) (BBR3464) belongs to the polynuclear class of platinum-based anticancer agents. These agents form in DNA long-range (Pt,Pt) interstrand cross-links, whose role in the antitumor effects of BBR3464 predominates. Our results show for the first time that the interstrand cross-links formed by BBR3464 between two guanine bases in opposite strands separated by two base pairs (1,4-interstrand cross-links) exist as two distinct conformers, which are not interconvertible, not only if these cross-links are formed in the 5'-5', but also in the less-usual 3'-3' direction. Analysis of the conformers by differential scanning calorimetry, chemical probes of DNA conformation, and minor groove binder Hoechst 33258 demonstrate that each of the four conformers affects DNA in a distinctly different way and adopts a different conformation. The results also support the thesis that the molecule of antitumor BBR3464 when forming DNA interstrand cross-links may adopt different global structures, including different configurations of the linker chain of BBR3464 in the minor groove of DNA. Our findings suggest that the multiple DNA interstrand cross-links available to BBR3464 may all contribute substantially to its cytotoxicity.

  12. Induction of a mutant phenotype in human repair proficient cells after overexpression of a mutated human DNA repair gene.

    NARCIS (Netherlands)

    P.B.G.M. Belt; M.F. van Oostenrijk; H. Odijk (Hanny); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude)

    1991-01-01

    textabstractAntisense and mutated cDNA of the human excision repair gene ERCC-1 were overexpressed in repair efficient HeLa cells by means of an Epstein-Barr-virus derived CDNA expression vector. Whereas antisense RNA did not influence the survival of the transfected cells, a mutated cDNA generating

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

    Science.gov (United States)

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

    2015-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Isao Kuraoka

    2015-09-01

    Full Text Available 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.

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

    DEFF Research Database (Denmark)

    Smith, Eric A; Gole, Boris; Willis, Nicholas A

    2017-01-01

    -deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout...... mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK...... filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition....

  16. DNA-repair measurements by use of the modified comet assay

    DEFF Research Database (Denmark)

    Godschalk, Roger W L; Ersson, Clara; Riso, Patrizia;

    2013-01-01

    The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity...... line as having the highest level of DNA-repair activity. The two laboratories that reported discordant results (with another cell line having the highest level of DNA-repair activity) were those that reported to have little experience with the modified comet assay to assess DNA repair. The laboratories...

  17. DNA repair mechanisms in eukaryotes: Special focus in Entamoeba histolytica and related protozoan parasites.

    Science.gov (United States)

    López-Camarillo, César; Lopez-Casamichana, Mavil; Weber, Christian; Guillen, Nancy; Orozco, Esther; Marchat, Laurence A

    2009-12-01

    Eukaryotic cell viability highly relies on genome stability and DNA integrity maintenance. The cellular response to DNA damage mainly consists of six biological conserved pathways known as homologous recombination repair (HRR), non-homologous end-joining (NHEJ), base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), and methyltransferase repair that operate in a concerted way to minimize genetic information loss due to a DNA lesion. Particularly, protozoan parasites survival depends on DNA repair mechanisms that constantly supervise chromosomes to correct damaged nucleotides generated by cytotoxic agents, host immune pressure or cellular processes. Here we reviewed the current knowledge about DNA repair mechanisms in the most relevant human protozoan pathogens. Additionally, we described the recent advances to understand DNA repair mechanisms in Entamoeba histolytica with special emphasis in the use of genomic approaches based on bioinformatic analysis of parasite genome sequence and microarrays technology.

  18. True Lies: The Double Life of the Nucleotide Excision Repair Factors in Transcription and DNA Repair

    Directory of Open Access Journals (Sweden)

    Nicolas Le May

    2010-01-01

    Full Text Available Nucleotide excision repair (NER is a major DNA repair pathway in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation or bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to three genetic disorders that result in predisposition to cancers, accelerated aging, neurological and developmental defects. During NER, more than 30 polypeptides cooperate to recognize, incise, and excise a damaged oligonucleotide from the genomic DNA. Recent papers reveal an additional and unexpected role for the NER factors. In the absence of a genotoxic attack, the promoters of RNA polymerases I- and II-dependent genes recruit XPA, XPC, XPG, and XPF to initiate gene expression. A model that includes the growth arrest and DNA damage 45α protein (Gadd45α and the NER factors, in order to maintain the promoter of active genes under a hypomethylated state, has been proposed but remains controversial. This paper focuses on the double life of the NER factors in DNA repair and transcription and describes the possible roles of these factors in the RNA synthesis process.

  19. An inverse switch in DNA base excision and strand break repair contributes to melphalan resistance in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Mirta M L Sousa

    Full Text Available Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs. Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ pathway of double-strand break (DSB repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel

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

    Science.gov (United States)

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

    2008-05-01

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

  1. P-glycoprotein attenuates DNA repair activity in multidrug-resistant cells by acting through the Cbp-Csk-Src cascade.

    Science.gov (United States)

    Lin, Li-Fang; Wu, Ming-Hsi; Pidugu, Vijaya Kumar; Ho, I-Ching; Su, Tsann-Long; Lee, Te-Chang

    2017-02-03

    Recent studies have demonstrated that P-glycoprotein (P-gp) expression impairs DNA interstrand cross-linking agent-induced DNA repair efficiency in multidrug-resistant (MDR) cells. To date, the detailed molecular mechanisms underlying how P-gp interferes with Src activation and subsequent DNA repair activity remain unclear. In this study, we determined that the C-terminal Src kinase-binding protein (Cbp) signaling pathway involved in the negative control of Src activation is enhanced in MDR cells. We also demonstrated that cells that ectopically express P-gp exhibit reduced activation of DNA damage response regulators, such as ATM, Chk2, Braca1 and Nbs1 and hence attenuated DNA double-strand break repair capacity and become more susceptible than vector control cells to DNA interstrand cross-linking (ICL) agents. Moreover, we demonstrated that P-gp can not only interact with Cbp and Src but also enhance the formation of inhibitory C-terminal Src kinase (Csk)-Cbp complexes that reduce phosphorylation of the Src activation residue Y416 and increase phosphorylation of the Src negative regulatory residue Y527. Notably, suppression of Cbp expression in MDR cells restores cisplatin-induced Src activation, improves DNA repair capacity, and increases resistance to ICL agents. Ectopic expression of Cbp attenuates cisplatin-induced Src activation and increases the susceptibility of cells to ICL agents. Together, the current results indicate that P-gp inhibits DNA repair activity by modulating Src activation via Cbp-Csk-Src cascade. These results suggest that DNA ICL agents are likely to have therapeutic potential against MDR cells with P-gp-overexpression.

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

    Science.gov (United States)

    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 O⁶-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

  3. Manipulating DNA repair for improved genetic engineering in Aspergillus

    DEFF Research Database (Denmark)

    Nødvig, Christina Spuur

    engineering strategies. Chapter 1 gives an introduction to the genus Aspergillus and some of the tools relevant to fungal genetic engineering. It also contains a short introduction to DNA repair and its interplay with gene targeting and finally an overview over the different genome editing technologies......Aspergillus is a genus of filamentous fungi, which members includes industrial producers of enzymes, organic acids and secondary metabolites, important pathogens and a model organism. As such no matter the specific area of interest there are many reasons to perform genetic engineering, whether...... it is metabolic engineering to create better performing cell factory, elucidating pathways to study secondary metabolism etc. In this thesis, the main focus is on different ways to manipulate DNA repair for optimizing gene targeting, ultimately improving the methods available for faster and better genetic...

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

    Science.gov (United States)

    Hu, Jinchuan; Adar, Sheera

    2017-01-01

    DNA damage presents a barrier to DNA-templated biochemical processes, including gene expression and faithful DNA replication. Compromised DNA repair leads to mutations, enhancing the risk for genetic diseases and cancer development. Conventional experimental approaches to study DNA damage required a researcher to choose between measuring bulk damage over the entire genome, with little or no resolution regarding a specific location, and obtaining data specific to a locus of interest, without a global perspective. Recent advances in high-throughput genomic tools overcame these limitations and provide high-resolution measurements simultaneously across the genome. In this review, we discuss the available methods for measuring DNA damage and their repair, focusing on genomewide assays for pyrimidine photodimers, the major types of damage induced by ultraviolet irradiation. These new genomic assays will be a powerful tool in identifying key components of genome stability and carcinogenesis. © 2016 The American Society of Photobiology.

  5. DNA Repair and Ethnic Differences in Prostate Cancer Risk

    Science.gov (United States)

    2007-03-01

    and oxidative DNA damage (14); and lycopene , vitaminE, and other antioxidants are suggested protective agents (15). Both OGG1 and XRCC1 repair...available for review. Temperature dependent equipment such as freezers, refrigerators and water baths are checked and recorded daily. Our repository...Products, Lycopene , and Prostate Cancer Risk. J.Natl.Cancer Inst. 3-6- 2002;94(5):391-8. 16. Xu, J., Zheng, S. L., Turner, A., Isaacs, S. D., Wiley

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  8. DNA repair inhibition by UVA photoactivated fluoroquinolones and vemurafenib

    Science.gov (United States)

    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

  9. Metabolism, genomics, and DNA repair in the mouse aging liver

    DEFF Research Database (Denmark)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ramesh C. Gupta

    2008-03-01

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

  11. Generation of Guanine – Thymidine Cross-links in DNA by Peroxynitrite/Carbon Dioxide

    Science.gov (United States)

    Yun, Byeong Hwa; Geacintov, Nicholas E.; Shafirovich, Vladimir

    2011-01-01

    Nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, is an important chemical mediator of inflammation. In aqueous solutions, it rapidly decomposes to the reactive species CO3•− and •NO2 radicals that are known to initiate the selective oxidation and nitration of guanine in DNA. We have previously demonstrated that the reactions of carbonate radical anions with guanine in 2′-deoxyoligoribonucleotides generate a previously unknown intrastrand cross-linked guanine-thymine product G*-T* with a covalent bond between the C8 (G*) and thymine N3 (T*) atoms (Crean et al., Nucleic Acids Res., 2008, 36, 742–755). In this work we demonstrate that G*-T* cross-linked products are also formed when peroxynitrite (0.1 mM) reacts with native DNA in aqueous solutions (pH 7.5–7.7) containing 25 mM carbon dioxide/bicarbonate, in addition to the well known nitration/oxidation products of guanine such as 8-nitroguanine (8-nitroG), 5-guanidino-4-nitroimidazole (NIm), 8-oxo-7,8-dehydroguanine (8-oxoG) and spiroiminodihydantoin (Sp). The yields of these products, after enzymatic digestion with P1 nuclease and alkaline phosphatase to the nucleotide level, and reversed phase HPLC separation, were compared with those obtained with the uniformly, isotopically labeled 15N,13C-labeled 2′-deoxy oligoribonucleotides 5′-dGpT and 5′-dGpCpT. The d(G*pT*) and d(G*-T*) cross-linked products derived from the di- and tri-oligonucleotides, respectively, were used as standards for identifying the analogous lesions in calf thymus DNA by isotope dilution LC-MS/MS methods in the selected reaction-monitoring mode. The Nim and 8nitroG are the major products formed (~ 0.05% each), and lesser amounts of 8-oxoG (~ 0.02%), and d(G*pT*) and d(G*-T*) enzymatic digestion products (~ 0.002% each) were found. It is shown that the formation of d(G*pT*) enzyme digestion product can arise only from intrastrand cross-links, whereas d(G*-T*) can arise from both

  12. Generation of guanine-thymidine cross-links in DNA by peroxynitrite/carbon dioxide.

    Science.gov (United States)

    Yun, Byeong Hwa; Geacintov, Nicholas E; Shafirovich, Vladimir

    2011-07-18

    Nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite is an important chemical mediator of inflammation. In aqueous solutions, it rapidly decomposes to the reactive species CO(3)(•-) and (•)NO(2) radicals that are known to initiate the selective oxidation and nitration of guanine in DNA. We have previously demonstrated that the reactions of carbonate radical anions with guanine in 2'-deoxyoligoribonucleotides generate a previously unknown intrastrand cross-linked guanine-thymine product G*-T* with a covalent bond between the C8 (G*) and the thymine N3 (T*) atoms (Crean Nucleic Acids Res. 2008, 36, 742-755). In this work, we demonstrate that G*-T* cross-linked products are also formed when peroxynitrite (0.1 mM) reacts with native DNA in aqueous solutions (pH 7.5-7.7) containing 25 mM carbon dioxide/bicarbonate, in addition to the well-known nitration/oxidation products of guanine such as 8-nitroguanine (8-nitro-G), 5-guanidino-4-nitroimidazole (NIm), 8-oxo-7,8-dehydroguanine (8-oxo-G), and spiroiminodihydantoin (Sp). The yields of these products, after enzymatic digestion with P1 nuclease and alkaline phosphatase to the nucleotide level and reversed phase HPLC separation, were compared with those obtained with the uniformly, isotopically labeled (15)N,(13)C-labeled 2'-deoxy oligoribonucleotides 5'-dGpT and 5'-dGpCpT. The d(G*pT*) and d(G*-T*) cross-linked products derived from the di- and trioligonucleotides, respectively, were used as standards for identifying the analogous lesions in calf thymus DNA by isotope dilution LC-MS/MS methods in the selected reaction monitoring mode. The NIm and 8-nitro-G are the major products formed (∼0.05% each), and lesser amounts of 8-oxo-G (∼0.02%) and d(G*pT*) and d(G*-T*) enzymatic digestion products (∼0.002% each) were found. It is shown that the formation of d(G*pT*) enzyme digestion product can arise only from intrastrand cross-links, whereas d(G*-T*) can arise from both interstrand

  13. Studies on bleomycin-induced repair DNA synthesis in permeable mouse ascites sarcoma cells.

    Directory of Open Access Journals (Sweden)

    Mori,Shigeru

    1989-04-01

    Full Text Available To study the mechanism of DNA excision repair, a DNA repair system employing permeable mouse sarcoma (SR-C3H/He cells was established and characterized. SR-C3H/He cells were permeabilized with a 0.0175% Triton X-100 solution. The permeable cells were treated with 1 mM ATP and 0.11 mM bleomycin, and then washed thoroughly to remove ATP and bleomycin. Repair DNA synthesis occurred in the bleomycin-damaged, permeable SR-C3H/He cells when incubated with ATP and four deoxyribonucleoside triphosphates. The repair nature of the DNA synthesis was confirmed by the BrdUMP density shift technique, and by the reduced sensitivity of the newly synthesized DNA to Escherichia coli exonuclease III. The DNA synthesis was optimally enhanced by addition of 0.08 M NaCl. Studies using selective inhibitors of DNA synthesis showed that aphidicolin-sensitive DNA polymerase (DNA polymerase alpha and/or delta and DNA polymerase beta were involved in the repair process. The present DNA repair system is thought to be useful to study nuclear DNA damage by bleomycin, removal of the damaged ends by an exonuclease, repair DNA synthesis by DNA polymerases and repair patch ligation by DNA ligase(s.

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

    OpenAIRE

    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 (i...

  15. HuCOP1 contributes to the regulation of DNA repair in keratinocytes.

    Science.gov (United States)

    Fazekas, B; Carty, M P; Németh, I; Kemény, L; Széll, M; Ádám, É

    2017-03-01

    We have previously demonstrated that the E3 ligase Human Constitutive Photomorphogenic Protein (huCOP1) is expressed in human keratinocytes and negatively regulates p53. The MutS homolog 2 (MSH2) protein plays a central role in DNA MMR mechanism and is implicated in the cellular response to anticancer agents, such as cisplatin. Our aim was to clarify whether huCOP1 plays a role in DNA MMR by affecting MSH2 protein level in human keratinocytes. To define the role of huCOP1 in DNA mismatch repair, we determined whether huCOP1 affects MSH2 abundance. MSH2 protein level was detected by immunocytochemical staining using a keratinocyte cell line in which the expression level of huCOP1 was stably decreased (siCOP1). To investigate whether huCOP1 silencing influences cisplatin-induced cell death, control and siCOP1 keratinocyte cells were treated with increasing concentrations of cisplatin and cell viability was recorded after 48 and 96 h. Stable silencing of huCOP1 in human keratinocytes resulted in a reduced level of MSH2 protein. huCOP1 silencing also sensitized keratinocytes to the interstrand crosslinking inducer cisplatin. Our results indicate that decreased huCOP1 correlates with lower MSH2 levels. These protein level changes lead to increased sensitivity toward cisplatin treatment, implicating that huCOP1 plays a positive role in maintaining genome integrity in human keratinocytes.

  16. Monitoring human lymphocytic DNA-protein cross-links as biomarkers of biologically active doses of chromate.

    Science.gov (United States)

    Costa, M; Zhitkovich, A; Toniolo, P; Taioli, E; Popov, T; Lukanova, A

    1996-10-01

    A simple and sensitive assay for DNA-protein cross-links has been used as a biomarker of chromate exposure and early carcinogenic effects. Pilot studies of DNA-protein cross-links in peripheral blood lymphocytes have been conducted with individuals who had higher exposure to chromate, including welders, and with individuals who had lower levels of exposure such as residents living in a chromium-contaminated area in Jersey City, New Jersey. Studies were also conducted in two Bulgarian cities (Jambol and Burgas) with different levels of air pollution and Cr(VI) exposure and in chrome platers in Bulgaria who had high exposure to chromate. DNA-protein cross-links in U.S. welders and in individuals living in Hudson County, New Jersey around chromium-contaminated areas were significantly higher compared to matched controls. Although blood and urinary levels of chromium were not extensively studied in these populations, we were able to obtain these measurements in the Bulgarian population. Chromium levels in red blood cells of controls living in Burgas were in the order of 1 to 2 ppb chromium, and these individuals had the lowest levels of DNA-protein cross-links. However, the chromium levels in Jambol ranged from about 2 to 7 ppb in red blood cells of city residents to about 22 ppb in chrome platers. DNA-protein cross-links were saturated at about 7 to 8 ppb chromium in the red blood cells, and cross-links correlated well only with chromium levels in red blood cells. Urinary chromium levels did not correlate well with either DNA-protein cross-links or chromium levels in with red blood cells.

  17. Mediator MED23 Links Pigmentation and DNA Repair through the Transcription Factor MITF

    Directory of Open Access Journals (Sweden)

    Min Xia

    2017-08-01

    Full Text Available DNA repair is related to many physiological and pathological processes, including pigmentation. Little is known about the role of the transcriptional cofactor Mediator complex in DNA repair and pigmentation. Here, we demonstrate that Mediator MED23 plays an important role in coupling UV-induced DNA repair to pigmentation. The loss of Med23 specifically impairs the pigmentation process in melanocyte-lineage cells and in zebrafish. Med23 deficiency leads to enhanced nucleotide excision repair (NER and less DNA damage following UV radiation because of the enhanced expression and recruitment of NER factors to chromatin for genomic stability. Integrative analyses of melanoma cells reveal that MED23 controls the expression of a melanocyte master regulator, Mitf, by modulating its distal enhancer activity, leading to opposing effects on pigmentation and DNA repair. Collectively, the Mediator MED23/MITF axis connects DNA repair to pigmentation, thus providing molecular insights into the DNA damage response and skin-related diseases.

  18. DNA Damage Response and DNA Repair in Skeletal Myocytes From a Mouse Model of Spinal Muscular Atrophy.

    Science.gov (United States)

    Fayzullina, Saniya; Martin, Lee J

    2016-09-01

    We studied DNA damage response (DDR) and DNA repair capacities of skeletal muscle cells from a mouse model of infantile spinal muscular atrophy (SMA) caused by loss-of-function mutation of survival of motor neuron (Smn). Primary myocyte cultures derived from skeletal muscle satellite cells of neonatal control and mutant SMN mice had similar myotube length, myonuclei, satellite cell marker Pax7 and differentiated myotube marker myosin, and acetylcholine receptor clustering. DNA damage was induced in differentiated skeletal myotubes by γ-irradiation, etoposide, and methyl methanesulfonate (MMS). Unexposed control and SMA myotubes had stable genome integrity. After γ-irradiation and etoposide, myotubes repaired most DNA damage equally. Control and mutant myotubes exposed to MMS exhibited equivalent DNA damage without repair. Control and SMA myotube nuclei contained DDR proteins phospho-p53 and phospho-H2AX foci that, with DNA damage, dispersed and then re-formed similarly after recovery. We conclude that mouse primary satellite cell-derived myotubes effectively respond to and repair DNA strand-breaks, while DNA alkylation repair is underrepresented. Morphological differentiation, genome stability, genome sensor, and DNA strand-break repair potential are preserved in mouse SMA myocytes; thus, reduced SMN does not interfere with myocyte differentiation, genome integrity, and DNA repair, and faulty DNA repair is unlikely pathogenic in SMA.

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

    Directory of Open Access Journals (Sweden)

    Jay C. Brown

    2017-01-01

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

  20. Structural basis for bacterial transcription-coupled DNA repair.

    Science.gov (United States)

    Deaconescu, Alexandra M; Chambers, Anna L; Smith, Abigail J; Nickels, Bryce E; Hochschild, Ann; Savery, Nigel J; Darst, Seth A

    2006-02-10

    Coupling of transcription and DNA repair in bacteria is mediated by transcription-repair coupling factor (TRCF, the product of the mfd gene), which removes transcription elongation complexes stalled at DNA lesions and recruits the nucleotide excision repair machinery to the site. Here we describe the 3.2 A-resolution X-ray crystal structure of Escherichia coli TRCF. The structure consists of a compact arrangement of eight domains, including a translocation module similar to the SF2 ATPase RecG, and a region of structural similarity to UvrB. Biochemical and genetic experiments establish that another domain with structural similarity to the Tudor-like domain of the transcription elongation factor NusG plays a critical role in TRCF/RNA polymerase interactions. Comparison with the translocation module of RecG as well as other structural features indicate that TRCF function involves large-scale conformational changes. These data, along with a structural model for the interaction of TRCF with the transcription elongation complex, provide mechanistic insights into TRCF function.

  1. Green tea and skin cancer: photoimmunology, angiogenesis and DNA repair.

    Science.gov (United States)

    Katiyar, Suchitra; Elmets, Craig A; Katiyar, Santosh K

    2007-05-01

    Human skin is constantly exposed to numerous noxious physical, chemical and environmental agents. Some of these agents directly or indirectly adversely affect the skin. Cutaneous overexposure to environmental solar ultraviolet (UV) radiation (290-400 nm) has a variety of adverse effects on human health, including the development of melanoma and nonmelanoma skin cancers. Therefore, there is a need to develop measures or strategies, and nutritional components are increasingly being explored for this purpose. The polyphenols present in green tea (Camellia sinensis) have been shown to have numerous health benefits, including protection from UV carcinogenesis. (-)-Epigallocatechin-3-gallate (EGCG) is the major and most photoprotective polyphenolic component of green tea. In this review article, we have discussed the most recent investigations and mechanistic studies that define and support the photoprotective efficacy of green tea polyphenols (GTPs) against UV carcinogenesis. The oral administration of GTPs in drinking water or the topical application of EGCG prevents UVB-induced skin tumor development in mice, and this prevention is mediated through: (a) the induction of immunoregulatory cytokine interleukin (IL) 12; (b) IL-12-dependent DNA repair following nucleotide excision repair mechanism; (c) the inhibition of UV-induced immunosuppression through IL-12-dependent DNA repair; (d) the inhibition of angiogenic factors; and (e) the stimulation of cytotoxic T cells in a tumor microenvironment. New mechanistic information strongly supports and explains the chemopreventive activity of GTPs against photocarcinogenesis.

  2. Targeting the DNA repair pathway in Ewing sarcoma.

    Science.gov (United States)

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

    2014-11-06

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

  3. ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor

    NARCIS (Netherlands)

    E. Citterio (Elisabetta); V. van den Boom (Vincent); G. Schnitzler; R. Kanaar (Roland); E. Bonte (Edgar); R.E. Kingston; W. Vermeulen (Wim); J.H.J. Hoeijmakers (Jan)

    2000-01-01

    textabstractThe Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Unraveling the Fanconi anaemia-DNA repair connection through DNA helicase and translocase activities

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, L H

    2005-08-16

    How the Fanconi anaemia (FA) chromosome stability pathway functions to cope with interstrand crosslinks and other DNA lesions has been elusive, even after FANCD1 proved to be BRCA2, a partner of Rad51 in homologous recombination. The identification and characterization of two new Fanconi proteins having helicase motifs, FANCM and FANCJ/BRIP1/BACH1, implicates the FANC nuclear core complex as a participant in recognizing or processing damaged DNA, and the BRIP1 helicase as acting independently of this complex.

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

    Science.gov (United States)

    Kakarougkas, A; Jeggo, P A

    2014-03-01

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

  7. UV-Induced DNA Interstrand Cross-Linking and Direct Strand Breaks from a New Type of Binitroimidazole Analogue.

    Science.gov (United States)

    Han, Yanyan; Chen, Wenbing; Kuang, Yunyan; Sun, Huabing; Wang, Zhiqiang; Peng, Xiaohua

    2015-05-18

    Four novel photoactivated binitroimidazole prodrugs were synthesized. These agents produced DNA interstrand cross-links (ICLs) and direct strand breaks (DSB) upon UV irradiation, whereas no or very few DNA ICLs and DSBs were observed without UV treatment. Although these four molecules (1-4) contain the same binitroimidazole moiety, they bear four different leaving groups, which resulted in their producing different yields of DNA damage. Compound 4, with nitrogen mustard as a leaving group, showed the highest ICL yield. Surprisingly, compounds 1-3, without any alkylating functional group, also induced DNA ICL formation, although they did so with lower yields, which suggested that the binitroimidazole moiety released from UV irradiation of 1-3 is capable of cross-linking DNA. The DNA cross-linked products induced by these compounds were completely destroyed upon 1.0 M piperidine treatment at 90 °C (leading to cleavage at dG sites), which revealed that DNA cross-linking mainly occurred via alkylation of dGs. We proposed a possible mechanism by which alkylating agents were released from these compounds. HRMS and NMR analysis confirmed that free nitrogen mustards were generated by UV irradiation of 4. Suppression of DNA ICL and DSB formation by a radical trap, TEMPO, indicated the involvement of free radicals in the photo reactions of 3 and 4 with DNA. On the basis of these data, we propose that UV irradiation of compounds 1-4 generated a binitroimidazole intermediate that cross-links DNA. The higher ICL yield observed with 4 resulted from the amine effector nitrogen mustard released from UV irradiation.

  8. Repair of surgically created diaphragmatic defect in rat with use of a crosslinked porous collagen scaffold

    NARCIS (Netherlands)

    Brouwer, K.M.; Daamen, W.F.; Reijnen, D.; Verstegen, R.H.J.; Lammers, G.; Hafmans, T.G.M.; Wismans, R.G.; Kuppevelt, A.H.M.S.M. van; Wijnen, R.M.H.

    2013-01-01

    Large defects in congenital diaphragmatic hernia are closed by patch repair, which is associated with a high complication risk and reherniation rate. New treatment modalities are warranted. We evaluated the feasibility of using an acellular biodegradable collagen bioscaffold for a regenerative medic

  9. Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases. delta. and. beta. are involved in DNA repair synthesis induced by N-methyl-N prime -nitro-N-nitrosoguanidine

    Energy Technology Data Exchange (ETDEWEB)

    Hammond, R.A.; Miller, M.R. (West Virginia Univ. Health Sciences Center, Morgantown (USA)); McClung, J.K. (Samuel Roberts Noble Foundation, Inc., East Ardmore, OK (USA))

    1990-01-09

    The involvement of DNA polymerases {alpha}, {beta}, and {delta} in DNA repair synthesis induced by N-methyl-N{prime}-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase {alpha}) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of ({sup 3}H)thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 {mu}g of aphidicolin/mL, 6% by 10 {mu}M BuPdGTP, 13% by anti-(DNA polymerse {alpha}) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 {mu}g of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase {alpha}) antibodies into HF nuclei. These results indicate that both DNA polymerase {delta} and {beta} are involved in repairing DNA damage caused by MNNG.

  10. Role of Rad54, Rad54b and Snm1 in DNA damage repair

    NARCIS (Netherlands)

    J. Wesoly (Joanna)

    2003-01-01

    textabstractThe aim of this thesis is to investigate the function of a number of genes involved in mammalian DNA damage repair, in particular in repair of DNA double-strand breaks (DSBs). Among a large number of different damages that can be introduced to DNA, DSBs are especially toxic. If left unre

  11. Role of Rad54, Rad54b and Snm1 in DNA damage repair

    NARCIS (Netherlands)

    J. Wesoly (Joanna)

    2003-01-01

    textabstractThe aim of this thesis is to investigate the function of a number of genes involved in mammalian DNA damage repair, in particular in repair of DNA double-strand breaks (DSBs). Among a large number of different damages that can be introduced to DNA, DSBs are especially toxic. If

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

    Science.gov (United States)

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

    2014-07-01

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

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

    Science.gov (United States)

    Neher, Tracy M; Turchi, John J

    2011-06-15

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

  14. Antibiotic persistence: the role of spontaneous DNA repair response.

    Science.gov (United States)

    Debbia, E A; Roveta, S; Schito, A M; Gualco, L; Marchese, A

    2001-01-01

    Persisters are a small proportion of a bacterial population that exists in a physiological state permitting survival despite the lethal activity of antibiotics. To explain this phenomenon, it has been suggested that persisters are bacteria repairing spontaneous errors of DNA synthesis. To verify this assumption, Escherichia coli AB1157 and its lexA3 derivative were exposed to a dose 6x MIC of various antibiotics representative of different molecular mechanisms of action (ampicillin, ceftriaxone, meropenem, amikacin, ciprofloxacin). Bacterial cell counts, after 24 hr of exposure to the antimicrobials, revealed a reduction of about 90% of viable organisms in the lexA3 strains in comparison to the lexA+. In several cases, the number of colony-forming units decreased below the limit of assay. This behavior was noted with all antibiotics used, alone or in combination (amikacin plus ceftriaxone and amikacin plus ciprofloxacin). The same experiments were repeated using E. coli AB1157 cultured in the presence of mitomycin C (0.25x MIC), and the number of survivors exceeded by about 90% the values found in the nonexposed control. In contrast, in the sulA background, mitomycin C reacted synergically with all the antibiotics tested causing a strong reduction of the survivors in comparison with the control. The addition of chloramphenicol (0.125x MIC), on the contrary, caused a reduction of the number of survivors of about 90%. These findings indicate that, when DNA repair is active (a mechanism known to block cell division), the number of survivors is greater than that observed with lexA3. Thus, in addition to other possible explanations, persisters might be a fraction of bacteria that during antibiotic treatment are not growing because they are repairing spontaneous errors of DNA synthesis.

  15. Effect of Manganese on DNA-Protein Crosslinks of Testicle in Chicken

    Institute of Scientific and Technical Information of China (English)

    ZHANG Liming; TANG Hongpeng; GU Xijuan; XU Shiwen

    2009-01-01

    In order to explore the effect of manganese on DNA-protein crosslinks (DPC) of testicle in chicken. 500, 800, 1 700 mg·kg-1MnCl2 were added to forage to establish the model of the sub-chronic manganese poisoning. After 30, 60 and 90 d, testicles were taken out to detect hydroxyl radical inhibiting capacity and DPC content. The results showed that compared with control group, hydroxyl radical inhibiting capacity significantly decreased and DPC content notably increased, and there was a time-dose relationship. It demonstrated that manganese could decrease the inhibitng capacity of hydroxyl radical, increase the content of hydroxyl and DPC,and induce DNA damage.

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

    Directory of Open Access Journals (Sweden)

    Scott A Lujan

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

  17. Homologous and non-homologous recombination differentially affect DNA damage repair in mice.

    NARCIS (Netherlands)

    J. Essers (Jeroen); H. van Steeg (Harry); J. de Wit (Jan); M. Vermeij (Marcel); J.H.J. Hoeijmakers (Jan); R. Kanaar (Roland); S.M.A. Swagemakers (Sigrid)

    2000-01-01

    textabstractIonizing radiation and interstrand DNA crosslinking compounds provide important treatments against cancer due to their extreme genotoxicity for proliferating cells. Both the efficacies of such treatments and the mutagenic potential of these agents are modulated by the a

  18. Effect of base sequence on the DNA cross-linking properties of pyrrolobenzodiazepine (PBD) dimers.

    Science.gov (United States)

    Rahman, Khondaker M; James, Colin H; Thurston, David E

    2011-07-01

    Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers are synthetic sequence-selective DNA minor-groove cross-linking agents that possess two electrophilic imine moieties (or their equivalent) capable of forming covalent aminal linkages with guanine C2-NH(2) functionalities. The PBD dimer SJG-136, which has a C8-O-(CH(2))(3)-O-C8'' central linker joining the two PBD moieties, is currently undergoing phase II clinical trials and current research is focused on developing analogues of SJG-136 with different linker lengths and substitution patterns. Using a reversed-phase ion pair HPLC/MS method to evaluate interaction with oligonucleotides of varying length and sequence, we recently reported (JACS, 2009, 131, 13 756) that SJG-136 can form three different types of adducts: inter- and intrastrand cross-linked adducts, and mono-alkylated adducts. These studies have now been extended to include PBD dimers with a longer central linker (C8-O-(CH(2))(5)-O-C8'), demonstrating that the type and distribution of adducts appear to depend on (i) the length of the C8/C8'-linker connecting the two PBD units, (ii) the positioning of the two reactive guanine bases on the same or opposite strands, and (iii) their separation (i.e. the number of base pairs, usually ATs, between them). Based on these data, a set of rules are emerging that can be used to predict the DNA-interaction behaviour of a PBD dimer of particular C8-C8' linker length towards a given DNA sequence. These observations suggest that it may be possible to design PBD dimers to target specific DNA sequences.

  19. Evidence for different mechanisms of ‘unhooking’ for melphalan and cisplatin-induced DNA interstrand cross-links in vitro and in clinical acquired resistant tumour samples

    Directory of Open Access Journals (Sweden)

    Spanswick Victoria J

    2012-09-01

    Full Text Available Abstract Background DNA interstrand cross-links (ICLs are critical lesions produced by several cancer chemotherapy agents including platinum drugs and nitrogen mustards. We have previously shown in haematological (multiple myeloma and solid tumours (ovarian cancer that clinical sensitivity to such agents can result from a defect in DNA ICL processing leading to their persistence. Conversely, enhanced repair can result in clinical acquired resistance following chemotherapy. The repair of ICLs is complex but it is assumed that the ‘unhooking’ step is common to all ICLs. Methods Using a modification of the single cell gel electrophoresis (Comet assay we measured the formation and unhooking of melphalan and cisplatin-induced ICLs in cell lines and clinical samples. DNA damage response in the form of γ-H2AX foci formation and the formation of RAD51 foci as a marker of homologous recombination were also determined. Real-time PCR of 84 genes involved in DNA damage signalling pathways was also examined pre- and post-treatment. Results Plasma cells from multiple myeloma patients known to be clinically resistant to melphalan showed significant unhooking of melphalan-induced ICLs at 48 hours, but did not unhook cisplatin-induced ICLs. In ovarian cancer cells obtained from patients following platinum-based chemotherapy, unhooking of cisplatin-induced ICLs was observed at 48 hours, but no unhooking of melphalan-induced ICLs. In vitro, A549 cells were proficient at unhooking both melphalan and cisplatin-induced ICLs. γ-H2AX foci formation closely followed the formation of ICLs for both drugs, and rapidly declined following the peak of formation. RPMI8226 cells unhooked melphalan, but not cisplatin-induced ICLs. In these cells, although cross-links form with cisplatin, the γ-H2AX response is weak. In A549 cells, addition of 3nM gemcitabine resulted in complete inhibition of cisplatin-induced ICL unhooking but no effect on repair of melphalan ICLs. The

  20. Systematic analysis of DNA damage induction and DNA repair pathway activation by continuous wave visible light laser micro-irradiation

    Directory of Open Access Journals (Sweden)

    Britta Muster

    2017-02-01

    Full Text Available Laser micro-irradiation can be used to induce DNA damage with high spatial and temporal resolution, representing a powerful tool to analyze DNA repair in vivo in the context of chromatin. However, most lasers induce a mixture of DNA damage leading to the activation of multiple DNA repair pathways and making it impossible to study individual repair processes. Hence, we aimed to establish and validate micro-irradiation conditions together with inhibition of several key proteins to discriminate different types of DNA damage and repair pathways using lasers commonly available in confocal microscopes. Using time-lapse analysis of cells expressing fluorescently tagged repair proteins and also validation of the DNA damage generated by micro-irradiation using several key damage markers, we show that irradiation with a 405 nm continuous wave laser lead to the activation of all repair pathways even in the absence of exogenous sensitization. In contrast, we found that irradiation with 488 nm laser lead to the selective activation of non-processive short-patch base excision and single strand break repair, which were further validated by PARP inhibition and metoxyamine treatment. We conclude that these low energy conditions discriminated against processive long-patch base excision repair, nucleotide excision repair as well as double strand break repair pathways.

  1. Screening for DNA Alkylation Mono and Cross-Linked Adducts with a Comprehensive LC-MS(3) Adductomic Approach.

    Science.gov (United States)

    Stornetta, Alessia; Villalta, Peter W; Hecht, Stephen S; Sturla, Shana J; Balbo, Silvia

    2015-12-01

    A high-resolution/accurate-mass DNA adductomic approach was developed to investigate anticipated and unknown DNA adducts induced by DNA alkylating agents in biological samples. Two new features were added to a previously developed approach to significantly broaden its scope, versatility, and selectivity. First, the neutral loss of a base (guanine, adenine, thymine, or cytosine) was added to the original methodology's neutral loss of the 2'-deoxyribose moiety to allow for the detection of all DNA base adducts. Second, targeted detection of anticipated DNA adducts based on the reactivity of the DNA alkylating agent was demonstrated by inclusion of an ion mass list for data dependent triggering of MS(2) fragmentation events and subsequent MS(3) fragmentation. Additionally, untargeted screening of the samples, based on triggering of an MS(2) fragmentation event for the most intense ions of the full scan, was included for detecting unknown DNA adducts. The approach was tested by screening for DNA mono and cross-linked adducts in purified DNA and in DNA extracted from cells treated with PR104A, an experimental DNA alkylating nitrogen mustard prodrug currently under investigation for the treatment of leukemia. The results revealed the ability of this new DNA adductomic approach to detect anticipated and unknown PR104A-induced mono and cross-linked DNA adducts in biological samples. This methodology is expected to be a powerful tool for screening for DNA adducts induced by endogenous or exogenous exposures.

  2. Quantitative DNA interstrand cross-link formation by coumarin and thymine: structure determination, sequence effect, and fluorescence detection.

    Science.gov (United States)

    Sun, Huabing; Fan, Heli; Peng, Xiaohua

    2014-12-01

    The coumarin analogues have been widely utilized in medicine, biology, biochemistry, and material sciences. Here, we report a detailed study on the reactivity of coumarins toward DNA. A series of coumarin analogues were synthesized and incorporated into oligodeoxynucleotides. A photoinduced [2 + 2] cycloaddition occurs between the coumarin moiety and the thymidine upon 350 nm irradiation forming both syn- and anti-cyclobutane adducts (17 and 18), which are photoreversible by 254/350 nm irradiation in DNA. Quantitative DNA interstrand cross-link (ICL) formation was observed with the coumarin moieties containing a flexible two-carbon or longer chain. DNA cross-linking by coumarins shows a kinetic preference when flanked by an A:T base pair as opposed to a G:C pair. An efficient photoinduced electron transfer between coumarin and dG slows down ICL formation. ICL formation quenches the fluorescence of coumarin, which, for the first time, enables fast, easy, and real-time monitoring of DNA cross-linking and photoreversibility via fluorescence spectroscopy. It can be used to detect the transversion mutation between pyrimidines and purines. Overall, this work provides new insights into the biochemical properties and possible toxicity of coumarins. A quantitative, fluorescence-detectable, and photoswitchable DNA cross-linking reaction of the coumarin moieties can potentially serve as mechanistic probes and tools for bioresearch without disrupting native biological environment.

  3. Repair of DNA Lesions by a Reductive Electron Tansfer

    Science.gov (United States)

    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

  4. Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: tryptophans 54 and 88 of E. coli SSB cross-link to DNA

    DEFF Research Database (Denmark)

    Steen, H; Petersen, J; Mann, M

    2001-01-01

    Protein-nucleic acid complexes are commonly studied by photochemical cross-linking. UV-induced cross-linking of protein to nucleic acid may be followed by structural analysis of the conjugated protein to localize the cross-linked amino acids and thereby identify the nucleic acid binding site. Mass....... coli single-stranded DNA binding protein (SSB) that was UV-cross-linked to a 5-iodouracil containing DNA oligomer. Two methods were optimized to circumvent the need for standard liquid chromatography and gel electrophoresis, thereby dramatically increasing the overall sensitivity of the analysis....... Enzymatic degradation of protein and oligonucleotide was combined with miniaturized sample preparation methods for enrichment and desalting of cross-linked peptide-nucleic acid heteroconjugates from complex mixtures prior to mass spectrometric analysis. Detailed characterization of the peptidic component...

  5. Mineral nutrient imbalance, DNA lesion and DNA-protein crosslink involved in growth retardation of Vicia faba L.seedlings exposed to lanthanum ions

    Institute of Scientific and Technical Information of China (English)

    Chengrun Wang; Kegui Zhang; Mei He; Chuanjun Jiang; Liumin Tian; Yuan Tian; Xiaorong Wang

    2012-01-01

    Effects of mineral nutrient imbalance,DNA lesion and DNA-protein crosslink on growth of Vicia faba L.seedlings hydroponically cultivated in concentrations of extraneous lanthanum (La) for 20 days were investigated in the present experiment.The results showed that contents of La,Cu or K elements in roots generally changed synchronously with those in leaves,while Ca,Fe,Zn,Mg,Mn or P in the roots altered inversely to those in the leaves.Thus,the extraneous La led to redistribution and imbalance of mineral nutrient elements in the roots and leaves.DNA lesion and DNA-protein crosslink were investigated by single cell gel electrophoresis (SCGE)and sodium dodecyl sulfate/potassium (SDS/K+) precipitation methods,respectively.The results demonstrated that the increasing La induced DNA break and DNA-protein crosslinks (DPCs) in the seedlings.These results suggested that mineral nutrient imbalance,DNA lesion and DNA-protein crosslink were involved in the growth retardation and growth alteration of the seedlings,which may help to understand the mechanisms of rare earth elements (REEs) on plant growth.

  6. Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: tryptophans 54 and 88 of E. coli SSB cross-link to DNA

    DEFF Research Database (Denmark)

    Steen, H; Petersen, J; Mann, M

    2001-01-01

    of two different peptide-DNA heteroconjugates was accomplished by matrix-assisted laser desorption/ionization mass spectrometry and allowed assignment of tryptophan-54 and tryptophan-88 as candidate cross-linked residues. Sequencing of those peptide-DNA heteroconjugates by nanoelectrospray quadrupole...

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

    Science.gov (United States)

    Bucholtz, Nina; Demuth, Ilja

    2013-10-01

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

  8. Attenuated XPC expression is not associated with impaired DNA repair in bladder cancer.

    Directory of Open Access Journals (Sweden)

    Kishan A T Naipal

    Full Text Available Bladder cancer has a high incidence with significant morbidity and mortality. Attenuated expression of the DNA damage response protein Xeroderma Pigmentosum complementation group C (XPC has been described in bladder cancer. XPC plays an essential role as the main initiator and damage-detector in global genome nucleotide excision repair (NER of UV-induced lesions, bulky DNA adducts and intrastrand crosslinks, such as those made by the chemotherapeutic agent Cisplatin. Hence, XPC protein might be an informative biomarker to guide personalized therapy strategies in a subset of bladder cancer cases. Therefore, we measured the XPC protein expression level and functional NER activity of 36 bladder tumors in a standardized manner. We optimized conditions for dissociation and in vitro culture of primary bladder cancer cells and confirmed attenuated XPC expression in approximately 40% of the tumors. However, NER activity was similar to co-cultured wild type cells in all but one of 36 bladder tumors. We conclude, that (i functional NER deficiency is a relatively rare phenomenon in bladder cancer and (ii XPC protein levels are not useful as biomarker for NER activity in these tumors.

  9. Architecture of the Human and Yeast General Transcription and DNA Repair Factor TFIIH.

    Science.gov (United States)

    Luo, Jie; Cimermancic, Peter; Viswanath, Shruthi; Ebmeier, Christopher C; Kim, Bong; Dehecq, Marine; Raman, Vishnu; Greenberg, Charles H; Pellarin, Riccardo; Sali, Andrej; Taatjes, Dylan J; Hahn, Steven; Ranish, Jeff

    2015-09-03

    TFIIH is essential for both RNA polymerase II transcription and DNA repair, and mutations in TFIIH can result in human disease. Here, we determine the molecular architecture of human and yeast TFIIH by an integrative approach using chemical crosslinking/mass spectrometry (CXMS) data, biochemical analyses, and previously published electron microscopy maps. We identified four new conserved "topological regions" that function as hubs for TFIIH assembly and more than 35 conserved topological features within TFIIH, illuminating a network of interactions involved in TFIIH assembly and regulation of its activities. We show that one of these conserved regions, the p62/Tfb1 Anchor region, directly interacts with the DNA helicase subunit XPD/Rad3 in native TFIIH and is required for the integrity and function of TFIIH. We also reveal the structural basis for defects in patients with xeroderma pigmentosum and trichothiodystrophy, with mutations found at the interface between the p62 Anchor region and the XPD subunit. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Identification of the first small-molecule inhibitor of the REV7 DNA repair protein interaction.

    Science.gov (United States)

    Actis, Marcelo L; Ambaye, Nigus D; Evison, Benjamin J; Shao, Youming; Vanarotti, Murugendra; Inoue, Akira; McDonald, Ezelle T; Kikuchi, Sotaro; Heath, Richard; Hara, Kodai; Hashimoto, Hiroshi; Fujii, Naoaki

    2016-09-15

    DNA interstrand crosslink (ICL) repair (ICLR) has been implicated in the resistance of cancer cells to ICL-inducing chemotherapeutic agents. Despite the clinical significance of ICL-inducing chemotherapy, few studies have focused on developing small-molecule inhibitors for ICLR. The mammalian DNA polymerase ζ, which comprises the catalytic subunit REV3L and the non-catalytic subunit REV7, is essential for ICLR. To identify small-molecule compounds that are mechanistically capable of inhibiting ICLR by targeting REV7, high-throughput screening and structure-activity relationship (SAR) analysis were performed. Compound 1 was identified as an inhibitor of the interaction of REV7 with the REV7-binding sequence of REV3L. Compound 7 (an optimized analog of compound 1) bound directly to REV7 in nuclear magnetic resonance analyses, and inhibited the reactivation of a reporter plasmid containing an ICL in between the promoter and reporter regions. The normalized clonogenic survival of HeLa cells treated with cisplatin and compound 7 was lower than that for cells treated with cisplatin only. These findings indicate that a small-molecule inhibitor of the REV7/REV3L interaction can chemosensitize cells by inhibiting ICLR.

  11. Enhanced base excision repair capacity in carotid atherosclerosis may protect nuclear DNA but not mitochondrial DNA

    DEFF Research Database (Denmark)

    Skarpengland, Tonje; B. Dahl, Tuva; Skjelland, Mona

    2016-01-01

    Lesional and systemic oxidative stress has been implicated in the pathogenesis of atherosclerosis, potentially leading to accumulation of DNA base lesions within atherosclerotic plaques. Although base excision repair (BER) is a major pathway counteracting oxidative DNA damage, our knowledge on BER...... and accumulation of DNA base lesions in clinical atherosclerosis is scarce. Here, we evaluated the transcriptional profile of a wide spectrum of BER components as well as DNA damage accumulation in atherosclerotic and non-atherosclerotic arteries. BER gene expression levels were analyzed in 162 carotid plaques, 8...... genes in atherosclerosis may contribute to lesional nuclear DNA stability but appears insufficient to maintain mtDNA integrity, potentially influencing mitochondrial function in cells within the atherosclerotic lesion....

  12. Azidopropylvinylsulfonamide as a New Bifunctional Click Reagent for Bioorthogonal Conjugations: Application for DNA-Protein Cross-Linking.

    Science.gov (United States)

    Dadová, Jitka; Vrábel, Milan; Adámik, Matej; Brázdová, Marie; Pohl, Radek; Fojta, Miroslav; Hocek, Michal

    2015-11-01

    N-(3-Azidopropyl)vinylsulfonamide was developed as a new bifunctional bioconjugation reagent suitable for the cross-linking of biomolecules through copper(I)-catalyzed azide-alkyne cycloaddition and thiol Michael addition reactions under biorthogonal conditions. The reagent is easily clicked to an acetylene-containing DNA or protein and then reacts with cysteine-containing peptides or proteins to form covalent cross-links. Several examples of bioconjugations of ethynyl- or octadiynyl-modified DNA with peptides, p53 protein, or alkyne-modified human carbonic anhydrase with peptides are given.

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

    Directory of Open Access Journals (Sweden)

    Akutsu Tatsuya

    2009-01-01

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

  14. DNA repair and gene therapy: implications for translational uses.

    Science.gov (United States)

    Limp-Foster, M; Kelley, M R

    2000-01-01

    Gene therapy has been proposed to have implications in the treatment of cancer. By genetically manipulating the hematopoietic stem cell compartment with genes that confer resistance to chemotherapeutic agents, the dose escalation that is necessary to effectively treat the cancers could potentially be achieved. DNA repair genes are some of the potential candidates to confer increased resistance to chemotherapeutic agents. Although initial focus in this area has been on the direct reversal protein (MGMT), its protective ability is limited to those agents that produce O(6)-methylGuanine cross-links-agents that are not extensively used clinically (e.g., nitrosoureas). Furthermore, most alkylating agents attack more sites in DNA other than O(6)-methylGuanine, such that the protections afforded by MGMT may prevent the initial cytotoxicity, but at a price of increased mutational burden and potential secondary leukemias. Therefore, some of the genes that are being tested as candidates for gene transfer are base excision repair (BER) genes. We and others have found that overexpression of selective BER genes confers resistance to chemotherapeutic agents such as thiotepa, ionizing radiation, bleomycin, and other agents. As these "proof of concept" analyses mature, many more clinically relevant chemotherapeutic agents can be tested for BER protective ability.

  15. DNA Repair in Despair-Vitamin D Is Not Fair.

    Science.gov (United States)

    Gocek, Elżbieta; Studzinski, George P

    2016-08-01

    The role of vitamin D as a treatment option for neoplastic diseases, once considered to have a bright future, remains controversial. The preclinical studies discussed herein show compelling evidence that Vitamin D Derivatives (VDDs) can convert some cancer and leukemia cells to a benign phenotype, by differentiation/maturation, cell cycle arrest, or induction of apoptosis. Furthermore, there is considerable, though still evolving, knowledge of the molecular mechanisms underlying these changes. However, the attempts to clearly document that the treatment outcomes of human neoplastic diseases can be positively influenced by VDDs have been, so far, disappointing. The clinical trials to date of VDDs, alone or combined with other agents, have not shown consistent results. It is our contention, shared by others, that there were limitations in the design or execution of these trials which have not yet been fully addressed. Based on the connection between upregulation of JNK by VDDs and DNA repair, we propose a new avenue of attack on cancer cells by increasing the toxicity of the current, only partially effective, cancer chemotherapeutic drugs by combining them with VDDs. This can impair DNA repair and thus kill the malignant cells, warranting a comprehensive study of this novel concept. J. Cell. Biochem. 117: 1733-1744, 2016. © 2016 Wiley Periodicals, Inc.

  16. Enhanced DNA repair of cyclobutane pyrimidine dimers changes the biological response to UV-B radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yarosh, Daniel B

    2002-11-30

    The goal of DNA repair enzyme therapy is the same as that for gene therapy: to rescue a defective proteome/genome by introducing a substitute protein/DNA. The danger of inadequate DNA repair is highlighted in the genetic disease xeroderma pigmentosum. These patients are hypersensitive to sunlight and develop multiple cutaneous neoplasms very early in life. The bacterial DNA repair enzyme T4 endonuclease V was shown over 25 years ago to be capable of reversing the defective repair in xeroderma pigmentosum cells. This enzyme, packaged in an engineered delivery vehicle, has been shown to traverse the stratum corneum, reach the nuclei of living cells of the skin, and enhance the repair of UV-induced cyclobutane pyrimidine dimers (CPD). In such a system, changes in DNA repair, mutagenesis, and cell signaling can be studied without manipulation of the genome.

  17. Repair of ultraviolet-damaged transforming DNA in a mismatch repair-deficient strain of Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Bagci, H.; Stuy, J.H. (Florida State Univ., Tallahassee (USA). Dept. of Biological Science)

    1982-03-01

    Ultraviolet inactivation of Haemophilus influenzae transforming DNA followed inverse square root kinetics in both mismatch repair-proficient (hex/sup +/) and deficient (hex-1) recipients. No DNA concentration effect was seen with UV-excision repair-deficient (uvr/sup -/) strains. Low-efficiency genetic markers remained more sensitive than high-efficiency ones when they were assayed on excision repair-deficient hex/sup +/ uvr/sup -/ strains. They were equally resistant when hex/sup -/ uvr/sup -/ recipients were used. This was explained by assuming that recombinational repair of UV lesions in the donor strand and mismatch repair of the recipient strand may overlap and cause double strand interruptions. This will eliminate low-efficiency transformants.

  18. Tendon Collagen Crosslinking Offers Potential to Improve Suture Pullout in Rotator Cuff Repair: An Ex Vivo Sheep Study.

    Science.gov (United States)

    Camenzind, Roland S; Wieser, Karl; Fessel, Gion; Meyer, Dominik C; Snedeker, Jess G

    2016-08-01

    The suture-tendon interface is often the weakest link in tendon to bone repair of massive rotator cuff tears. Genipin is a low-toxicity collagen crosslinker derived from the gardenia fruit that has been shown to augment collagen tissue strength and mechanically arrest tendon-tear progression. The purpose of the current study was to evaluate whether genipin crosslinking can sufficiently augment the suture-tendon interface to improve suture pullout strength using simple single-loop sutures and the modified Mason-Allen technique. The study also aimed to assess whether time of genipin treatment is a relevant factor in efficacy. In an ex vivo (cadaveric) sheep rotator cuff tendon model, a total of 142 suture pullout tests were performed on 32 infraspinatus tendons. Each tendon was prepared with three single-loop stitches. Two groups were pretreated by incubation in genipin solution for either 4 hours or 24 hours. Two corresponding control groups were incubated in phosphate buffered saline for the same periods. The same test protocol was applied to tendons using modified Mason-Allen technique stitch patterns. Each suture was loaded to failure on a universal materials testing machine. Suture pullout force, stiffness, and work to failure were calculated from force-displacement data, and then compared among the groups. Median single-loop pullout force on tendons incubated for 24 hours in genipin yielded an approximately 30% increase in maximum pullout force for single-loop stitches with a median of 73 N (range, 56-114 N) compared with 56 N (range, 40-69 N; difference of medians = 17 N; p = 0.028), with corresponding increases in the required work to failure but not stiffness. Genipin treatment for 4 hours showed no added benefit for suture-pullout behavior (46 N, [range, 35-95 N] versus 45 N, [range, 28-63 N]; difference of medians, 1 N; p = 1). No tested genipin crosslinking conditions indicated benefit for tendons grasped using the modified Mason-Allen technique after 4

  19. Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase.

    NARCIS (Netherlands)

    B.P. Engelward (Bevin); G. Weeda (Geert); M.D. Wyatt; J.L.M. Broekhof (Jose'); J. de Wit (Jan); I. Donker (Ingrid); J.M. Allan (James); B. Gold (Bert); J.H.J. Hoeijmakers (Jan); L.D. Samson (Leona)

    1997-01-01

    textabstract3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA glyc

  20. Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase.

    NARCIS (Netherlands)

    B.P. Engelward (Bevin); G. Weeda (Geert); M.D. Wyatt; J.L.M. Broekhof (Jose'); J. de Wit (Jan); I. Donker (Ingrid); J.M. Allan (James); B. Gold (Bert); J.H.J. Hoeijmakers (Jan); L.D. Samson (Leona)

    1997-01-01

    textabstract3-methyladenine (3MeA) DNA glycosylases remove 3MeAs from alkylated DNA to initiate the base excision repair pathway. Here we report the generation of mice deficient in the 3MeA DNA glycosylase encoded by the Aag (Mpg) gene. Alkyladenine DNA glycosylase turns out to be the major DNA glyc

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

    Science.gov (United States)

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

    2010-07-07

    Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process. Copyright 2010 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2016-08-18

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

  3. Cisplatin GG-crosslinks within single-stranded DNA: origin of the preference for left-handed helicity.

    Science.gov (United States)

    Monnet, Jordan; Kozelka, Jiří

    2012-10-01

    Molecular dynamics (MD) simulations of the single-stranded DNA trinucleotide TG*G*, with the G* guanines crosslinked by the antitumor drug cisplatin, were performed with explicit representation of the water as solvent. The purpose of the simulations was to explain previous NMR observations indicating that in single-stranded cisplatin-DNA adducts, the crosslinked guanines adopt a left-handed helical orientation, whereas in duplexes, the orientation is right-handed. The analysis of the MD trajectory of TG*G* has ascribed a crucial role to hydrogen-bonding (direct or through-water) interactions of the 5'-oriented NH(3) ligand of platinum with acceptor groups at the 5'-side of the crosslink, namely the TpG* phosphate and the terminal 5'-OH group. These interactions bring about some strain into the trinucleotide which is slightly but significantly (1-1.5 kcal.mol(-1)) higher for the right-handed orientation than for the left-handed one. During the unconstrained, 3 ns long MD simulation, left-handed conformations were ~15 times more abundant than the right-handed ones. This sampling difference agrees roughly with the calculated energy difference in strain energy. Overall, these results show that the Pt-GG crosslink within single-stranded DNA is malleable and can access different conformations at a moderate energy cost. This malleability could be of importance in interactions between the platinated DNA and cellular proteins, in which the DNA is locally unwound.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ames, B.N.; Shigenaga, M.K. [Univ. of California, Berkeley, CA (United States); Gold, L.S. [Lawrence Berkeley National Lab., CA (United States)

    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).

  5. DNA ligase 1 deficient plants display severe growth defects and delayed repair of both DNA single and double strand breaks

    Directory of Open Access Journals (Sweden)

    Bray Clifford M

    2009-06-01

    Full Text Available Abstract Background DNA ligase enzymes catalyse the joining of adjacent polynucleotides and as such play important roles in DNA replication and repair pathways. Eukaryotes possess multiple DNA ligases with distinct roles in DNA metabolism, with clear differences in the functions of DNA ligase orthologues between animals, yeast and plants. DNA ligase 1, present in all eukaryotes, plays critical roles in both DNA repair and replication and is indispensable for cell viability. Results Knockout mutants of atlig1 are lethal. Therefore, RNAi lines with reduced levels of AtLIG1 were generated to allow the roles and importance of Arabidopsis DNA ligase 1 in DNA metabolism to be elucidated. Viable plants were fertile but displayed a severely stunted and stressed growth phenotype. Cell size was reduced in the silenced lines, whilst flow cytometry analysis revealed an increase of cells in S-phase in atlig1-RNAi lines relative to wild type plants. Comet assay analysis of isolated nuclei showed atlig1-RNAi lines displayed slower repair of single strand breaks (SSBs and also double strand breaks (DSBs, implicating AtLIG1 in repair of both these lesions. Conclusion Reduced levels of Arabidopsis DNA ligase 1 in the silenced lines are sufficient to support plant development but result in retarded growth and reduced cell size, which may reflect roles for AtLIG1 in both replication and repair. The finding that DNA ligase 1 plays an important role in DSB repair in addition to its known function in SSB repair, demonstrates the existence of a previously uncharacterised novel pathway, independent of the conserved NHEJ. These results indicate that DNA ligase 1 functions in both DNA replication and in repair of both ss and dsDNA strand breaks in higher plants.

  6. A PHF8 homolog in C. elegans promotes DNA repair via homologous recombination.

    Directory of Open Access Journals (Sweden)

    Changrim Lee

    Full Text Available PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation. In this study, we examined the roles of two PHF8 homologs, JMJD-1.1 and JMJD-1.2, in the model organism C. elegans in response to DNA damage. A deletion mutation in either of the genes led to hypersensitivity to interstrand DNA crosslinks (ICLs, while only mutation of jmjd-1.1 resulted in hypersensitivity to double-strand DNA breaks (DSBs. In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway. However, the dynamic behavior of RPA-1 and RAD-51 was affected by the mutation: the accumulations of both proteins at ICLs appeared normal, but their subsequent disappearance was retarded, suggesting that later steps of homologous recombination were defective. Similar changes in the dynamic behavior of RPA-1 and RAD-51 were seen in response to DSBs, supporting a role of JMJD-1.1 in homologous recombination. Such a role was also supported by our finding that the hypersensitivity of jmjd-1.1 worms to ICLs was rescued by knockdown of lig-4, a homolog of Ligase 4 active in nonhomologous end-joining. The hypersensitivity of jmjd-1.1 worms to ICLs was increased by rad-54 knockdown, suggesting that JMJD-1.1 acts in parallel with RAD-54 in modulating chromatin structure. Indeed, the level of histone H3 Lys9 tri-methylation, a marker of heterochromatin, was higher in jmjd-1.1 cells than in wild-type cells. We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.

  7. Detection of DNA damage induced by topoisomerase II inhibitors, gamma radiation and crosslinking agents using the comet assay.

    Science.gov (United States)

    Hazlehurst, Lori A

    2009-01-01

    The comet assay is a simple gel electrophoresis method for visualizing and quantifying DNA damage. The comet assay is sensitive and reproducible and can be used to detect single-strand DNA breaks, double-strand DNA breaks, protein-associated DNA strand breaks and DNA crosslinks. The comet assay uses fluorescent DNA-binding dyes to detect both damaged DNA that resides in the tail region and undamaged DNA that is retained in the head region following gel electrophoresis. This assay is a single cell-based assay and thus is highly adaptable for measuring DNA damage in clinical samples. Furthermore, unlike other assays the detection of DNA damage is not dependent on the random incorporation of radiolabeled nucleotides. Again this can be problematic with clinical samples as proliferation rates are often slow and culturing of primary patient specimens for 48 h required to randomly label DNA is often not possible. In this chapter we will outline the comet assay for the detection of DNA damage induced by topoisomerase II inhibitors, cross-linking agents and gamma radiation.

  8. Double-strand break repair and G4 DNA stability in Caenorhabditis elegans

    NARCIS (Netherlands)

    Pontier, D.B.

    2010-01-01

    DNA double-strand breaks (DSBs) can be repaired by three canonical repair pathways. Homologous recombination (HR) uses the sister chromatid or homologous chromosome as a template to repair the DSB in an error-free manner. In non-homologous end-joining (NHEJ), the broken ends are ligated with little

  9. Meiotic and mitotic functions of mammalian RAD 18 in DNA double-strand break repair

    NARCIS (Netherlands)

    A. Inagaki (Akiko)

    2010-01-01

    textabstractThis thesis focuses on the role of RAD 18 in DNA double-strand break (DSB ) repair. Much is known about the role of RAD 18, and its critical substrate PCNA in replication damage bypass (RDB ) repair. However, the roles of RAD 18 in DSB repair are still elusive, although several

  10. Double-strand break repair and G4 DNA stability in Caenorhabditis elegans

    NARCIS (Netherlands)

    Pontier, D.B.

    2010-01-01

    DNA double-strand breaks (DSBs) can be repaired by three canonical repair pathways. Homologous recombination (HR) uses the sister chromatid or homologous chromosome as a template to repair the DSB in an error-free manner. In non-homologous end-joining (NHEJ), the broken ends are ligated with little

  11. DNA repair and gene targeting in plant end-joining mutants

    NARCIS (Netherlands)

    Jia, Qi

    2011-01-01

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or by non-homologous end joining (NHEJ). The latter mechanism is the major route for DSB repair in the somatic cells of higher eukaryotes, including plants. If we could manipulate the balance of the DSB repair pathways

  12. Meiotic and mitotic functions of mammalian RAD 18 in DNA double-strand break repair

    NARCIS (Netherlands)

    A. Inagaki (Akiko)

    2010-01-01

    textabstractThis thesis focuses on the role of RAD 18 in DNA double-strand break (DSB ) repair. Much is known about the role of RAD 18, and its critical substrate PCNA in replication damage bypass (RDB ) repair. However, the roles of RAD 18 in DSB repair are still elusive, although several interacti

  13. DNA repair and gene targeting in plant end-joining mutants

    NARCIS (Netherlands)

    Jia, Qi

    2011-01-01

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or by non-homologous end joining (NHEJ). The latter mechanism is the major route for DSB repair in the somatic cells of higher eukaryotes, including plants. If we could manipulate the balance of the DSB repair pathways

  14. Sharpening the ends for repair: mechanisms and regulation of DNA resection

    Institute of Scientific and Technical Information of China (English)

    Sharad C.Paudyal; Zhongsheng You

    2016-01-01

    DNA end resection is a key process in the cellular response to DNA double-strand break damage that is essential for genome maintenance and cell survival.Resection involves selective processing of 5' ends of broken DNA to generate ssDNA overhangs,which in turn control both DNA repair and checkpoint signaling.DNA resection is the first step in homologous recombination-mediated repair and a prerequisite for the activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent checkpoint that coordinates repair with cell cycle progression and other cellular processes.Resection occurs in a cell cycle-dependent manner and is regulated by multiple factors to ensure an optimal amount of ssDNA required for proper repair and genome stability.Here,we review the latest findings on the molecular mechanisms and regulation of the DNA end resection process and their implications for cancer formation and treatment.

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

    DEFF Research Database (Denmark)

    Garm, Christian; Moreno-Villanueva, Maria; Bürkle, Alexander

    2013-01-01

    factors are likely to influence DNA repair capacity. In order to gain more insight into the genetic and environmental contribution to the molecular basis of DNA repair, we have performed a human twin study, where we focused on the consequences of some of the most abundant types of DNA damage (single......Accumulation of DNA damage deriving from exogenous and endogenous sources has significant consequences for cellular survival, and is implicated in aging, cancer, and neurological diseases. Different DNA repair pathways have evolved in order to maintain genomic stability. Genetic and environmental......-strand breaks), and some of the most hazardous lesions (DNA double-strand breaks). DNA damage signaling response (Gamma-H2AX signaling), relative amount of endogenous damage, and DNA-strand break repair capacities were studied in peripheral blood mononuclear cells from 198 twins (94 monozygotic and 104...

  16. Photoprotection by topical DNA repair enzymes: molecular correlates of clinical studies.

    Science.gov (United States)

    Yarosh, D B; O'Connor, A; Alas, L; Potten, C; Wolf, P

    1999-02-01

    A new approach to photoprotection is to repair DNA damage after UV exposure. This can be accomplished by delivery of a DNA repair enzyme with specificity to UV-induced cyclobutane pyrimidine dimers into skin by means of specially engineered liposomes. Treatment of DNA-repair-deficient xeroderma pigmentosum patients or skin cancer patients with T4N5 liposome lotion containing such DNA repair liposomes increases the removal of DNA damage in the first few hours after treatment. In these studies, a DNA repair effect was observed in some patients treated with heat-inactivated enzyme. Unexpectedly, it was discovered that the heat-inactivated T4 endonuclease V enzyme refolds and recovers enzymatic activity. These studies demonstrate that measurements of molecular changes induced by biological drugs are useful adjuvants to clinical studies.

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

    Directory of Open Access Journals (Sweden)

    Erhong Meng

    2015-07-01

    Full Text Available 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Erhong; Hanna, Ann; Samant, Rajeev S.; Shevde, Lalita A., E-mail: lsamant@uab.edu [Department of Pathology, Comprehensive Cancer Center, University of Alabama at Birmingham, WTI320D, 1824 6th Avenue South, Birmingham, AL 35233 (United States)

    2015-07-21

    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.

  19. Chemical structure and properties of interstrand cross-links formed by reaction of guanine residues with abasic sites in duplex DNA.

    Science.gov (United States)

    Catalano, Michael J; Liu, Shuo; Andersen, Nisana; Yang, Zhiyu; Johnson, Kevin M; Price, Nathan E; Wang, Yinsheng; Gates, Kent S

    2015-03-25

    A new type of interstrand cross-link resulting from the reaction of a DNA abasic site with a guanine residue on the opposing strand of the double helix was recently identified, but the chemical connectivity of the cross-link was not rigorously established. The work described here was designed to characterize the chemical structure and properties of dG-AP cross-links generated in duplex DNA. The approach involved characterization of the nucleoside cross-link "remnant" released by enzymatic digestion of DNA duplexes containing the dG-AP cross-link. We first carried out a chemical synthesis and complete spectroscopic structure determination of the putative cross-link remnant 9b composed of a 2-deoxyribose adduct attached to the exocyclic N(2)-amino group of dG. A reduced analogue of the cross-link remnant was also prepared (11b). Liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis revealed that the retention times and mass spectral properties of synthetic standards 9b and 11b matched those of the authentic cross-link remnants released by enzymatic digestion of duplexes containing the native and reduced dG-AP cross-link, respectively. These results establish the chemical connectivity of the dG-AP cross-link released from duplex DNA and provide a foundation for detection of this lesion in biological samples. The dG-AP cross-link in duplex DNA was remarkably stable, decomposing with a half-life of 22 days at pH 7 and 23 °C. The intrinsic chemical stability of the dG-AP cross-link suggests that this lesion in duplex DNA may have the power to block DNA-processing enzymes involved in transcription and replication.

  20. DNA Single-Strand Break Repair and Spinocerebellar Ataxia with Axonal Neuropathy-1

    OpenAIRE

    Caldecott, K. W.

    2007-01-01

    DNA single-strand breaks (SSBs) are the commonest DNA lesions arising spontaneously in cells, and if not repaired may block transcription or may be converted into potentially lethal/clastogenic DNA double-strand breaks (DSBs). Recently, evidence has emerged that defects in the rapid repair of SSBs preferentially impact the nervous system. In particular, spinocerebellar ataxia with axonal neuropathy (SCAN1) is a human disease that is associated with mutation of TDP1 (tyrosyl DNA phosphodiester...

  1. DNA single-strand break repair and spinocerebellar ataxia with axonal neuropathy-1

    OpenAIRE

    El-Khamisy, S.F.; Caldecott, K. W.

    2007-01-01

    DNA single-strand breaks (SSBs) are the commonest DNA lesions arising spontaneously in cells, and if not repaired may block transcription or may be converted into potentially lethal/clastogenic DNA double-strand breaks (DSBs). Recently, evidence has emerged that defects in the rapid repair of SSBs preferentially impact the nervous system. In particular, spinocerebellar ataxia with axonal neuropathy (SCAN1) is a human disease that is associated with mutation of TDP1 (tyrosyl DNA phosphodiester...

  2. The modulation of topoisomerase I-mediated DNA cleavage and the induction of DNA–topoisomerase I crosslinks by crotonaldehyde-derived DNA adducts

    OpenAIRE

    2008-01-01

    Crotonaldehyde is a representative α,β-unsaturated aldehyde endowed of mutagenic and carcinogenic properties related to its propensity to react with DNA. Cyclic crotonaldehyde-derived deoxyguanosine (CrA-PdG) adducts can undergo ring opening in duplex DNA to yield a highly reactive aldehydic moiety. Here, we demonstrate that site-specifically modified DNA oligonucleotides containing a single CrA-PdG adduct can form crosslinks with topoisomerase I (Top1), both directly and indirectly. Direct c...

  3. Corneal Wound Repair After Rose Bengal and Green Light Crosslinking: Clinical and Histologic Study.

    Science.gov (United States)

    Gallego-Muñoz, Patricia; Ibares-Frías, Lucía; Lorenzo, Elvira; Marcos, Susana; Peréz-Merino, Pablo; Bekesi, Nandor; Kochevar, Irene E; Martínez-García, M Carmen

    2017-07-01

    To evaluate corneal wound healing after treatment with a new collagen crosslinking protocol using rose bengal dye and green light (RGX). One cornea of 20 New Zealand rabbits was de-epithelialized (DE) in an 8-mm diameter circle and, in another group (n = 25), the DE corneas were then stained with 0.1% rose bengal for 2 minutes and exposed to green light (532 nm) for 7 minutes (RGX). The contralateral eyes without treatment acted as controls. The animals were clinically followed including fluorescein staining and pachymetry. Healing events were analyzed after euthanasia at 2, 30, and 60 days. Cell death (TUNEL assay), cell proliferation (5-bromo-2'-deoxyuridine incorporation), and cell differentiation to myofibroblasts (α-SMA labeling) were carried out. In addition, loss of keratocytes and subsequent repopulation of the corneal stroma were quantified on hematoxylin-eosin-stained sections. Wound closure was slower after RGX (4.4 days) then after DE (3.3 days). Cell death was restricted to the anterior central stroma, and the cellular decrease did not differ significantly between RGX and DE corneas. Cell proliferation in the epithelium and stroma appeared at 2 days. In both DE and RGX corneas, recovery of the epithelium was complete at day 30, although cell repopulation of the stroma was not complete at 60 days. The healing response in corneas after RGX is very similar to that observed after DE alone, suggesting that, along with its short treatment time and limited effect on keratocytes, RGX displays good potential for clinical cornea stiffening.

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

    Science.gov (United States)

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

    2016-01-15

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations.

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

    Science.gov (United States)

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

    2015-01-01

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

  6. Evaluation of mutagenic effects of formocresol: detection of DNA-protein cross-links and micronucleus in mouse bone marrow.

    Science.gov (United States)

    Ramos, Maria Emília Santos Pereira; Cavalcanti, Bruno Coêlho; Lotufo, Letícia Veras Costa; de Moraes, Manoel Odorico; Cerqueira, Eneida de Moraes Marcílio; Pessoa, Cláudia

    2008-03-01

    The genotoxic potential of formocresol was assessed by comet assay on human peripheral blood lymphocytes and in vivo micronucleus in mice. Peripheral blood lymphocytes, obtained from healthy donors, were exposed directly with different dilutions of formocresol for 45 minutes at 37 degrees C. To verify the possibility of formocresol to induce DNA-protein cross-links, treated lymphocytes were incubated with proteinase K. Micronucleus test was performed on male Swiss mice treated with several dilutions of formocresol by single intraperitoneal injection. After treatment, bone marrow was sampled 24 and 48 hours after formocresol administration. Formocresol did not produce detectable DNA damage as evaluated by comet assay. However, after proteinase K exposure, a dose-dependent increase of DNA migration was observed. Formocresol induced a significant increase in micronucleus frequencies at the highest dilution only at 24 hours after administration. Formocresol induced DNA-protein cross-links and an increased frequency of micronucleus.

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

    Directory of Open Access Journals (Sweden)

    Nadine Schuler

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

  8. Isolation and study of two mutants of Streptomyces cattleya affected in DNA repair and genetic instability.

    Science.gov (United States)

    Hromic, A; Kirby, R

    1989-01-15

    Two mutants of Streptomyces cattleya affecting DNA repair were isolated. These mutants were analysed using spore survival curves and phage reactivation curves in the presence and absence of caffeine and arsenite. Two DNA repair systems (uvr1 and uvr2) were identified, the latter of which seems to influence genetic instability.

  9. ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor

    NARCIS (Netherlands)

    E. Citterio (Elisabetta); V. van den Boom (Vincent); G. Schnitzler; R. Kanaar (Roland); E. Bonte (Edgar); R.E. Kingston; W. Vermeulen (Wim); J.H.J. Hoeijmakers (Jan)

    2000-01-01

    textabstractThe Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-d

  10. Functions and Dynamics of DNA Repair Proteins in Mitosis and Meiosis

    NARCIS (Netherlands)

    E.J. Uringa

    2005-01-01

    textabstractMy PhD project encompassed studies on the functions of several different proteins, all involved in DNA repair, in somatic and germ-line cells. Hr6b and Rad18Sc are involved in a DNA repair mechanism called ‘Replicative Damage Bypass’ (RDB), and function as ubiquitin conjugating enzym

  11. 53BP1 fosters fidelity of homology-directed DNA repair

    DEFF Research Database (Denmark)

    Ochs, Fena; Somyajit, Kumar; Altmeyer, Matthias

    2016-01-01

    Repair of DNA double-strand breaks (DSBs) in mammals is coordinated by the ubiquitin-dependent accumulation of 53BP1 at DSB-flanking chromatin. Owing to its ability to limit DNA-end processing, 53BP1 is thought to promote nonhomologous end-joining (NHEJ) and to suppress homology-directed repair...

  12. Analysis of DNA repair gene polymorphisms and survival in low-grade and anaplastic gliomas

    DEFF Research Database (Denmark)

    Berntsson, Shala Ghaderi; Wibom, Carl; Sjöström, Sara;

    2011-01-01

    The purpose of this study was to explore the variation in DNA repair genes in adults with WHO grade II and III gliomas and their relationship to patient survival. We analysed a total of 1,458 tagging single-nucleotide polymorphisms (SNPs) that were selected to cover DNA repair genes, in 81 grade ...

  13. DNA repair pathways in radiation induced cellular damage: a molecular approach

    NARCIS (Netherlands)

    L.R. van Veelen (Lieneke)

    2005-01-01

    markdownabstract__Abstract__ DNA damage, especially double-strand breaks, can be induced by endogenous or exogenous darnaging agents, such as ionizing radiation. Repair of DNA damage is very important in maintaining genomic stability. Incorrect repair may lead to chromosomal aberrations,

  14. DNA repair pathways in radiation induced cellular damage: a molecular approach

    NARCIS (Netherlands)

    L.R. van Veelen (Lieneke)

    2005-01-01

    markdownabstract__Abstract__ DNA damage, especially double-strand breaks, can be induced by endogenous or exogenous darnaging agents, such as ionizing radiation. Repair of DNA damage is very important in maintaining genomic stability. Incorrect repair may lead to chromosomal aberrations, translocat

  15. RTEL1 contributes to DNA replication and repair and telomere maintenance

    NARCIS (Netherlands)

    Uringa, Evert-Jan; Lisaingo, Kathleen; Pickett, Hilda A.; Brind'Amour, Julie; Rohde, Jan-Hendrik; Zelensky, Alex; Essers, Jeroen; Lansdorp, Peter M.

    2012-01-01

    Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance

  16. Targeting the DNA Repair Pathway in Ewing Sarcoma

    Directory of Open Access Journals (Sweden)

    Elizabeth Stewart

    2014-11-01

    Full Text Available 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.

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

    Science.gov (United States)

    Lu, Jian; Matunis, Michael J

    2013-12-01

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

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

    Science.gov (United States)

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

    2011-02-16

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Levine, E.; Thiel, T.

    1987-09-01

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

  20. Complex DNA repair pathways as possible therapeutic targets to overcome temozolomide resistance in glioblastoma

    Directory of Open Access Journals (Sweden)

    Koji eYoshimoto

    2012-12-01

    Full Text Available Many conventional chemotherapeutic drugs exert their cytotoxic function by inducing DNA damage in the tumor cell. Therefore, a cell-inherent DNA repair pathway, which reverses the DNA-damaging effect of the cytotoxic drugs, can mediate therapeutic resistance to chemotherapy. The monofunctional DNA-alkylating agent temozolomide (TMZ is a commonly used chemotherapeutic drug and the gold standard treatment for glioblastoma. Although the activity of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT has been described as the main modulator to determine the sensitivity of glioblastoma to TMZ, a subset of glioblastoma does not respond despite MGMT inactivation, suggesting that another DNA repair mechanism may also modulate the tolerance to TMZ. Considerable interest has focused on MGMT, mismatch repair (MMR, and the base-excision repair (BER pathway in the mechanism of mediating TMZ resistance, but emerging roles for the DNA strand-break repair pathway have been demonstrated. In the first part of this review article, we briefly review the significant role of MGMT, MMR, and the BER pathway in the tolerance to TMZ; in the last part, we review the recent publications that demonstrate possible roles of DNA strand-break repair pathways, such as single-strand break (SSB repair and double-strand break (DSB repair, as well as the Fanconi anemia pathway in the repair process after alkylating agent-based therapy. It is possible that all of these repair pathways have a potential to modulate the sensitivity to TMZ and aid in overcoming the therapeutic resistance in the clinic.

  1. Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases.

    Science.gov (United States)

    Fekairi, Samira; Scaglione, Sarah; Chahwan, Charly; Taylor, Ewan R; Tissier, Agnès; Coulon, Stéphane; Dong, Meng-Qiu; Ruse, Cristian; Yates, John R; Russell, Paul; Fuchs, Robert P; McGowan, Clare H; Gaillard, Pierre-Henri L

    2009-07-10

    Structure-specific endonucleases resolve DNA secondary structures generated during DNA repair and recombination. The yeast 5' flap endonuclease Slx1-Slx4 has received particular attention with the finding that Slx4 has Slx1-independent key functions in genome maintenance. Although Slx1 is a highly conserved protein in eukaryotes, no orthologs of Slx4 were reported other than in fungi. Here we report the identification of Slx4 orthologs in metazoa, including fly MUS312, essential for meiotic recombination, and human BTBD12, an ATM/ATR checkpoint kinase substrate. Human SLX1-SLX4 displays robust Holliday junction resolvase activity in addition to 5' flap endonuclease activity. Depletion of SLX1 and SLX4 results in 53BP1 foci accumulation and H2AX phosphorylation as well as cellular hypersensitivity to MMS. Furthermore, we show that SLX4 binds the XPF(ERCC4) and MUS81 subunits of the XPF-ERCC1 and MUS81-EME1 endonucleases and is required for DNA interstrand crosslink repair. We propose that SLX4 acts as a docking platform for multiple structure-specific endonucleases.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

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

    Directory of Open Access Journals (Sweden)

    Katarzyna A. Wojcik

    2014-10-01

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

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

    Science.gov (United States)

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

    2006-01-01

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

  5. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

    DEFF Research Database (Denmark)

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott

    2014-01-01

    slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I...... by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional......Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial...

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

    Science.gov (United States)

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

    2014-11-01

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

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

    Science.gov (United States)

    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.

  8. A role for the malignant brain tumour (MBT domain protein LIN-61 in DNA double-strand break repair by homologous recombination.

    Directory of Open Access Journals (Sweden)

    Nicholas M Johnson

    Full Text Available Malignant brain tumour (MBT domain proteins are transcriptional repressors that function within Polycomb complexes. Some MBT genes are tumour suppressors, but how they prevent tumourigenesis is unknown. The Caenorhabditis elegans MBT protein LIN-61 is a member of the synMuvB chromatin-remodelling proteins that control vulval development. Here we report a new role for LIN-61: it protects the genome by promoting homologous recombination (HR for the repair of DNA double-strand breaks (DSBs. lin-61 mutants manifest numerous problems associated with defective HR in germ and somatic cells but remain proficient in meiotic recombination. They are hypersensitive to ionizing radiation and interstrand crosslinks but not UV light. Using a novel reporter system that monitors repair of a defined DSB in C. elegans somatic cells, we show that LIN-61 contributes to HR. The involvement of this MBT protein in HR raises the possibility that MBT-deficient tumours may also have defective DSB repair.

  9. Direct inhibition of excision/synthesis DNA repair activities by cadmium: Analysis on dedicated biochips

    Energy Technology Data Exchange (ETDEWEB)

    Candeias, S., E-mail: serge.candeias@cea.fr [CEA, INAC, SCIB, UJF and CNRS, LCIB (UMR-E 3 CEA-UJF and FRE 3200), Laboratoire Lesions des Acides Nucleiques, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9 (France); CEA, DSV, iRTSV, LBBSI, UMR 5092 CNRS, F-38054 Grenoble Cedex 9 (France); Pons, B.; Viau, M.; Caillat, S.; Sauvaigo, S. [CEA, INAC, SCIB, UJF and CNRS, LCIB (UMR-E 3 CEA-UJF and FRE 3200), Laboratoire Lesions des Acides Nucleiques, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9 (France)

    2010-12-10

    The well established toxicity of cadmium and cadmium compounds results from their additive effects on several key cellular processes, including DNA repair. Mammalian cells have evolved several biochemical pathways to repair DNA lesions and maintain genomic integrity. By interfering with the homeostasis of redox metals and antioxidant systems, cadmium promotes the development of an intracellular environment that results in oxidative DNA damage which can be mutagenic if unrepaired. Small base lesions are recognised by specialized glycosylases and excised from the DNA molecule. The resulting abasic sites are incised, and the correct sequences restored by DNA polymerases using the opposite strands as template. Bulky lesions are recognised by a different set of proteins and excised from DNA as part of an oligonucleotide. As in base repair, the resulting gaps are filled by DNA polymerases using the opposite strands as template. Thus, these two repair pathways consist in excision of the lesion followed by DNA synthesis. In this study, we analysed in vitro the direct effects of cadmium exposure on the functionality of base and nucleotide DNA repair pathways. To this end, we used recently described dedicated microarrays that allow the parallel monitoring in cell extracts of the repair activities directed against several model base and/or nucleotide lesions. Both base and nucleotide excision/repair pathways are inhibited by CdCl{sub 2}, with different sensitivities. The inhibitory effects of cadmium affect mainly the recognition and excision stages of these processes. Furthermore, our data indicate that the repair activities directed against different damaged bases also exhibit distinct sensitivities, and the direct comparison of cadmium effects on the excision of uracile in different sequences even allows us to propose a hierarchy of cadmium sensibility within the glycosylases removing U from DNA. These results indicate that, in our experimental conditions, cadmium is a

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

    Science.gov (United States)

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

    2016-03-01

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

  11. The new base excision repair pathway in mammals mediated by tyrosyl-DNA-phosphodiesterase 1

    Directory of Open Access Journals (Sweden)

    Lavrik O. I.

    2012-06-01

    Full Text Available Human tyrosyl-DNA phosphodiesterase 1 (Tdp1 hydrolyzes the phosphodiester bond at a DNA 3' end linked to a tyrosyl moiety and has been implicated in the repair of Topoisomerase I (TopI-DNA covalent complexes. Tdp1 can also hydrolyze other 3' end DNA alterations including 3' phosphoglycolate and 3' abasic (AP sites, and exhibits the 3' nucleosidase activity indicating that it may function as a general 3' end-processing DNA repair enzyme. Recently we have shown a new Tdp1 activity generating DNA strand break with the 3' phosphate termini from the AP site. AP sites are formed spontaneously and are inevitable intermediates during base excision repair of DNA base damages. AP sites are both mutagenic and cytotoxic, and key enzymes for their removal are AP endonucleases. However, AP endonuclease independent repair, initiated by DNA glycosylases performing beta, delta-elimination cleavage of the AP sites, has been described in mammalian cells. Here, we describe another AP endonuclease independent repair pathway for removal of AP sites that is initiated by tyrosyl phosphodiesterase Tdp1. We propose that repair is completed by the action of a polynucleotide kinase, a DNA polymerase and finally a DNA ligase to seal the gap.

  12. Excision repair of UV radiation-induced DNA damage in Caenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Hartman, P.S.; Hevelone, J.; Dwarakanath, V.; Mitchell, D.L. (Texas Christian Univ., Fort Worth (USA))

    1989-06-01

    Radioimmunoassays were used to monitor the removal of antibody-binding sites associated with the two major UV radiation-induced DNA photoproducts (cyclobutane dimers and (6-4) photoproducts). Unlike with cultured human cells, where (6-4) photoproducts are removed more rapidly than cyclobutane dimers, the kinetics of repair were similar for both lesions. Repair capacity in wild type diminished throughout development. The radioimmunoassays were also employed to confirm the absence of photoreactivation in C. elegans. In addition, three radiation-sensitive mutants (rad-1, rad-2, rad-7) displayed normal repair capacities. An excision defect was much more pronounced in larvae than embryos in the fourth mutant tested (rad-3). This correlates with the hypersensitivity pattern of this mutant and suggests that DNA repair may be developmentally regulated in C. elegans. The mechanism of DNA repair in C. elegans as well as the relationship between the repair of specific photoproducts and UV radiation sensitivity during development are discussed.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre

    DEFF Research Database (Denmark)

    Lisby, M.; Mortensen, Uffe Hasbro; Rothstein, R.

    2003-01-01

    DNA double-strand break repair (DSBR) is an essential process for preserving genomic integrity in all organisms. To investigate this process at the cellular level, we engineered a system of fluorescently marked DNA double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae to visualize...... in vivo DSBR in single cells. Using this system, we demonstrate for the first time that Rad52 DNA repair foci and DSBs colocalize. Time-lapse microscopy reveals that the relocalization of Rad52 protein into a focal assembly is a rapid and reversible process. In addition, analysis of DNA damage checkpoint......-deficient cells provides direct evidence for coordination between DNA repair and subsequent release from checkpoint arrest. Finally, analyses of cells experiencing multiple DSBs demonstrate that Rad52 foci are centres of DNA repair capable of simultaneously recruiting more than one DSB....

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

    Science.gov (United States)

    Santos, Juliana Carvalho; Ribeiro, Marcelo Lima

    2015-08-14

    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.

  16. DNA repair in Haemophilus influenzae: isolation and characterization of an ultraviolet sensitive mutator mutant

    Energy Technology Data Exchange (ETDEWEB)

    Walter, R.B.

    1985-01-01

    DNA repair in Haemophilus influenzae appears to be quite different from that seen in Escherichia coli in that H. influenzae shows neither SOS nor adaptation phenomena. Repair of DNA lesions in H. influenzae has been seen to occur via recombinational, excision, and mismatch repair pathways acting independently of one another. The author has isolated an ultraviolet (UV)-sensitive mutator mutant (mutB1) of H. influenzae Rd which shows deficiencies in both recombinational and mismatch repair pathways. This mutant is sensitive to a variety of DNA damaging agents as well as being hypermutable by alkylating agents and base analogues. MutB1 cells do not show post-UV DNA breakdown but do begin excision after UV irradiation. Genetic transformation with UV-irradiated DNA on mut B1 recipients shows that high (HE) and low (LE) efficiency markers are transformed at a ratio of 1.0 as in the mismatch repair deficient hex 1 mutant; however, kinetics of UV-inactivation experiments indicate that HE markers are sensitized and act as LE markers do on wild type recipients. Thus, the mutB gene product appears to play a role in both DNA repair and genetic transformation. A model is outlined which presents a role for a DNA helicase in both DNA repair and genetic transformation of H. influenzae.

  17. PARP-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis?

    Energy Technology Data Exchange (ETDEWEB)

    Swindall, Amanda F.; Stanley, Jennifer A. [Department of Radiation Oncology Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, 176F HSROC Suite 2232B, 1700 6th Avenue South, Birmingham, AL 35249 (United States); Yang, Eddy S., E-mail: eyang@uab.edu [Department of Radiation Oncology Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, 176F HSROC Suite 2232B, 1700 6th Avenue South, Birmingham, AL 35249 (United States); Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35249 (United States); Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35249 (United States)

    2013-07-26

    Oxidative stress induced by reactive oxygen species can result in DNA damage within cells and subsequently increase risk for carcinogenesis. This may be averted by repair of DNA damage through the base or nucleotide excision repair (BER/NER) pathways. PARP, a BER protein, is known for its role in DNA-repair. However, multiple lesions can occur within a small range of DNA, known as oxidative clustered DNA lesions (OCDLs), which are difficult to repair and may lead to the more severe DNA double-strand break (DSB). Inefficient DSB repair can then result in increased mutagenesis and neoplastic transformation. OCDLs occur more frequently within a variety of tumor tissues. Interestingly, PARP is highly expressed in several human cancers. Additionally, chronic inflammation may contribute to tumorigenesis through ROS-induced DNA damage. Furthermore, PARP can modulate inflammation through interaction with NFκB and regulating the expression of inflammatory signaling molecules. Thus, the upregulation of PARP may present a double-edged sword. PARP is needed to repair ROS-induced DNA lesions, but PARP expression may lead to increased inflammation via upregulation of NFκB signaling. Here, we discuss the role of PARP in the repair of oxidative damage versus the formation of OCDLs and speculate on the feasibility of PARP inhibition for the treatment and prevention of cancers by exploiting its role in inflammation.

  18. New tools to study DNA double-strand break repair pathway choice.

    Directory of Open Access Journals (Sweden)

    Daniel Gomez-Cabello

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

  19. Collagen based film with well epithelial and stromal regeneration as corneal repair materials: Improving mechanical property by crosslinking with citric acid

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xuan; Liu, Yang; Li, Weichang; Long, Kai; Wang, Lin; Liu, Sa; Wang, Yingjun [School of Materials Science and Engineering, South China University of Technology, Guangzhou (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou (China); Ren, Li, E-mail: psliren@scut.edu.cn [School of Materials Science and Engineering, South China University of Technology, Guangzhou (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou (China)

    2015-10-01

    Corneal disease can lead to vision loss. It has become the second greatest cause of blindness in the world, and keratoplasty is considered as an effective treatment method. This paper presents the crosslinked collagen (Col)–citric acid (CA) films developed by making use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The results showed that the Col–CA films had necessary optical performance, water content. The collagenase resistance of CA crosslinked films was superior to that of EDC crosslinked films. And CA5 film (Col:CA:EDC:NHS = 60:3:10:10) had the best mechanical properties. Cell experiments showed that CA5 film was non-cytotoxic and human corneal epithelial cells could proliferate well on the films. Lamellar keratoplasty showed that the CA5 film could be sutured in the rabbit eyes and was epithelialized completely in about 10 days, and the transparency was restored quickly in 30 ± 5 days. No inflammation and corneal neovascularization were observed at 6 months. Corneal stroma had been repaired; stromal cells and neo-stroma could be seen in the area of operation from the hematoxylin–eosin stained histologic sections and anterior segment optical coherence tomography images. These results indicated that Col–CA films were highly promising biomaterials that could be used in corneal tissue engineering and a variety of other tissue engineering applications. - Highlights: • Adding different amounts of citric acid could change the properties of films. • The crosslinked films had better mechanical property than non-modified films. • Crosslinked collagen–citric acid films could tolerate suture during operation. • The films showed good ability of epithelial and stromal repair.

  20. Synthesis of G-N2-(CH2)3-N2-G Trimethylene DNA interstrand cross-links

    Science.gov (United States)

    Gruppi, Francesca; Salyard, Tracy L. Johnson; Rizzo, Carmelo J.

    2014-01-01

    The synthesis of G-N2-(CH2)3-N2-G trimethylene DNA interstrand cross-links (ICLs) in a 5′-CG-3′ and 5′-GC-3′ sequence from oligodeoxynucleotides containing N2-(3-aminopropyl)-2′-deoxyguanosine and 2-fluoro-O6-(trimethylsilylethyl)inosine is presented. Automated solid-phase DNA synthesis was used for unmodified bases and modified nucleotides were incorporated via their corresponding phosphoramidite reagent by a manual coupling protocol. The preparation of the phosphoramidite reagents for incorporation of N2-(3-aminopropyl)-2′-deoxyguanosine is reported. The high-purity trimethylene DNA interstrand cross-link product is obtained through a nucleophilic aromatic substitution reaction between the N2-(3-aminopropyl)-2′-deoxyguanosine and 2-fluoro-O6-(trimethylsilylethyl)inosine containing oligodeoxynucleotides. PMID:25431636

  1. Both hMutSα and hMutSß DNA mismatch repair complexes participate in 5-fluorouracil cytotoxicity.

    Directory of Open Access Journals (Sweden)

    Akihiro Tajima

    Full Text Available BACKGROUND: Patients with advanced microsatellite unstable colorectal cancers do not show a survival benefit from 5-fluorouracil (5-FU-based chemotherapy. We and others have shown that the DNA mismatch repair (MMR complex hMutSα binds 5-FU incorporated into DNA. Although hMutSß is known to interact with interstrand crosslinks (ICLs induced by drugs such as cisplatin and psoralen, it has not been demonstrated to interact with 5-FU incorporated into DNA. Our aim was to examine if hMutSß plays a role in 5-FU recognition. METHODS: We compared the normalized growth of 5-FU treated cells containing either or both mismatch repair complexes using MTT and clonogenic assays. We utilized oligonucleotides containing 5-FU and purified baculovirus-synthesized hMutSα and hMutSß in electromobility shift assays (EMSA and further analyzed binding using surface plasmon resonance. RESULTS: MTT and clonogenic assays after 5-FU treatment demonstrated the most cytotoxicity in cells with both hMutSα and hMutSß, intermediate cytotoxicity in cells with hMutSα alone, and the least cytotoxicity in cells with hMutSß alone, hMutSß binds 5-FU-modified DNA, but its relative binding is less than the binding of 5-FU-modified DNA by hMutSα. CONCLUSION: Cytotoxicity induced by 5-FU is dependent on intact DNA MMR, with relative cell death correlating directly with hMutSα and/or hMutSß 5-FU binding ability (hMutSα>hMutSß. The MMR complexes provide a hierarchical chemosensitivity for 5-FU cell death, and may have implications for treatment of patients with certain MMR-deficient tumors.

  2. Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and aminoacid homology with the yeast DNA repair gene RAD10.

    NARCIS (Netherlands)

    M. van Duin (Mark); J. de Wit (Jan); H. Odijk (Hanny); A. Westerveld (Andries); A. Yasui (Akira); M.H.M. Koken (Marcel); J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

    1986-01-01

    textabstractThe human excision repair gene ERCC-7 was cloned after DNA mediated gene transfer to the CHO mutant 43-38, which is sensitive to ultraviolet light and mitomycin-C. We describe the cloning and sequence analysis of the ERCC-7 cDNA and partial characterization of the gene. ERCC.1 has a size

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

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, Ann-Sofie, E-mail: ann-sofie.gustafsson@bms.uu.se; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-15

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

  4. Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining

    OpenAIRE

    Bowater, Richard; Doherty, Aidan J.

    2006-01-01

    DNA double-strand breaks (DSBs) are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ). Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHE...

  5. RECQL4 Promotes DNA End Resection in Repair of DNA Double-Strand Breaks.

    Science.gov (United States)

    Lu, Huiming; Shamanna, Raghavendra A; Keijzers, Guido; Anand, Roopesh; Rasmussen, Lene Juel; Cejka, Petr; Croteau, Deborah L; Bohr, Vilhelm A

    2016-06-28

    The RecQ helicase RECQL4, mutated in Rothmund-Thomson syndrome, regulates genome stability, aging, and cancer. Here, we identify a crucial role for RECQL4 in DNA end resection, which is the initial and an essential step of homologous recombination (HR)-dependent DNA double-strand break repair (DSBR). Depletion of RECQL4 severely reduces HR-mediated repair and 5' end resection in vivo. RECQL4 physically interacts with MRE11-RAD50-NBS1 (MRN), which senses DSBs and initiates DNA end resection with CtIP. The MRE11 exonuclease regulates the retention of RECQL4 at laser-induced DSBs. RECQL4 also directly interacts with CtIP via its N-terminal domain and promotes CtIP recruitment to the MRN complex at DSBs. Moreover, inactivation of RECQL4's helicase activity impairs DNA end processing and HR-dependent DSBR without affecting its interaction with MRE11 and CtIP, suggesting an important role for RECQL4's unwinding activity in the process. Thus, we report that RECQL4 is an important participant in HR-dependent DSBR.

  6. RECQL4 Promotes DNA End Resection in Repair of DNA Double-Strand Breaks

    Directory of Open Access Journals (Sweden)

    Huiming Lu

    2016-06-01

    Full Text Available The RecQ helicase RECQL4, mutated in Rothmund-Thomson syndrome, regulates genome stability, aging, and cancer. Here, we identify a crucial role for RECQL4 in DNA end resection, which is the initial and an essential step of homologous recombination (HR-dependent DNA double-strand break repair (DSBR. Depletion of RECQL4 severely reduces HR-mediated repair and 5′ end resection in vivo. RECQL4 physically interacts with MRE11-RAD50-NBS1 (MRN, which senses DSBs and initiates DNA end resection with CtIP. The MRE11 exonuclease regulates the retention of RECQL4 at laser-induced DSBs. RECQL4 also directly interacts with CtIP via its N-terminal domain and promotes CtIP recruitment to the MRN complex at DSBs. Moreover, inactivation of RECQL4’s helicase activity impairs DNA end processing and HR-dependent DSBR without affecting its interaction with MRE11 and CtIP, suggesting an important role for RECQL4’s unwinding activity in the process. Thus, we report that RECQL4 is an important participant in HR-dependent DSBR.

  7. RECQL4 Promotes DNA End Resection in Repair of DNA Double-Strand Breaks

    DEFF Research Database (Denmark)

    Lu, Huiming; Shamanna, Raghavendra A; Keijzers, Guido

    2016-01-01

    The RecQ helicase RECQL4, mutated in Rothmund-Thomson syndrome, regulates genome stability, aging, and cancer. Here, we identify a crucial role for RECQL4 in DNA end resection, which is the initial and an essential step of homologous recombination (HR)-dependent DNA double-strand break repair (DSBR......). Depletion of RECQL4 severely reduces HR-mediated repair and 5' end resection in vivo. RECQL4 physically interacts with MRE11-RAD50-NBS1 (MRN), which senses DSBs and initiates DNA end resection with CtIP. The MRE11 exonuclease regulates the retention of RECQL4 at laser-induced DSBs. RECQL4 also directly...... interacts with CtIP via its N-terminal domain and promotes CtIP recruitment to the MRN complex at DSBs. Moreover, inactivation of RECQL4's helicase activity impairs DNA end processing and HR-dependent DSBR without affecting its interaction with MRE11 and CtIP, suggesting an important role for RECQL4's...

  8. 1999 Gordon Research Conference on Mammalian DNA Repair. Final Progress Report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1999-02-12

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

  9. Association between age and repair of oxidatively damaged DNA in human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Løhr, Mille; Jensen, Annie; Eriksen, Louise

    2015-01-01

    It has been hypothesised that positive associations between age and levels of oxidative stress-generated damage to DNA may be related to an age-dependent decline in DNA repair activity. The objective of this study was to investigate the association between age and repair activity of oxidatively...... assay. There was an inverse association between age and DNA repair activity with a 0.65% decline in activity per year from age 18 to 83 (95% confidence interval: 0.16-1.14% per year). Univariate regression analysis also indicated inverse associations between DNA repair activity and waist-hip ratio (P...... indicating that the decline in repair activity was not mediated by metabolic risk factors. In summary...

  10. The 2015 Nobel Prize in Chemistry The Discovery of Essential Mechanisms that Repair DNA Damage.

    Science.gov (United States)

    Lindahl, Tomas; Modrich, Paul; Sancar, Aziz

    2016-01-01

    The Royal Swedish Academy awarded the Nobel Prize in Chemistry for 2015 to Tomas Lindahl, Paul Modrich and Aziz Sancar for their discoveries in fundamental mechanisms of DNA repair. This pioneering research described three different essential pathways that correct DNA damage, safeguard the integrity of the genetic code to ensure its accurate replication through generations, and allow proper cell division. Working independently of each other, Tomas Lindahl, Paul Modrich and Aziz Sancar delineated the mechanisms of base excision repair, mismatch repair and nucleotide excision repair, respectively. These breakthroughs challenged and dismissed the early view that the DNA molecule was very stable, paving the way for the discovery of human hereditary diseases associated with distinct DNA repair deficiencies and a susceptibility to cancer. It also brought a deeper understanding of cancer as well as neurodegenerative or neurological diseases, and let to novel strategies to treat cancer.

  11. The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

    Science.gov (United States)

    Leem, S H; Ropp, P A; Sugino, A

    1994-08-11

    We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.

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

    Science.gov (United States)

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

    2009-06-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. DNA-PK-dependent RPA2 hyperphosphorylation facilitates DNA repair and suppresses sister chromatid exchange.

    Directory of Open Access Journals (Sweden)

    Hungjiun Liaw

    Full Text Available Hyperphosphorylation of RPA2 at serine 4 and serine 8 (S4, S8 has been used as a marker for activation of the DNA damage response. What types of DNA lesions cause RPA2 hyperphosphorylation, which kinase(s are responsible for them, and what is the biological outcome of these phosphorylations, however, have not been fully investigated. In this study we demonstrate that RPA2 hyperphosphorylation occurs primarily in response to genotoxic stresses that cause high levels of DNA double-strand breaks (DSBs and that the DNA-dependent protein kinase complex (DNA-PK is responsible for the modifications in vivo. Alteration of S4, S8 of RPA2 to alanines, which prevent phosphorylations at these sites, caused increased mitotic entry with concomitant increases in RAD51 foci and homologous recombination. Taken together, our results demonstrate that RPA2 hyperphosphorylation by DNA-PK in response to DSBs blocks unscheduled homologous recombination and delays mitotic entry. This pathway thus permits cells to repair DNA damage properly and increase cell viability.

  15. Cetuximab Induces Eme1-Mediated DNA Repair: a Novel Mechanism for Cetuximab Resistance

    Directory of Open Access Journals (Sweden)

    Agnieszka Weinandy

    2014-03-01

    Full Text Available Overexpression of the epidermal growth factor receptor (EGFR is observed in a large number of neoplasms. The monoclonal antibody cetuximab/Erbitux is frequently applied to treat EGFR-expressing tumors. However, the application of cetuximab alone or in combination with radio- and/or chemotherapy often yields only little benefit for patients. In the present study, we describe a mechanism that explains resistance of both tumor cell lines and cultured primary human glioma cells to cetuximab. Treatment of these cells with cetuximab promoted DNA synthesis in the absence of increased proliferation, suggesting that DNA repair pathways were activated. Indeed, we observed that cetuximab promoted the activation of the DNA damage response pathway and prevented the degradation of essential meiotic endonuclease 1 homolog 1 (Eme1, a heterodimeric endonuclease involved in DNA repair. The increased levels of Eme1 were necessary for enhanced DNA repair, and the knockdown of Eme1 was sufficient to prevent efficient DNA repair in response to ultraviolet-C light or megavoltage irradiation. These treatments reduced the survival of tumor cells, an effect that was reversed by cetuximab application. Again, this protection was dependent on Eme1. Taken together, these results suggest that cetuximab initiates pathways that result in the stabilization of Eme1, thereby resulting in enhanced DNA repair. Accordingly, cetuximab enhances DNA repair, reducing the effectiveness of DNA-damaging therapies. This aspect should be considered when using cetuximab as an antitumor agent and suggests that Eme1 is a negative predictive marker.

  16. Production, Purification, and Characterization of 15N-Labeled DNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements

    Science.gov (United States)

    Jaruga, Pawel; Nelson, Bryant C.; Lowenthal, Mark S.; Jemth, Ann-Sofie; Loseva, Olga; Coskun, Erdem; Helleday, Thomas

    2016-01-01

    Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA–protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length 15N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of 15N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented. PMID:26791985

  17. Cross-linked Polyethylenimine as Potential DNA Vector for Gene Delivery with High Efficiency and Low Cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    Wei DONG; Guang-Hui JIN; Shu-Feng LI; Qi-Ming SUN; Ding-Yuan MA; Zi-Chun HUA

    2006-01-01

    Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on its molecular weight. To enhance its gene delivery efficiency and minimize cytotoxicity, we have synthesized small cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro. In this study, branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate [ 1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h. The efficiencies of the cross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein (EGFP) reporter gene were assessed in melanoma B 16F10 cell line and other cell lines. Flow cytometry was used to quantify the cellular entry efficiency of plasmid and the transgene expression level. The cytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay. EGDMA-PEI 800-4h, a typical cross-linked PEI reported here, mediated a more efficient expression of reporter gene than the commercially available 25-kDa branched PEI control, and resulted in a 9-fold increase in gene delivery in B16F10 cells and a 16-fold increase in 293T cells, while no cytotoxicity was found at the optimized condition for gene delivery. Furthermore, the transfection activity of polyplexes was preserved in the presence of serum proteins.

  18. Homologous recombination is a primary pathway to repair DNA double-strand breaks generated during DNA rereplication.

    Science.gov (United States)

    Truong, Lan N; Li, Yongjiang; Sun, Emily; Ang, Katrina; Hwang, Patty Yi-Hwa; Wu, Xiaohua

    2014-10-17

    Re-initiation of DNA replication at origins within a given cell cycle would result in DNA rereplication, which can lead to genome instability and tumorigenesis. DNA rereplication can be induced by loss of licensing control at cellular replication origins, or by viral protein-driven multiple rounds of replication initiation at viral origins. DNA double-strand breaks (DSBs) are generated during rereplication, but the mechanisms of how these DSBs are repaired to maintain genome stability and cell viability are poorly understood in mammalian cells. We generated novel EGFP-based DSB repair substrates, which specifically monitor the repair of rereplication-associated DSBs. We demonstrated that homologous recombination (HR) is an important mechanism to repair rereplication-associated DSBs, and sister chromatids are used as templates for such HR-mediated DSB repair. Micro-homology-mediated non-homologous end joining (MMEJ) can also be used but to a lesser extent compared to HR, whereas Ku-dependent classical non-homologous end joining (C-NHEJ) has a minimal role to repair rereplication-associated DSBs. In addition, loss of HR activity leads to severe cell death when rereplication is induced. Therefore, our studies identify HR, the most conservative repair pathway, as the primary mechanism to repair DSBs upon rereplication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Proteasome inhibition enhances resistance to DNA damage via upregulation of Rpn4-dependent DNA repair genes.

    Science.gov (United States)

    Karpov, Dmitry S; Spasskaya, Daria S; Tutyaeva, Vera V; Mironov, Alexander S; Karpov, Vadim L

    2013-09-17

    The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.

  20. Beyond repair foci: DNA double-strand break repair in euchromatic and heterochromatic compartments analyzed by transmission electron microscopy.

    Directory of Open Access Journals (Sweden)

    Yvonne Lorat

    Full Text Available PURPOSE: DNA double-strand breaks (DSBs generated by ionizing radiation pose a serious threat to the preservation of genetic and epigenetic information. The known importance of local chromatin configuration in DSB repair raises the question of whether breaks in different chromatin environments are recognized and repaired by the same repair machinery and with similar efficiency. An essential step in DSB processing by non-homologous end joining is the high-affinity binding of Ku70-Ku80 and DNA-PKcs to double-stranded DNA ends that holds the ends in physical proximity for subsequent repair. METHODS AND MATERIALS: Using transmission electron microscopy to localize gold-labeled pKu70 and pDNA-PKcs within nuclear ultrastructure, we monitored the formation and repair of actual DSBs within euchromatin (electron-lucent and heterochromatin (electron-dense in cortical neurons of irradiated mouse brain. RESULTS: While DNA lesions in euchromatin (characterized by two pKu70-gold beads, reflecting the Ku70-Ku80 heterodimer are promptly sensed and rejoined, DNA packaging in heterochromatin appears to retard DSB processing, due to the time needed to unravel higher-order chromatin structures. Complex pKu70-clusters formed in heterochromatin (consisting of 4 or ≥ 6 gold beads may represent multiple breaks in close proximity caused by ionizing radiation of highly-compacted DNA. All pKu70-clusters disappeared within 72 hours post-irradiation, indicating efficient DSB rejoining. However, persistent 53BP1 clusters in heterochromatin (comprising ≥ 10 gold beads, occasionally co-localizing with γH2AX, but not pKu70 or pDNA-PKcs, may reflect incomplete or incorrect restoration of chromatin structure rather than persistently unrepaired DNA damage. DISCUSSION: Higher-order organization of chromatin determines the accessibility of DNA lesions to repair complexes, defining how readily DSBs are detected and processed. DNA lesions in heterochromatin appear to be more

  1. How to Relate Complex DNA Repair Genotypes to Pathway Function and, Ultimately, Health Risk

    Energy Technology Data Exchange (ETDEWEB)

    Jones, IM

    2002-01-09

    Exposure to ionizing radiation increases the incidence of cancer. However, predicting which individuals are at most risk from radiation exposure is a distant goal. Predictive ability is needed to guide policies that regulate radiation exposure and ensure that medical treatments have maximum benefit and minimum risk. Differences between people in susceptibility to radiation are largely based on their genotype, the genes inherited from their parents. Among the important genes are those that produce proteins that repair DNA damaged by radiation. Base Excision Repair (BER) proteins repair single strand breaks and oxidized bases in DNA. Double Strand Break Repair proteins repair broken chromosomes. Using technologies and information from the Human Genome Project, we have previously determined that the DNA sequence of DNA repair genes varies within the human population. An average of 3-4 different variants were found that affect the protein for each of 37 genes studied. The average frequency of these variants is 5%. Given the many genes in each DNA repair pathway and their many variants, technical ability to determine an individual's repair genotype greatly exceeds ability to interpret the information. A long-term goal is to relate DNA repair genotypes to health risk from radiation. This study focused on the BER pathway. The BER genes are known, variants of the genes have been identified at LLNL, and LLNL had recently developed an assay for BER function using white blood cells. The goal of this initial effort was to begin developing data that could be used to test the hypothesis that many different genotypes have similar DNA repair capacity phenotypes (function). Relationships between genotype and phenotype could then be used to group genotypes with similar function and ultimately test the association of groups of genotypes with health risk from radiation. Genotypes with reduced repair function are expected to increase risk of radiation-induced health effects. The

  2. Thermodynamics of the DNA damage repair steps of human 8-oxoguanine DNA glycosylase.

    Directory of Open Access Journals (Sweden)

    Nikita A Kuznetsov

    Full Text Available 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.

  3. Analysis of DNA double-strand break repair pathways in mice

    Energy Technology Data Exchange (ETDEWEB)

    Brugmans, Linda [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands); Kanaar, Roland [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands); Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam (Netherlands); Essers, Jeroen [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands) and Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam (Netherlands)]. E-mail: j.essers@erasmusmc.nl

    2007-01-03

    During the last years significant new insights have been gained into the mechanism and biological relevance of DNA double-strand break (DSB) repair in relation to genome stability. DSBs are a highly toxic DNA lesion, because they can lead to chromosome fragmentation, loss and translocations, eventually resulting in cancer. DSBs can be induced by cellular processes such as V(D)J recombination or DNA replication. They can also be introduced by exogenous agents DNA damaging agents such as ionizing radiation or mitomycin C. During evolution several pathways have evolved for the repair of these DSBs. The most important DSB repair mechanisms in mammalian cells are nonhomologous end-joining and homologous recombination. By using an undamaged repair template, homologous recombination ensures accurate DSB repair, whereas the untemplated nonhomologous end-joining pathway does not. Although both pathways are active in mammals, the relative contribution of the two repair pathways to genome stability differs in the different cell types. Given the potential differences in repair fidelity, it is of interest to determine the relative contribution of homologous recombination and nonhomologous end-joining to DSB repair. In this review, we focus on the biological relevance of DSB repair in mammalian cells and the potential overlap between nonhomologous end-joining and homologous recombination in different tissues.

  4. Influence of XRCC1 Genetic Polymorphisms on Ionizing Radiation-Induced DNA Damage and Repair

    Science.gov (United States)

    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

  5. Influence of XRCC1 Genetic Polymorphisms on Ionizing Radiation-Induced DNA Damage and Repair

    Directory of Open Access Journals (Sweden)

    Silvia Sterpone

    2010-01-01

    Full Text Available 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.

  6. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

  7. Mutagenic roles of DNA "repair" proteins in antibody diversity and disease-associated trinucleotide repeat instability.

    Science.gov (United States)

    Slean, Meghan M; Panigrahi, Gagan B; Ranum, Laura P; Pearson, Christopher E

    2008-07-01

    While DNA repair proteins are generally thought to maintain the integrity of the whole genome by correctly repairing mutagenic DNA intermediates, there are cases where DNA "repair" proteins are involved in causing mutations instead. For instance, somatic hypermutation (SHM) and class switch recombination (CSR) require the contribution of various DNA repair proteins, including UNG, MSH2 and MSH6 to mutate certain regions of immunoglobulin genes in order to generate antibodies of increased antigen affinity and altered effector functions. Another instance where "repair" proteins drive mutations is the instability of gene-specific trinucleotide repeats (TNR), the causative mutations of numerous diseases including Fragile X mental retardation syndrome (FRAXA), Huntington's disease (HD), myotonic dystrophy (DM1) and several spinocerebellar ataxias (SCAs) all of which arise via various modes of pathogenesis. These healthy and deleterious mutations that are induced by repair proteins are distinct from the genome-wide mutations that arise in the absence of repair proteins: they occur at specific loci, are sensitive to cis-elements (sequence context and/or epigenetic marks) and transcription, occur in specific tissues during distinct developmental windows, and are age-dependent. Here we review and compare the mutagenic role of DNA "repair" proteins in the processes of SHM, CSR and TNR instability.

  8. Recent progress with the DNA repair mutants of Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-04-02

    Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs.

  9. Repair of ultraviolet-irradiated transforming DNA in A recA mutant of Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Stuy, J.H.; Walter, R.B. (Florida State Univ., Tallahassee (USA). Dept. of Biological Science)

    1983-04-01

    Ultraviolet-irradiated transforming DNA was assayed on a wild-type strain of Haemophilus influenzae strain Rd, on an excision repair-deficient (uvr-2) mutant, on a recombination repair-deficient (recA4) mutant, and on a strain carrying both mutations. The donor DNA had a point mutation genetic marker (strAl) and a long nonhomologous plasmid-derived DNA segment inserted in the HPl prophage. The shape of the inactivation curves suggested that only recombination was responsible for the inverse square root kinetics observed with excision repair-proficient recipients.

  10. Immunochemical approach to the study of DNA repair. Proposed technical program and technical progress report

    Energy Technology Data Exchange (ETDEWEB)

    1982-01-01

    A simple immunochemical assay to quantify DNA lesions is being developed in order to facilitate the study of DNA repair. Antibodies have been raised to 5,6-dihydroxy-dihydrothymine and to thymine dimers and these have been used to measure DNA damages produced by osmium tetroxide and ultraviolet light, respectively. An enzyme immunoassay has been developed and the sensitivity of this method will be compared to physical, enzymatic, and chemical methods using PM2 bacteriophage DNA. Finally DNA repair will be assayed in several model systems.

  11. Prevalence of Germline Mutations in Genes Engaged in DNA Damage Repair by Homologous Recombination in Patients with Triple-Negative and Hereditary Non-Triple-Negative Breast Cancers.

    Directory of Open Access Journals (Sweden)

    Pawel Domagala

    Full Text Available This study sought to assess the prevalence of common germline mutations in several genes engaged in the repair of DNA double-strand break by homologous recombination in patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers. Tumors deficient in this type of DNA damage repair are known to be especially sensitive to DNA cross-linking agents (e.g., platinum drugs and to poly(ADP-ribose polymerase (PARP inhibitors.Genetic testing was performed for 36 common germline mutations in genes engaged in the repair of DNA by homologous recombination, i.e., BRCA1, BRCA2, CHEK2, NBN, ATM, PALB2, BARD1, and RAD51D, in 202 consecutive patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers.Thirty five (22.2% of 158 patients in the triple-negative group carried mutations in genes involved in DNA repair by homologous recombination, while 10 (22.7% of the 44 patients in the hereditary non-triple-negative group carried such mutations. Mutations in BRCA1 were most frequent in patients with triple-negative breast cancer (18.4%, and mutations in CHEK2 were most frequent in patients with hereditary non-triple-negative breast cancers (15.9%. In addition, in the triple-negative group, mutations in CHEK2, NBN, and ATM (3.8% combined were found, while mutations in BRCA1, NBN, and PALB2 (6.8% combined were identified in the hereditary non-triple-negative group.Identifying mutations in genes engaged in DNA damage repair by homologous recombination other than BRCA1/2 can substantially increase the proportion of patients with triple-negative breast cancer and hereditary non-triple-negative breast cancer who may be eligible for therapy using PARP inhibitors and platinum drugs.

  12. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells.

    Science.gov (United States)

    Ganesan, Shanthi; Keating, Aileen F

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6μM) for 24 or 48h. Cell viability was reduced (Padduct was detected after 24h of 6μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response.

  13. The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

    Science.gov (United States)

    Jette, Nicholas; Lees-Miller, Susan P.

    2015-01-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes. PMID:25550082

  14. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease

    Energy Technology Data Exchange (ETDEWEB)

    Dupuy, Aurélie [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Sarasin, Alain, E-mail: alain.sarasin@gustaveroussy.fr [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Service de Génétique, Institut Gustave Roussy (France)

    2015-06-15

    Graphical abstract: - Highlights: • Full correction of mutation in the XPC gene by engineered nucleases. • Meganucleases and TALENs are inhibited by 5-MeC for inducing double strand breaks. • Gene therapy of XP cells is possible using homologous recombination for DSB repair. - Abstract: 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.

  15. Genotoxic stress and DNA repair in plants: emerging functions and tools for improving crop productivity.

    Science.gov (United States)

    Balestrazzi, Alma; Confalonieri, Massimo; Macovei, Anca; Donà, Mattia; Carbonera, Daniela

    2011-03-01

    Crop productivity is strictly related to genome stability, an essential requisite for optimal plant growth/development. Genotoxic agents (e.g., chemical agents, radiations) can cause both chemical and structural damage to DNA. In some cases, they severely affect the integrity of plant genome by inducing base oxidation, which interferes with the basal processes of replication and transcription, eventually leading to cell death. The cell response to oxidative stress includes several DNA repair pathways, which are activated to remove the damaged bases and other lesions. Information concerning DNA repair in plants is still limited, although results from gene profiling and mutant analysis suggest possible differences in repair mechanisms between plants and other eukaryotes. The present review focuses on the base- and nucleotide excision repair (BER, NER) pathways, which operate according to the most common DNA repair rule (excision of damaged bases and replacement by the correct nucleotide), highlighting the most recent findings in plants. An update on DNA repair in organelles, chloroplasts and mitochondria is also provided. Finally, it is generally acknowledged that DNA repair plays a critical role during seed imbibition, preserving seed vigor. Despite this, only a limited number of studies, described here, dedicated to seeds are currently available.

  16. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    Science.gov (United States)

    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.

  17. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    Science.gov (United States)

    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.

  18. A symphony on C : orchestrating DNA repair for gene expression via cytosine modification The 2012 IMB Conference: DNA Demethylation, Repair and Beyond Institute of Molecular Biology, Mainz, Germany, 18-21 October 2012

    NARCIS (Netherlands)

    Rots, Marianne G.; Petersen-Mahrt, Svend K.

    2013-01-01

    Headline-grabbing attention has been given to DNA demethylation pathways as new epigenetic mechanisms, with reviews and hypotheses outnumbering research papers. As candidate proteins for DNA demethylation include well-known DNA repair enzymes, it was timely to join epigenetics and DNA repair experts

  19. Divergent Requirement for a DNA Repair Enzyme during Enterovirus Infections

    Directory of Open Access Journals (Sweden)

    Sonia Maciejewski

    2015-12-01

    Full Text Available Viruses of the Enterovirus genus of picornaviruses, including poliovirus, coxsackievirus B3 (CVB3, and human rhinovirus, commandeer the functions of host cell proteins to aid in the replication of their small viral genomic RNAs during infection. One of these host proteins is a cellular DNA repair enzyme known as 5′ tyrosyl-DNA phosphodiesterase 2 (TDP2. TDP2 was previously demonstrated to mediate the cleavage of a unique covalent linkage between a viral protein (VPg and the 5′ end of picornavirus RNAs. Although VPg is absent from actively translating poliovirus mRNAs, the removal of VPg is not required for the in vitro translation and replication of the RNA. However, TDP2 appears to be excluded from replication and encapsidation sites during peak times of poliovirus infection of HeLa cells, suggesting a role for TDP2 during the viral replication cycle. Using a mouse embryonic fibroblast cell line lacking TDP2, we found that TDP2 is differentially required among enteroviruses. Our single-cycle viral growth analysis shows that CVB3 replication has a greater dependency on TDP2 than does poliovirus or human rhinovirus replication. During infection, CVB3 protein accumulation is undetectable (by Western blot analysis in the absence of TDP2, whereas poliovirus protein accumulation is reduced but still detectable. Using an infectious CVB3 RNA with a reporter, CVB3 RNA could still be replicated in the absence of TDP2 following transfection, albeit at reduced levels. Overall, these results indicate that TDP2 potentiates viral replication during enterovirus infections of cultured cells, making TDP2 a potential target for antiviral development for picornavirus infections.

  20. Writers, Readers, and Erasers of Histone Ubiquitylation in DNA Double-Strand Break Repair

    DEFF Research Database (Denmark)

    Smeenk, Godelieve; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose faulty repair may alter the content and organization of cellular genomes. To counteract this threat, numerous signaling and repair proteins are recruited hierarchically to the chromatin areas surrounding DSBs to facilitate...... accurate lesion repair and restoration of genome integrity. In vertebrate cells, ubiquitin-dependent modifications of histones adjacent to DSBs by RNF8, RNF168, and other ubiquitin ligases have a key role in promoting the assembly of repair protein complexes, serving as direct recruitment platforms...... for a range of genome caretaker proteins and their associated factors. These DNA damage-induced chromatin ubiquitylation marks provide an essential component of a histone code for DSB repair that is controlled by multifaceted regulatory circuits, underscoring its importance for genome stability maintenance...

  1. Triple Negative Breast Cancers Have a Reduced Expression of DNA Repair Genes

    Science.gov (United States)

    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

  2. The RSC and INO80 chromatin-remodeling complexes in DNA double-strand break repair.

    Science.gov (United States)

    Chambers, Anna L; Downs, Jessica A

    2012-01-01

    In eukaryotes, DNA is packaged into chromatin and is therefore relatively inaccessible to DNA repair enzymes. In order to perform efficient DNA repair, ATP-dependent chromatin-remodeling enzymes are required to alter the chromatin structure near the site of damage to facilitate processing and allow access to repair enzymes. Two of the best-studied remodeling complexes involved in repair are RSC (Remodels the Structure of Chromatin) and INO80 from Saccharomyces cerevisiae, which are both conserved in higher eukaryotes. RSC is very rapidly recruited to breaks and mobilizes nucleosomes to promote phosphorylation of H2A S129 and resection. INO80 enrichment at a break occurs later and is dependent on phospho-S129 H2A. INO80 activity at the break site also facilitates resection. Consequently, both homologous recombination and nonhomologous end-joining are defective in rsc mutants, while subsets of these repair pathways are affected in ino80 mutants.

  3. DNA repair in lymphocytes from patients with secondary leukemia as measured by strand rejoining and unscheduled DNA synthesis

    DEFF Research Database (Denmark)

    Bohr, V; Køber, L

    1985-01-01

    deficiencies as measured by their ability to rejoin strand breaks, and 5 out of 7 had increased unscheduled DNA synthesis compared to treated and normal controls. All patients with SL and 4 out of 8 treated controls had inherent strand breaks in their DNA as compared to the normal controls when measured...... in isolated peripheral lymphocytes from the patients by measuring the rejoining of strand breaks following alkylation damage to the lymphocytes or by measuring unscheduled DNA synthesis. Day-to-day variability in the assays was considerable, but findings were that 5 out of 7 SL patients had repair......The ability to repair damage to DNA was compared in 2 groups of patients having undergone treatment for leukemia, one of which developed secondary leukemia (SL), and the other without signs of secondary malignancy (treated controls). Both were related to normal controls. DNA repair was assessed...

  4. Ultraviolet mutagenesis and inducible DNA repair in Caulobacter crescentus

    Energy Technology Data Exchange (ETDEWEB)

    Bender, R.A.

    1984-11-19

    The ability to reactivate ultraviolet (UV) damaged phage phiCbK (W-reactivation) is induced by UV irradiation of Caulobacter crescentus cells. Induction of W-reactivation potential is specific for phage phiCbK, requires protein synthesis, and is greatly reduced in the presence of the rec-526 mutation. The induction signal generated by UV irradiation is transient, lasting about 1 1/2 - 2 h at 30/sup 0/C; if chloramphenicol is present during early times after UV irradiation, induction of W-reactivation does not occur. Induction is maximal when cells are exposed to 5-10 J/m/sup 2/ of UV, a dose that also results in considerable mutagenesis of the cells. Taken together, these observations demonstrate the existence of a UV inducible, protein synthesis requiring, transiently signalled, rec-requiring DNA repair system analogous to W-reactivation in Escherichia coli. In addition, C. crescentus also has an efficient photoreactivation system that reverses UV damage in the presence of strong visible light.

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

    Directory of Open Access Journals (Sweden)

    Menck Carlos FM

    2007-03-01

    Full Text Available Abstract 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

  6. Quantification of DNA repair protein kinetics after γ-irradiation using number and brightness analysis

    Science.gov (United States)

    Abdisalaam, Salim; Poudel, Milan; Chen, David J.; Alexandrakis, George

    2011-03-01

    The kinetics of most proteins involved in DNA damage sensing, signaling and repair following ionizing radiation exposure cannot be quantified by current live cell fluorescence microscopy methods. This is because most of these proteins, with only few notable exceptions, do not attach in large numbers at DNA damage sites to form easily detectable foci in microscopy images. As a result a high fluorescence background from freely moving and immobile fluorescent proteins in the nucleus masks the aggregation of proteins at sparse DNA damage sites. Currently, the kinetics of these repair proteins are studied by laser-induced damage and Fluorescence Recovery After Photobleaching that rely on the detectability of high fluorescence intensity spots of clustered DNA damage. We report on the use of Number and Brightness (N&B) analysis methods as a means to monitor kinetics of DNA repair proteins during sparse DNA damage created by γ-irradiation, which is more relevant to cancer treatment than laser-induced clustered damage. We use two key double strand break repair proteins, namely Ku 70/80 and the DNA-dependent protein kinase catalytic subunit (DNA-PKCS), as specific examples to showcase the feasibility of the proposed methods to quantify dose-dependent kinetics for DNA repair proteins after exposure to γ-rays.

  7. Influence of glutathione and glutathione S-transferases on DNA interstrand cross-link formation by 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine, the active anticancer moiety generated by laromustine.

    Science.gov (United States)

    Penketh, Philip G; Patridge, Eric; Shyam, Krishnamurthy; Baumann, Raymond P; Zhu, Rui; Ishiguro, Kimiko; Sartorelli, Alan C

    2014-08-18

    Prodrugs of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) are promising anticancer agents. The 90CE moiety is a readily latentiated, short-lived (t1/2 ∼ 30 s) chloroethylating agent that can generate high yields of oxophilic electrophiles responsible for the chloroethylation of the O-6 position of guanine in DNA. These guanine O-6 alkylations are believed to be responsible for the therapeutic effects of 90CE and its prodrugs. Thus, 90CE demonstrates high selectivity toward tumors with diminished levels of O(6)-alkylguanine-DNA alkyltransferase (MGMT), the resistance protein responsible for O(6)-alkylguanine repair. The formation of O(6)-(2-chloroethyl)guanine lesions ultimately leads to the generation of highly cytotoxic 1-(N(3)-cytosinyl),-2-(N(1)-guaninyl)ethane DNA interstrand cross-links via N(1),O(6)-ethanoguanine intermediates. The anticancer activity arising from this sequence of reactions is thus identical to this component of the anticancer activity of the clinically used chloroethylnitrosoureas. Herein, we evaluate the ability of glutathione (GSH) and other low molecular weight thiols, as well as GSH coupled with various glutathione S-transferase enzymes (GSTs) to attenuate the final yields of cross-links generated by 90CE when added prior to or immediately following the initial chloroethylation step to determine the major point(s) of interaction. In contrast to studies utilizing BCNU as a chloroethylating agent by others, GSH (or GSH/GST) did not appreciably quench DNA interstrand cross-link precursors. While thiols alone offered little protection at either alkylation step, the GSH/GST couple was able to diminish the initial yields of cross-link precursors. 90CE exhibited a very different GST isoenzyme susceptibility to that reported for BCNU, this could have important implications in the relative resistance of tumor cells to these agents. The protection afforded by GSH/GST was compared to that produced by MGMT.

  8. A requirement for polymerized actin in DNA double-strand break repair.

    Science.gov (United States)

    Andrin, Christi; McDonald, Darin; Attwood, Kathleen M; Rodrigue, Amélie; Ghosh, Sunita; Mirzayans, Razmik; Masson, Jean-Yves; Dellaire, Graham; Hendzel, Michael J

    2012-07-01

    Nuclear actin is involved in several nuclear processes from chromatin remodeling to transcription. Here we examined the requirement for actin polymerization in DNA double-strand break repair. Double-strand breaks are considered the most dangerous type of DNA lesion. Double-strand break repair consists of a complex set of events that are tightly regulated. Failure at any step can have catastrophic consequences such as genomic instability, oncogenesis or cell death. Many proteins involved in this repair process have been identified and their roles characterized. We discovered that some DNA double-strand break repair factors are capable of associating with polymeric actin in vitro and specifically, that purified Ku70/80 interacts with polymerized actin under these conditions. We find that the disruption of polymeric actin inhibits DNA double strand break repair both in vitro and in vivo. Introduction of nuclear targeted mutant actin that cannot polymerize, or the depolymerization of endogenous actin filaments by the addition of cytochalasin D, alters the retention of Ku80 at sites of DNA damage in live cells. Our results suggest that polymeric actin is required for proper DNA double-strand break repair and may function through the stabilization of the Ku heterodimer at the DNA damage site.

  9. Structural basis for a novel mechanism of DNA bridging and alignment in eukaryotic DSB DNA repair.

    Science.gov (United States)

    Gouge, Jérôme; Rosario, Sandrine; Romain, Félix; Poitevin, Frédéric; Béguin, Pierre; Delarue, Marc

    2015-04-15

    Eukaryotic DNA polymerase mu of the PolX family can promote the association of the two 3'-protruding ends of a DNA double-strand break (DSB) being repaired (DNA synapsis) even in the absence of the core non-homologous end-joining (NHEJ) machinery. Here, we show that terminal deoxynucleotidyltransferase (TdT), a closely related PolX involved in V(D)J recombination, has the same property. We solved its crystal structure with an annealed DNA synapsis containing one micro-homology (MH) base pair and one nascent base pair. This structure reveals how the N-terminal domain and Loop 1 of Tdt cooperate for bridging the two DNA ends, providing a templating base in trans and limiting the MH search region to only two base pairs. A network of ordered water molecules is proposed to assist the incorporation of any nucleotide independently of the in trans templating base. These data are consistent with a recent model that explains the statistics of sequences synthesized in vivo by Tdt based solely on this dinucleotide step. Site-directed mutagenesis and functional tests suggest that this structural model is also valid for Pol mu during NHEJ.

  10. Bisdemethoxycurcumin induces DNA damage and inhibits DNA repair associated protein expressions in NCI-H460 human lung cancer cells.

    Science.gov (United States)

    Yu, Chien-Chih; Yang, Su-Tso; Huang, Wen-Wen; Peng, Shu-Fen; Huang, An-Cheng; Tang, Nou-Ying; Liu, Hsin-Chung; Yang, Mei-Due; Lai, Kuang-Chi; Chung, Jing-Gung

    2016-12-01

    Nonsmall cell lung carcinoma (NSCLC) is a devastating primary lung tumor resistant to conventional therapies. Bisdemethoxycurcumin (BDMC) is one of curcumin derivate from Turmeric and has been shown to induce NSCLC cell death. Although there is one report to show BDMC induced DNA double strand breaks, however, no available information to show BDMC induced DNA damage action with inhibited DNA repair protein in lung cancer cells in detail. In this study, we tested BDMC-induced DNA damage and condensation in NCI-H460 cells by using Comet assay and DAPI staining examinations, respectively and we found BDMC induced DNA damage and condension. Western blotting was used to examine the effects of BDMC on protein expression associated with DNA damage and repair and results indicated that BDMC suppressed the protein levels associated with DNA damage and repair, such as 14-3-3σ (an important checkpoint keeper of DDR), O6-methylguanine-DNA methyltransferase, DNA repair proteins breast cancer 1, early onset, mediator of DNA damage checkpoint 1 but activate phosphorylated p53 and p-H2A.X (phospho Ser140) in NCI-H460 cells. Confocal laser systems microscopy was used for examining the protein translocation and results show that BDMC increased the translocation of p-p53 and p-H2A.X (phospho Ser140) from cytosol to nuclei in NCI-H460 cells. In conclusion, BDMC induced DNA damage and condension and affect DNA repair proteins in NCI-H460 cells in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1859-1868, 2016. © 2015 Wiley Periodicals, Inc.

  11. Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease.

    NARCIS (Netherlands)

    A.M. Sijbers (Anneke); W.L. de Laat (Wouter); R.A. Ariza (Rafael); M. Biggerstaff (Maureen); Y-F. Wei; J.G. Moggs (Jonathan); K.C. Carter (Kenneth); B.K. Shell (Brenda); E. Evans (Elizabeth); M.C. de Jong (Mariska); S. Rademakers (Suzanne); J.D. de Rooij (Johan); N.G.J. Jaspers (Nicolaas); J.H.J. Hoeijmakers (Jan); R.D. Wood (Richard)

    1996-01-01

    textabstractNucleotide excision repair, which is defective in xeroderma pigmentosum (XP), involves incision of a DNA strand on each side of a lesion. We isolated a human gene homologous to yeast Rad1 and found that it corrects the repair defects of XP group F as well as rodent groups 4 and 11. Causa

  12. Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange

    NARCIS (Netherlands)

    H.B. Beverloo (Berna); R.D. Johnson (Roger); M. Jasin (Maria); R. Kanaar (Roland); J.H.J. Hoeijmakers (Jan); M.L.G. Dronkert (Mies)

    2000-01-01

    textabstractCells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we

  13. ERCC1-XPF endonuclease facilitates DNA double-strand break repair

    NARCIS (Netherlands)

    R.A. Ahmad (Riris); A.R. Robinson (Andria Rasile); A. Duensing (Anette); E. van Drunen (Ellen); H.B. Beverloo (Berna); D.B. Weisberg (David); P. Hasty (Paul); J.H.J. Hoeijmakers (Jan); L.J. Niedernhofer (Laura)

    2008-01-01

    textabstractERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyce

  14. Deletion-bias in DNA double-strand break repair differentially contributes to plant genome shrinkage.

    Science.gov (United States)

    Vu, Giang T H; Cao, Hieu X; Reiss, Bernd; Schubert, Ingo

    2017-02-28

    In order to prevent genome instability, cells need to be protected by a number of repair mechanisms, including DNA double-strand break (DSB) repair. The extent to which DSB repair, biased towards deletions or insertions, contributes to evolutionary diversification of genome size is still under debate. We analyzed mutation spectra in Arabidopsis thaliana and in barley (Hordeum vulgare) by PacBio sequencing of three DSB-targeted loci each, uncovering repair via gene conversion, single strand annealing (SSA) or nonhomologous end-joining (NHEJ). Furthermore, phylogenomic comparisons between A. thaliana and two related species were used to detect naturally occurring deletions during Arabidopsis evolution. Arabidopsis thaliana revealed significantly more and larger deletions after DSB repair than barley, and barley displayed more and larger insertions. Arabidopsis displayed a clear net loss of DNA after DSB repair, mainly via SSA and NHEJ. Barley revealed a very weak net loss of DNA, apparently due to less active break-end resection and easier copying of template sequences into breaks. Comparative phylogenomics revealed several footprints of SSA in the A. thaliana genome. Quantitative assessment of DNA gain and loss through DSB repair processes suggests deletion-biased DSB repair causing ongoing genome shrinking in A. thaliana, whereas genome size in barley remains nearly constant.

  15. On the role of baculovirus photolyases in DNA repair upon UV damage of occlusion bodies

    NARCIS (Netherlands)

    Biernat, M.A.; Caballero, P.; Vlak, J.M.; Oers, van M.M.

    2013-01-01

    The use of baculoviruses in insect biocontrol is hampered by their sensitivity to ultraviolet (UV) light. This irradiation induces cyclobutane pyrimidine dimers (CPDs) in DNA. CPD-photolyases repair CPDs using visible light. Plusiine baculoviruses encode photolyases, which could potentially repair

  16. Cell resistance to the Cytolethal Distending Toxin involves an association of DNA repair mechanisms

    Science.gov (United States)

    Bezine, Elisabeth; Malaisé, Yann; Loeuillet, Aurore; Chevalier, Marianne; Boutet-Robinet, Elisa; Salles, Bernard; Mirey, Gladys; Vignard, Julien

    2016-01-01

    The Cytolethal Distending Toxin (CDT), produced by many bacteria, has been associated with various diseases including cancer. CDT induces DNA double-strand breaks (DSBs), leading to cell death or mutagenesis if misrepaired. At low doses of CDT, other DNA lesions precede replication-dependent DSB formation, implying that non-DSB repair mechanisms may contribute to CDT cell resistance. To address this question, we developed a proliferation assay using human cell lines specifically depleted in each of the main DNA repair pathways. Here, we validate the involvement of the two major DSB repair mechanisms, Homologous Recombination and Non Homologous End Joining, in the management of CDT-induced lesions. We show that impairment of single-strand break repair (SSBR), but not nucleotide excision repair, sensitizes cells to CDT, and we explore the interplay of SSBR with the DSB repair mechanisms. Finally, we document the role of the replicative stress response and demonstrate the involvement of the Fanconi Anemia repair pathway in response to CDT. In conclusion, our work indicates that cellular survival to CDT-induced DNA damage involves different repair pathways, in particular SSBR. This reinforces a model where CDT-related genotoxicity primarily involves SSBs rather than DSBs, underlining the importance of cell proliferation during CDT intoxication and pathogenicity. PMID:27775089

  17. DNA repair in human cells: from genetic complementation to isolation of genes.

    NARCIS (Netherlands)

    D. Bootsma (Dirk); A. Westerveld (Andries); J.H.J. Hoeijmakers (Jan)

    1988-01-01

    textabstractThe genetic disease xeroderma pigmentosum (XP) demonstrates the association between defective repair of DNA lesions and cancer. Complementation analysis performed on XP cell strains and on repair deficient rodent cell lines has revealed that at least nine and possibly more than 13 genes

  18. Cloning and characterization of the human DNA-excision repair gene ERCC-1

    NARCIS (Netherlands)

    M. van Duin (Michel)

    1988-01-01

    textabstractIt is the aim of the work described in this thesis to isolate and characterize human genes involved DNA excision repair. This will facilitate the understanding of the mechanism of this repair process whereas it also provides an important step to better understand the relationship

  19. Cloning and characterization of the human DNA-excision repair gene ERCC-1

    NARCIS (Netherlands)

    M. van Duin (Michel)

    1988-01-01

    textabstractIt is the aim of the work described in this thesis to isolate and characterize human genes involved DNA excision repair. This will facilitate the understanding of the mechanism of this repair process whereas it also provides an important step to better understand the relationship between

  20. DNA repair in human cells: from genetic complementation to isolation of genes.

    NARCIS (Netherlands)

    D. Bootsma (Dirk); A. Westerveld (Andries); J.H.J. Hoeijmakers (Jan)

    1988-01-01

    textabstractThe genetic disease xeroderma pigmentosum (XP) demonstrates the association between defective repair of DNA lesions and cancer. Complementation analysis performed on XP cell strains and on repair deficient rodent cell lines has revealed that at least nine and possibly more than 13 genes

  1. Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair

    NARCIS (Netherlands)

    N. Hermans (Nicolaas); C. Laffeber; M. Cristovao (Michele); Artola-Borán, M. (Mariela); Mardenborough, Y. (Yannicka); P. Ikpa (Pauline); Jaddoe, A. (Aruna); H.H.K. Winterwerp (Herrie); C. Wyman (Claire); J. Jiricny (Josef); R. Kanaar (Roland); P. Friedhoff (Peter); J.H.G. Lebbink (Joyce)

    2016-01-01

    textabstractDNA mismatch repair (MMR) is an evolutionarily-conserved process responsible for the repair of replication errors. In Escherichia coli, MMR is initiated by MutS and MutL, which activate MutH to incise transiently-hemimethylated GATC sites. MMR efficiency depends on the distribution of th

  2. A baculovirus photolyase with DNA repair activity and circadian clock regulatory function

    NARCIS (Netherlands)

    Biernat, M.A.; Eker, A.P.M.; Oers, van M.M.; Vlak, J.M.; Horst, van der G.T.J.; Chaves, I.

    2012-01-01

    Cryptochromes and photolyases belong to the same family of flavoproteins but, despite being structurally conserved, display distinct functions. Photolyases use visible light to repair ultraviolet-induced DNA damage. Cryptochromes, however, function as blue-light receptors, circadian photoreceptors,

  3. Alkylation damage in DNA and RNA--repair mechanisms and medical significance

    DEFF Research Database (Denmark)

    Drabløs, Finn; Feyzi, Emadoldin; Aas, Per Arne

    2004-01-01

    Alkylation lesions in DNA and RNA result from endogenous compounds, environmental agents and alkylating drugs. Simple methylating agents, e.g. methylnitrosourea, tobacco-specific nitrosamines and drugs like temozolomide or streptozotocin, form adducts at N- and O-atoms in DNA bases. These lesions...... are mainly repaired by direct base repair, base excision repair, and to some extent by nucleotide excision repair (NER). The identified carcinogenicity of O(6)-methylguanine (O(6)-meG) is largely caused by its miscoding properties. Mutations from this lesion are prevented by O(6)-alkylG-DNA alkyltransferase......, inactivation of the MMR system in an AGT-defective background causes resistance to the killing effects of O(6)-alkylating agents, but not to the mutagenic effect. Bifunctional alkylating agents, such as chlorambucil or carmustine (BCNU), are commonly used anti-cancer drugs. DNA lesions caused by these agents...

  4. Comparative DNA Damage and Repair in Echinoderm Coelomocytes Exposed to Genotoxicants

    OpenAIRE

    El-Bibany, Ameena H.; Bodnar, Andrea G.; Reinardy, Helena C.

    2014-01-01

    The capacity to withstand and repair DNA damage differs among species and plays a role in determining an organism's resistance to genotoxicity, life history, and susceptibility to disease. Environmental stressors that affect organisms at the genetic level are of particular concern in ecotoxicology due to the potential for chronic effects and trans-generational impacts on populations. Echinoderms are valuable organisms to study the relationship between DNA repair and resistance to genotoxic st...

  5. DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch Syndrome

    OpenAIRE

    Poulogiannis, George; Frayling, Ian; Arends, Mark

    2009-01-01

    Abstract DNA mismatch repair (MMR) deficiency is one of the best understood forms of genetic instability in colorectal cancer (CRC), and is characterised by the loss of function of the MMR pathway. Failure to repair replication-associated errors due to a defective MMR system allows persistence of mismatch mutations all over the genome, but especially in regions of repetitive DNA known as microsatellites, giving rise to the phenomenon of microsatellite instability (MSI). A high freq...

  6. Emerging models for DNA repair: Dictyostelium discoideum as a model for nonhomologous end-joining.

    Science.gov (United States)

    Pears, Catherine J; Lakin, Nicholas D

    2014-05-01

    DNA double strand breaks (DSBs) are a particularly cytotoxic variety of DNA lesion that can be repaired by homologous recombination (HR) or nonhomologous end-joining (NHEJ). HR utilises sequences homologous to the damage DNA template to facilitate repair. In contrast, NHEJ does not require homologous sequences for repair but instead functions by directly re-joining DNA ends. These pathways are critical to resolve DSBs generated intentionally during processes such as meiotic and site-specific recombination. However, they are also utilised to resolve potentially pathological DSBs generated by mutagens and errors during DNA replication. The importance of DSB repair is underscored by the findings that defects in these pathways results in chromosome instability that contributes to a variety of disease states including malignancy. The general principles of NHEJ are conserved in eukaryotes. As such, relatively simple model organisms have been instrumental in identifying components of these pathways and providing a mechanistic understanding of repair that has subsequently been applied to vertebrates. However, certain components of the NHEJ pathway are absent or show limited conservation in the most commonly used invertebrate models exploited to study DNA repair. Recently, however, it has become apparent that vertebrate DNA repair pathway components, including those involved in NHEJ, are unusually conserved in the amoeba Dictyostelium discoideum. Traditionally, this genetically tractable organism has been exploited to study the molecular basis of cell type specification, cell motility and chemotaxis. Here we discuss the use of this organism as an additional model to study DNA repair, with specific reference to NHEJ. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. DNA double strand break repair via non-homologous end-joining

    OpenAIRE

    Davis, Anthony J.; Chen, David J.

    2013-01-01

    DNA double-stranded breaks (DSB) are among the most dangerous forms of DNA damage. Unrepaired DSBs results in cells undergoing apoptosis or senescence whereas mis-processing of DSBs can lead to genomic instability and carcinogenesis. One important pathway in eukaryotic cells responsible for the repair of DSBs is non-homologous end-joining (NHEJ). In this review we will discuss the interesting new insights into the mechanism of the NHEJ pathway and the proteins which mediate this repair proces...

  8. Kaempferol induces DNA damage and inhibits DNA repair associated protein expressions in human promyelocytic leukemia HL-60 cells.

    Science.gov (United States)

    Wu, Lung-Yuan; Lu, Hsu-Feng; Chou, Yu-Cheng; Shih, Yung-Luen; Bau, Da-Tian; Chen, Jaw-Chyun; Hsu, Shu-Chun; Chung, Jing-Gung

    2015-01-01

    Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60

  9. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair.

    Science.gov (United States)

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott; Scheibye-Knudsen, Morten; Desler, Claus; Hickson, Ian D; Bohr, Vilhelm A

    2014-04-01

    Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy.

  10. Cell cycle phase dependent role of DNA polymerase beta in DNA repair and survival after ionizing radiation.

    NARCIS (Netherlands)

    Vermeulen, C.; Verwijs-Janssen, M.; Begg, A.C.; Vens, C.

    2008-01-01

    PURPOSE: The purpose of the present study was to determine the role of DNA polymerase beta in repair and response after ionizing radiation in different phases of the cell cycle. METHODS AND MATERIALS: Synchronized cells deficient and proficient in DNA polymerase beta were irradiated in different pha

  11. Mechanistic Modelling of DNA Repair and Cellular Survival Following Radiation-Induced DNA Damage

    Science.gov (United States)

    McMahon, Stephen J.; Schuemann, Jan; Paganetti, Harald; Prise, Kevin M.

    2016-09-01

    Characterising and predicting the effects of ionising radiation on cells remains challenging, with the lack of robust models of the underlying mechanism of radiation responses providing a significant limitation to the development of personalised radiotherapy. In this paper we present a mechanistic model of cellular response to radiation that incorporates the kinetics of different DNA repair processes, the spatial distribution of double strand breaks and the resulting probability and severity of misrepair. This model enables predictions to be made of a range of key biological endpoints (DNA repair kinetics, chromosome aberration and mutation formation, survival) across a range of cell types based on a set of 11 mechanistic fitting parameters that are common across all cells. Applying this model to cellular survival showed its capacity to stratify the radiosensitivity of cells based on aspects of their phenotype and experimental conditions such as cell cycle phase and plating delay (correlation between modelled and observed Mean Inactivation Doses R2 > 0.9). By explicitly incorporating underlying mechanistic factors, this model can integrate knowledge from a wide range of biological studies to provide robust predictions and may act as a foundation for future calculations of individualised radiosensitivity.

  12. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease.

    Science.gov (United States)

    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.

  13. Exome-wide somatic microsatellite variation is altered in cells with DNA repair deficiencies.

    Directory of Open Access Journals (Sweden)

    Zalman Vaksman

    Full Text Available 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.

  14. Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    M. Pádula

    1999-09-01

    Full Text Available In the present study, we analyzed DNA damage induced by phycocyanin (PHY in the presence of visible light (VL using a set of repair endonucleases purified from Escherichia coli. We demonstrated that the profile of DNA damage induced by PHY is clearly different from that induced by molecules that exert deleterious effects on DNA involving solely singlet oxygen as reactive species. Most of PHY-induced lesions are single strand breaks and, to a lesser extent, base oxidized sites, which are recognized by Nth, Nfo and Fpg enzymes. High pressure liquid chromatography coupled to electrochemical detection revealed that PHY photosensitization did not induce 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo at detectable levels. DNA repair after PHY photosensitization was also investigated. Plasmid DNA damaged by PHY photosensitization was used to transform a series of Saccharomyces cerevisiae DNA repair mutants. The results revealed that plasmid survival was greatly reduced in rad14 mutants, while the ogg1 mutation did not modify the plasmid survival when compared to that in the wild type. Furthermore, plasmid survival in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant. The results reported here indicate that lethal lesions induced by PHY plus VL are repaired differently by prokaryotic and eukaryotic cells. Morever, nucleotide excision repair seems to play a major role in the recognition and repair of these lesions in Saccharomyces cerevisiae.

  15. DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

    Science.gov (United States)

    Morrical, Scott W.

    2015-01-01

    The formation of heteroduplex DNA is a central step in the exchange of DNA sequences via homologous recombination, and in the accurate repair of broken chromosomes via homology-directed repair pathways. In cells, heteroduplex DNA largely arises through the activities of recombination proteins that promote DNA-pairing and annealing reactions. Classes of proteins involved in pairing and annealing include RecA-family DNA-pairing proteins, single-stranded DNA (ssDNA)-binding proteins, recombination mediator proteins, annealing proteins, and nucleases. This review explores the properties of these pairing and annealing proteins, and highlights their roles in complex recombination processes including the double Holliday junction (DhJ) formation, synthesis-dependent strand annealing, and single-strand annealing pathways—DNA transactions that are critical both for genome stability in individual organisms and for the evolution of species. PMID:25646379

  16. DSB (Im)mobility and DNA repair compartmentalization in mammalian cells.

    Science.gov (United States)

    Lemaître, Charlène; Soutoglou, Evi

    2015-02-13

    Chromosomal translocations are considered as causal in approximately 20% of cancers. Therefore, understanding their mechanisms of formation is crucial in the prevention of carcinogenesis. The first step of translocation formation is the concomitant occurrence of double-strand DNA breaks (DSBs) in two different chromosomes. DSBs can be repaired by different repair mechanisms, including error-free homologous recombination (HR), potentially error-prone non-homologous end joining (NHEJ) and the highly mutagenic alternative end joining (alt-EJ) pathways. Regulation of DNA repair pathway choice is crucial to avoid genomic instability. In yeast, DSBs are mobile and can scan the entire nucleus to be repaired in specialized DNA repair centers or if they are persistent, in order to associate with the nuclear pores or the nuclear envelope where they can be repaired by specialized repair pathways. DSB mobility is limited in mammals; therefore, raising the question of whether the position at which a DSB occurs influences its repair. Here, we review the recent literature addressing this question. We first present the reports describing the extent of DSB mobility in mammalian cells. In a second part, we discuss the consequences of non-random gene positioning on chromosomal translocations formation. In the third part, we discuss the mobility of heterochromatic DSBs in light of our recent data on DSB repair at the nuclear lamina, and finally, we show that DSB repair compartmentalization at the nuclear periphery is conserved from yeast to mammals, further pointing to a role for gene positioning in the outcome of DSB repair. When regarded as a whole, the different studies reviewed here demonstrate the importance of nuclear architecture on DSB repair and reveal gene positioning as an important parameter in the study of tumorigenesis.

  17. Identification of the DNA repair defects in a case of Dubowitz syndrome.

    Directory of Open Access Journals (Sweden)

    Jingyin Yue

    Full Text Available Dubowitz Syndrome is an autosomal recessive disorder with a unique set of clinical features including microcephaly and susceptibility to tumor formation. Although more than 140 cases of Dubowitz syndrome have been reported since 1965, the genetic defects of this disease has not been identified. In this study, we systematically analyzed the DNA damage response and repair capability of fibroblasts established from a Dubowitz Syndrome patient. Dubowitz syndrome fibroblasts are hypersensitive to ionizing radiation, bleomycin, and doxorubicin. However, they have relatively normal sensitivities to mitomycin-C, cisplatin, and camptothecin. Dubowitz syndrome fibroblasts also have normal DNA damage signaling and cell cycle checkpoint activations after DNA damage. These data implicate a defect in repair of DNA double strand break (DSB likely due to defective non-homologous end joining (NHEJ. We further sequenced several genes involved in NHEJ, and identified a pair of novel compound mutations in the DNA Ligase IV gene. Furthermore, expression of wild type DNA ligase IV completely complement the DNA repair defects in Dubowitz syndrome fibroblasts, suggesting that the DNA ligase IV mutation is solely responsible for the DNA repair defects. These data suggests that at least subset of Dubowitz syndrome can be attributed to DNA ligase IV mutations.

  18. The NF90/NF45 complex participates in DNA break repair via nonhomologous end joining.

    Science.gov (United States)

    Shamanna, Raghavendra A; Hoque, Mainul; Lewis-Antes, Anita; Azzam, Edouard I; Lagunoff, David; Pe'ery, Tsafi; Mathews, Michael B

    2011-12-01

    Nuclear factor 90 (NF90), an RNA-binding protein implicated in the regulation of gene expression, exists as a heterodimeric complex with NF45. We previously reported that depletion of the NF90/NF45 complex results in a multinucleated phenotype. Time-lapse microscopy revealed that binucleated cells arise by incomplete abscission of progeny cells followed by fusion. Multinucleate cells arose through aberrant division of binucleated cells and displayed abnormal metaphase plates and anaphase chromatin bridges suggestive of DNA repair defects. NF90 and NF45 are known to interact with the DNA-dependent protein kinase (DNA-PK), which is involved in telomere maintenance and DNA repair by nonhomologous end joining (NHEJ). We hypothesized that NF90 modulates the activity of DNA-PK. In an in vitro NHEJ assay system, DNA end joining was reduced by NF90/NF45 immunodepletion or by RNA digestion to an extent similar to that for catalytic subunit DNA-PKcs immunodepletion. In vivo, NF90/NF45-depleted cells displayed increased γ-histone 2A.X foci, indicative of an accumulation of double-strand DNA breaks (DSBs), and increased sensitivity to ionizing radiation consistent with decreased DSB repair. Further, NF90/NF45 knockdown reduced end-joining activity in vivo. These results identify the NF90/NF45 complex as a regulator of DNA damage repair mediated by DNA-PK and suggest that structured RNA may modulate this process.

  19. DNA repair polymorphisms and cancer risk in non-smokers in a cohort study.

    NARCIS (Netherlands)

    Matullo, G; Dunning, A M; Guarrera, S; Baynes, C; Polidoro, S; Garte, S; Autrup, H; Malaveille, C; Peluso, M; Airoldi, L; Veglia, F; Gormally, E; Hoek, G; Krzyzanowski, M; Overvad, K; Raaschou-Nielsen, O; Clavel-Chapelon, F; Linseisen, J; Boeing, H; Trichopoulou, A; Palli, D; Krogh, V; Tumino, R; Panico, S; Bueno-De-Mesquita, H Bas; Peeters, Petra H M; Lund, E; Pera, G; Martinez, C; Dorronsoro, M; Barricarte, A; Tormo, M J; Quiros, J R; Day, N E; Key, T J; Saracci, R; Kaaks, R; Riboli, E; Vineis, P

    2006-01-01

    Environmental carcinogens contained in air pollution, such as polycyclic aromatic hydrocarbons, aromatic amines or N-nitroso compounds, predominantly form DNA adducts but can also generate interstrand cross-links and reactive oxygen species. If unrepaired, such lesions increase the risk of somatic

  20. DNA repair polymorphisms and cancer risk in non-smokers in a cohort study.

    NARCIS (Netherlands)

    Matullo, G; Dunning, A M; Guarrera, S; Baynes, C; Polidoro, S; Garte, S; Autrup, H; Malaveille, C; Peluso, M; Airoldi, L; Veglia, F; Gormally, E; Hoek, G; Krzyzanowski, M; Overvad, K; Raaschou-Nielsen, O; Clavel-Chapelon, F; Linseisen, J; Boeing, H; Trichopoulou, A; Palli, D; Krogh, V; Tumino, R; Panico, S; Bueno-De-Mesquita, H Bas; Peeters, Petra H M; Lund, E; Pera, G; Martinez, C; Dorronsoro, M; Barricarte, A; Tormo, M J; Quiros, J R; Day, N E; Key, T J; Saracci, R; Kaaks, R; Riboli, E; Vineis, P

    2006-01-01

    Environmental carcinogens contained in air pollution, such as polycyclic aromatic hydrocarbons, aromatic amines or N-nitroso compounds, predominantly form DNA adducts but can also generate interstrand cross-links and reactive oxygen species. If unrepaired, such lesions increase the risk of somatic m

  1. DNA repair polymorphisms and cancer risk in non-smokers in a cohort study.

    NARCIS (Netherlands)

    Matullo, G; Dunning, A M; Guarrera, S; Baynes, C; Polidoro, S; Garte, S; Autrup, H; Malaveille, C; Peluso, M; Airoldi, L; Veglia, F; Gormally, E; Hoek, G; Krzyzanowski, M; Overvad, K; Raaschou-Nielsen, O; Clavel-Chapelon, F; Linseisen, J; Boeing, H; Trichopoulou, A; Palli, D; Krogh, V; Tumino, R; Panico, S; Bueno-De-Mesquita, H Bas; Peeters, Petra H M; Lund, E; Pera, G; Martinez, C; Dorronsoro, M; Barricarte, A; Tormo, M J; Quiros, J R; Day, N E; Key, T J; Saracci, R; Kaaks, R; Riboli, E; Vineis, P

    2006-01-01

    Environmental carcinogens contained in air pollution, such as polycyclic aromatic hydrocarbons, aromatic amines or N-nitroso compounds, predominantly form DNA adducts but can also generate interstrand cross-links and reactive oxygen species. If unrepaired, such lesions increase the risk of somatic m

  2. DNA Repair Gene Polymorphisms in Hereditary and Sporadic Breast Cancer

    Science.gov (United States)

    2006-03-01

    DNA polymerase beta, and DNA ligase 3. Alternatively, in long patch BER, few bases are excised and removed by FEN-1, including bases adjacent to...the damaged base, and incorporation of new nucleotides are mediated by PCNA, Polymerase delta or epsilon and DNA ligase I. 7 The nucleotide...requires the DNA-end-binding protein Ku, which binds free DNA ends and recruits DNA-PKcs. Xrcc4 is then recruited along with DNA ligase IV. The Rad50

  3. Human POLD1 modulates cell cycle progression and DNA damage repair

    OpenAIRE

    Song, Jing; Hong, Ping; Liu, Chengeng; Zhang, Yueqi; Wang, Jinling; Wang, Peichang

    2015-01-01

    Background The activity of eukaryotic DNA polymerase delta (Pol ?) plays an essential role in genome stability through its effects on DNA replication and repair. The p125 catalytic subunit of Pol ? is encoded by POLD1 gene in human cells. To clarify biological functions of POLD1, we investigated the effects of POLD1 overexpression or downregulation on cell proliferation, cell cycle progression, DNA synthesis and oxidative DNA damage induced by H2O2. Methods HEK293 cells were transfected with ...

  4. Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination

    DEFF Research Database (Denmark)

    Gao, Min; Wei, Wei; Li, Ming Hua

    2014-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute...... (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian...

  5. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    Science.gov (United States)

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1.

  6. Adriamycin does not affect the repair of X-ray induced DNA single strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Cantoni, O.; Sestili, P.; Cattabeni, F.

    1985-06-01

    The ability of the antitumor antibiotic adriamycin (Ad) to inhibit the rejoining of DNA single strand breaks produced by X-rays was investigated in cultured cells. Chinese hamster ovary cells were given 400 rad and were allowed to repair in the presence or absence of Ad for 60 min at 37degC. The drug did not affect the ability of cells to repair DNA breaks and residual breaks found after the repair period were attributed to those induced by Ad alone. (author). 16 refs.

  7. Genetic characterization of cells of homocystinuria patients with disrupted DNA repair system

    Energy Technology Data Exchange (ETDEWEB)

    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.

  8. Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines.

    Science.gov (United States)

    Yamamoto, Kimiyo N; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P; Witt, Kristine L; Tice, Raymond R

    2011-08-01

    Included among the quantitative high throughput screens (qHTS) conducted in support of the US Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in seven isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis.

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

    DEFF Research Database (Denmark)

    Vyas, R; Kumar, R; Clermont, F

    2013-01-01

    for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation-induced DNA damage and signaling......Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial......, and that Rnf4-deficient cells and mice exhibit increased sensitivity to genotoxic stress. Mechanistically, we show that Rnf4 targets SUMOylated MDC1 and SUMOylated BRCA1, and is required for the loading of Rad51, an enzyme required for HR repair, onto sites of DNA damage. Similarly to inactivating mutations...

  10. Impact of nanoparticles on DNA repair processes: current knowledge and working hypotheses.

    Science.gov (United States)

    Carriere, Marie; Sauvaigo, Sylvie; Douki, Thierry; Ravanat, Jean-Luc

    2017-01-01

    The potential health effects of exposure to nanomaterials (NMs) is currently heavily studied. Among the most often reported impact is DNA damage, also termed genotoxicity. While several reviews relate the DNA damage induced by NMs and the techniques that can be used to prove such effects, the question of impact of NMs on DNA repair processes has never been specifically reviewed. The present review article proposes to fill this gap of knowledge by critically describing the DNA repair processes that could be affected by nanoparticle (NP) exposure, then by reporting the current state of the art on effects of NPs on DNA repair, at the level of protein function, gene induction and post-transcriptional modifications, and taking into account the advantages and limitations of the different experimental approaches. Since little is known about this impact, working hypothesis for the future are then proposed.

  11. [Study on repair capacity of DNA damage associated with chronic benzene poisoning].

    Science.gov (United States)

    Xing, Cai-hong; Ji, Zhi-ying; Li, Gui-lan; Yin, Song-nian

    2006-07-01

    To explore the repair capacity of DNA damage associated with chronic benzene poisonings. 63 workers suffered from chronic benzene poisonings and 45 workers exposed to benzene, who were engaged in the same job title, were investigated. Comet assay and cytokinesis-block micronucleus (CBMN) detection were used to evaluate gamma-radiation-induced DNA and chromosomal damage and repair capacity in peripheral blood lymphocyte. The comet tail length difference of the benzene poisoning group (4.64 +/- 1.57 microm) was significantly higher than that of the control group (3.77 +/- 1.30 microm) (P = 0.0029). There was no significant difference of the 3AB index between the poisoning group and the control group. The relative risk of benzene poisoning in the subject with comet tail length difference > 3.81 was significantly higher than that in the subject with comet tail length difference poisoning in the subject with 3AB index or = 0.20. DNA repair capacity on DNA-strand level might tightly associate with chronic benzene poisoning. The DNA repair capacity on DNA-strand level would be worse, and the benzene poisoning risk could be higher. There was no clear relation between the DNA repair capacity on chromosome level and the benzene poisoning risk.

  12. Types, Causes, Detection and Repair of DNA Fragmentation in Animal and Human Sperm Cells

    Directory of Open Access Journals (Sweden)

    Rosa Roy

    2012-10-01

    Full Text Available Concentration, motility and morphology are parameters commonly used to determine the fertilization potential of an ejaculate. These parameters give a general view on the quality of sperm but do not provide information about one of the most important components of the reproductive outcome: DNA. Either single or double DNA strand breaks can set the difference between fertile and infertile males. Sperm DNA fragmentation can be caused by intrinsic factors like abortive apoptosis, deficiencies in recombination, protamine imbalances or oxidative stress. Damage can also occur due to extrinsic factors such as storage temperatures, extenders, handling conditions, time after ejaculation, infections and reaction to medicines or post-testicular oxidative stress, among others. Two singular characteristics differentiate sperm from somatic cells: Protamination and absence of DNA repair. DNA repair in sperm is terminated as transcription and translation stops post-spermiogenesis, so these cells have no mechanism to repair the damage occurred during their transit through the epididymis and post-ejaculation. Oocytes and early embryos have been shown to repair sperm DNA damage, so the effect of sperm DNA fragmentation depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it. In this contribution we review some of these issues.

  13. Types, Causes, Detection and Repair of DNA Fragmentation in Animal and Human Sperm Cells

    Science.gov (United States)

    González-Marín, Clara; Gosálvez, Jaime; Roy, Rosa

    2012-01-01

    Concentration, motility and morphology are parameters commonly used to determine the fertilization potential of an ejaculate. These parameters give a general view on the quality of sperm but do not provide information about one of the most important components of the reproductive outcome: DNA. Either single or double DNA strand breaks can set the difference between fertile and infertile males. Sperm DNA fragmentation can be caused by intrinsic factors like abortive apoptosis, deficiencies in recombination, protamine imbalances or oxidative stress. Damage can also occur due to extrinsic factors such as storage temperatures, extenders, handling conditions, time after ejaculation, infections and reaction to medicines or post-testicular oxidative stress, among others. Two singular characteristics differentiate sperm from somatic cells: Protamination and absence of DNA repair. DNA repair in sperm is terminated as transcription and translation stops post-spermiogenesis, so these cells have no mechanism to repair the damage occurred during their transit through the epididymis and post-ejaculation. Oocytes and early embryos have been shown to repair sperm DNA damage, so the effect of sperm DNA fragmentation depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it. In this contribution we review some of these issues. PMID:23203048

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

    Directory of Open Access Journals (Sweden)

    Celina Janion

    2008-01-01

    Full Text Available 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.

  15. Estimating the effect of human base excision repair protein variants on the repair of oxidative DNA base damage.

    Science.gov (United States)

    Sokhansanj, Bahrad A; Wilson, David M

    2006-05-01

    Epidemiologic studies have revealed a complex association between human genetic variance and cancer risk. Quantitative biological modeling based on experimental data can play a critical role in interpreting the effect of genetic variation on biochemical pathways relevant to cancer development and progression. Defects in human DNA base excision repair (BER) proteins can reduce cellular tolerance to oxidative DNA base damage caused by endogenous and exogenous sources, such as exposure to toxins and ionizing radiation. If not repaired, DNA base damage leads to cell dysfunction and mutagenesis, consequently leading to cancer, disease, and aging. Population screens have identified numerous single-nucleotide polymorphism variants in many BER proteins and some have been purified and found to exhibit mild kinetic defects. Epidemiologic studies have led to conflicting conclusions on the association between single-nucleotide polymorphism variants in BER proteins and cancer risk. Using experimental data for cellular concentration and the kinetics of normal and variant BER proteins, we apply a previously developed and tested human BER pathway model to (i) estimate the effect of mild variants on BER of abasic sites and 8-oxoguanine, a prominent oxidative DNA base modification, (ii) identify ranges of variation associated with substantial BER capacity loss, and (iii) reveal nonintuitive consequences of multiple simultaneous variants. Our findings support previous work suggesting that mild BER variants have a minimal effect on pathway capacity whereas more severe defects and simultaneous variation in several BER proteins can lead to inefficient repair and potentially deleterious consequences of cellular damage.

  16. Preventing damage limitation: targeting DNA-PKcs and DNA double strand break repair pathways for ovarian cancer therapy

    Directory of Open Access Journals (Sweden)

    Daniela A Dungl

    2015-10-01

    Full Text Available 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 are associated with resistance to platinum, and defects in DNA repair pathways play critical roles in determining patient response to chemotherapy. In ovarian cancer, tumour cell defects in homologous recombination - a repair pathway activated in response to DNA double strand 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-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 signalling, 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.

  17. Comparison of Achilles tendon repair techniques in a sheep model using a cross-linked acellular porcine dermal patch and platelet-rich plasma fibrin matrix for augmentation.

    Science.gov (United States)

    Sarrafian, Tiffany L; Wang, Hali; Hackett, Eileen S; Yao, Jian Q; Shih, Mei-Shu; Ramsay, Heather L; Turner, A Simon

    2010-01-01

    The primary goal of this study was to evaluate a cross-linked acellular porcine dermal patch (APD), as well as platelet-rich plasma fibrin matrix (PRPFM), for repair of acute Achilles tendon rupture in a sheep model. The 2 surgically transected tendon ends were reapproximated in groups 1 and 2, whereas a gap was left between the tendon ends in group 3. APD was used to reinforce the repair in group 2, and autologous PRPFM was used to fill the gap, which was also reinforced with APD, in group 3. All sheep were humanely euthanized at 24 weeks after the repair, and biomechanical and histological testing were performed. Tensile strength testing showed a statistically significant difference in elongation between the operated limb and the unoperated contralateral limb in groups 1 and 3, but not in group 2. All operated tendons appeared healed with no apparent fibrosis under light and polarized microscopy. In group 1, all surgical separation sites were identifiable, and healing occurred via increasing tendon thickness. In group 2, healing occurred with new tendon fibers across the separation, without increasing tendon thickness in 2 out of 6 animals. Group 3 showed complete bridging of the gap, with no change in tendon thickness in 2 out of 6 animals. In groups 2 and 3, peripheral integration of the APD to tendon fibers was observed. These findings support the use of APD, alone or with PRPFM, to augment Achilles tendon repair in a sheep model.

  18. Protein phosphatase 5 is necessary for ATR-mediated DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Yoonsung [Department of Pharmacology, DNA Repair Research Center, Chosun University School of Medicine, 375 Seosuk-Dong, Gwangju 501-759 (Korea, Republic of); Cheong, Hyang-Min [Department of Life Science, College of Natural Science, Chung-Ang University, 221 Heuksuk-Dong, Dongjak-Ku, Seoul 156-756 (Korea, Republic of); Lee, Jung-Hee [Department of Pharmacology, DNA Repair Research Center, Chosun University School of Medicine, 375 Seosuk-Dong, Gwangju 501-759 (Korea, Republic of); Song, Peter I. [Department of Dermatology, University of Arkansas for Medical Science, 4301 West Markham, Slot 576, Little Rock, AR 72205 (Korea, Republic of); Lee, Kwang-Ho [Department of Life Science, College of Natural Science, Chung-Ang University, 221 Heuksuk-Dong, Dongjak-Ku, Seoul 156-756 (Korea, Republic of); Kim, Sang-Yong [Division of Endocrinology, Department of Internal Medicine, Chosun University School of Medicine, 375 Seosuk-Dong, Gwangju 501-759 (Korea, Republic of); Jun, Jae Yeoul [Department of Physiology, Chosun University School of Medicine, 375 Seosuk-Dong, Gwangju 501-759 (Korea, Republic of); You, Ho Jin, E-mail: hjyou@chosun.ac.kr [Department of Pharmacology, DNA Repair Research Center, Chosun University School of Medicine, 375 Seosuk-Dong, Gwangju 501-759 (Korea, Republic of)

    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.

  19. Genetic variants of the DNA repair genes from Exome Aggregation Consortium (EXAC) database: significance in cancer.

    Science.gov (United States)

    Das, Raima; Ghosh, Sankar Kumar

    2017-04-01

    DNA repair pathway is a primary defense system that eliminates wide varieties of DNA damage. Any deficiencies in them are likely to cause the chromosomal instability that leads to cell malfunctioning and tumorigenesis. Genetic polymorphisms in DNA repair genes have demonstrated a significant association with cancer risk. Our study attempts to give a glimpse of the overall scenario of the germline polymorphisms in the DNA repair genes by taking into account of the Exome Aggregation Consortium (ExAC) database as well as the Human Gene Mutation Database (HGMD) for evaluating the disease link, particularly in cancer. It has been found that ExAC DNA repair dataset (which consists of 228 DNA repair genes) comprises 30.4% missense, 12.5% dbSNP reported and 3.2% ClinVar significant variants. 27% of all the missense variants has the deleterious SIFT score of 0.00 and 6% variants carrying the most damaging Polyphen-2 score of 1.00, thus affecting the protein structure and function. However, as per HGMD, only a fraction (1.2%) of ExAC DNA repair variants was found to be cancer-related, indicating remaining variants reported in both the databases to be further analyzed. This, in turn, may provide an increased spectrum of the reported cancer linked variants in the DNA repair genes present in ExAC database. Moreover, further in silico functional assay of the identified vital cancer-associated variants, which is essential to get their actual biological significance, may shed some lights in the field of targeted drug development in near future. Copyright © 2017. Published by Elsevier B.V.

  20. On-bead fluorescent DNA nanoprobes to analyze base excision repair activities

    Energy Technology Data Exchange (ETDEWEB)

    Gines, Guillaume; Saint-Pierre, Christine; Gasparutto, Didier, E-mail: didier.gasparutto@cea.fr

    2014-02-17

    Graphical abstract: -- Highlights: •On magnetic beads fluorescent enzymatic assays. •Simple, easy, non-radioactive and electrophoresis-free functional assay. •Lesion-containing hairpin DNA probes are selective for repair enzymes. •The biosensing platform allows the measurement of DNA repair activities from purified enzymes or within cell free extracts. -- Abstract: DNA integrity is constantly threatened by endogenous and exogenous agents that can modify its physical and chemical structure. Changes in DNA sequence can cause mutations sparked by some genetic diseases or cancers. Organisms have developed efficient defense mechanisms able to specifically repair each kind of lesion (alkylation, oxidation, single or double strand break, mismatch, etc). Here we report the adjustment of an original assay to detect enzymes’ activity of base excision repair (BER), that supports a set of lesions including abasic sites, alkylation, oxidation or deamination products of bases. The biosensor is characterized by a set of fluorescent hairpin-shaped nucleic acid probes supported on magnetic beads, each containing a selective lesion targeting a specific BER enzyme. We have studied the DNA glycosylase alkyl-adenine glycosylase (AAG) and the human AP-endonuclease (APE1) by incorporating within the DNA probe a hypoxanthine lesion or an abasic site analog (tetrahydrofuran), respectively. Enzymatic repair activity induces the formation of a nick in the damaged strand, leading to probe's break, that is detected in the supernatant by fluorescence. The functional assay allows the measurement of DNA repair activities from purified enzymes or in cell-free extracts in a fast, specific, quantitative and sensitive way, using only 1 pmol of probe for a test. We recorded a detection limit of 1 μg mL{sup −1} and 50 μg mL{sup −1} of HeLa nuclear extracts for APE1 and AAG enzymes, respectively. Finally, the on-bead assay should be useful to screen inhibitors of DNA repair

  1. The DNA repair-ubiquitin-associated HR23 proteins are constituents of neuronal inclusions in specific neurodegenerative disorders without hampering DNA repair

    NARCIS (Netherlands)

    Bergink, Steven; Severijnen, Lies-Anne; Wijgers, Nils; Sugasawa, Kaoru; Yousaf, Humaira; Kros, Johan M.; van Swieten, John; Oostra, Ben A.; Hoeijmakers, Jan H.; Vermeulen, Wim; Willemsen, Rob

    2006-01-01

    Intracellular inclusions play a profound role in many neurodegenerative diseases. Here, we report that HR23B and HR23A, proteins that are involved in both DNA repair and shuttling proteins to the 26S proteasome for degradation, accumulate in neuronal inclusions in brain from a mouse model for FXTAS,

  2. Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining.

    Directory of Open Access Journals (Sweden)

    Richard Bowater

    2006-02-01

    Full Text Available DNA double-strand breaks (DSBs are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ. Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHEJ apparatus has now been identified and characterized in the prokarya. Here we review the recent discoveries concerning bacterial NHEJ and discuss the possible origins of this repair system. We also examine the insights gained from the recent cellular and biochemical studies of this DSB-repair process and discuss the possible cellular roles of an NHEJ pathway in the life-cycle of prokaryotes and phages.

  3. Polymorphisms in human DNA repair genes and head and neck squamous cell carcinoma

    Indian Academy of Sciences (India)

    Rim Khlifi; Ahmed Rebai; Amel Hamza-Chaffai

    2012-12-01

    Genetic polymorphisms in some DNA repair proteins are associated with a number of malignant transformations like head and neck squamous cell carcinoma (HNSCC). Xeroderma pigmentosum group D (XPD) and X-ray repair cross-complementing proteins 1 (XRCC1) and 3 (XRCC3) genes are involved in DNA repair and were found to be associated with HNSCC in numerous studies. To establish our overall understanding of possible relationships between DNA repair gene polymorphisms and development of HNSCC, we surveyed the literature on epidemiological studies that assessed potential associations with HNSCC risk in terms of gene–environment interactions, genotype-induced functional defects in enzyme activity and/or protein expression, and the influence of ethnic origin on these associations.We conclude that large, well-designed studies of common polymorphisms in DNA repair genes are needed. Such studies may benefit from analysis of multiple genes or polymorphisms and from the consideration of relevant exposures that may influence the likelihood of HNSCC when DNA repair capacity is reduced.

  4. Radiation- and drug-induced DNA repair in mammalian oocytes and embryos

    Energy Technology Data Exchange (ETDEWEB)

    Pedersen, R A; Brandriff, B

    1979-01-01

    A review of studies showing ultraviolet- or drug-induced unscheduled DNA synthesis in mammalian oocytes and embryos suggests that the female gamete has an excision repair capacity from the earliest stages of oocyte growth. The oocyte's demonstrable excision repair capacity decreases at the time of meiotic maturation for unknown reasons, but the fully mature oocyte maintans a repair capacity, in contrast to the mature sperm, and contributes this to the zygote. Early embryo cells maintain relatively constant levels of excision repair until late fetal stages, when they lose their capacity for excision repair. These apparent changes in excision repair capacity do not have a simple relationship to known differences in radiation sensitivity of germ cells and embryos.

  5. Chromosomal Aberrations and DNA Repair Gene Variants in a Radon-exposed Population

    Energy Technology Data Exchange (ETDEWEB)

    Kiuru, A.; Lindholm, C.; Koivistoinen, A.; Salomaa, S.

    2004-07-01

    Polymorphisms of XRCC1 (X-ray repair cross-complementing group 1), XRCC3 (X-ray repair cross-complementing group 3), and hOGG1 (the human homologue of the yeast OGG1 gene) DNA repair genes have been associated with altered DNA repair capacity and risk of various cancers. In the present study our goal was to clarify the influence of various DNA repair gene variants on the frequency of chromosomal aberrations (CA) in subjects exposed to residential radon. The study group of 84 non-smoking, healthy individuals exposed to domestic radon were analysed using the fluorescence in-situ hybridization (FISH) technique. No association between radon concentration and CA frequencies was observed. However, a significant increase with age was shown as well as a large variability in translocation frequencies between individuals within the same age group. In order to investigate the role of individual susceptibility to this variation genotypes of DNA repair genes XRCC1 (codons 194, 280 and 399), XRCC3 (codon 241) and hOGG1 (codon 326) were determined from leukocyte DNA using methods based on polymerase chain reaction. Multiple regression analysis was applied to evaluate the effect of the polymorphisms and the other confounding factors (age, exposure to randon etc) to the frequency of CA. The preliminary statistical analyses showed that the different gene appeared not to be related to a pronounced increase in chromosome aberration frequencies observed by FISH painting. However, the analysis indicated that the homozygous variant of XRCC3 codon 241 was associated (P<0.05) with two-ways translocations in conjunction with age. Larger studies, both with regard to the cohort and the number of gene variants are needed to elucidate the influence of other DNA repair variants to the yield of chromosomal aberrations. The results indicate that the chromosomal translocations accumulated by age (spontaneous background) may be partly explained by defects in homologous recombination repair. (Author

  6. FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair

    Directory of Open Access Journals (Sweden)

    Stephen eDownes

    2014-08-01

    Full Text Available Thymidine kinase 1 (TK1 is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1+ and TK1- clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumour suppressor (TP53 and human telomerase reverse transcriptase (hTERT gene regions, over 1 hour after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1+ compared to TK1- cells, while levels of genomic DNA repair were consistant between the two cell-lines. The targeted gene-specific repair in TK+ cells occurred rapidly, mainly over the first 15 minute repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1+ cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents.

  7. Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

    Science.gov (United States)

    Martins-Pinheiro, Marinalva; Schons-Fonseca, Luciane; da Silva, Josefa B; Domingos, Renan H; Momo, Leonardo Hiroyuki Santos; Simões, Ana Carolina Quirino; Ho, Paulo Lee; da Costa, Renata M A

    2016-04-01

    Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen.

  8. FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair

    Science.gov (United States)

    McAllister, Katherine A.; Yasseen, Akeel A.; McKerr, George; Downes, C. S.; McKelvey-Martin, Valerie J.

    2014-01-01

    Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1+ and TK1- clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1+ compared to TK1- cells, a trend also reflected to a lesser degree in genomic DNA repair between the cell-lines. The targeted gene-specific repair in TK+ cells occurred rapidly, mainly over the first 15 min repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1+ cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents. PMID:25152750

  9. Ultraviolet cross-linking of helical oligonucleotides to two monoclonal MRL-1pr/1pr anti-DNA autoantibodies. Variations in H and L chain binding to DNA

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Y.J.; Stollar, B.D. (Tufts Univ., Boston, MA (USA))

    1990-11-15

    Experiments were performed to determine whether both H and L chains of different anti-native DNA autoantibodies are uniformly involved in binding to DNA. Two purified monoclonal mouse (MRL-1pr/1pr) IgG autoantibodies, H241 and 2C10, were tested. They both bound synthetic helical oligonucleotides of 10 to 20 base pairs in a gel electrophoresis retardation assay but differed in their preferences for given base sequences. Exposure of antibody-radiolabeled oligonucleotide mixtures to UV light (254 nm) for 10 min led to specific covalent cross-linking of oligonucleotide to both the H and the L chains of H241 but only to the H chain of 2C10. Single labeling events were detected without higher aggregation. The oligonucleotides were not cross-linked to unrelated IgG, even after 2 h of irradiation. Cross-linked (radioactively labeled) H and L chains of H241 and 2C10 were isolated from denaturing electrophoresis gels and digested with lysyl endopeptidase and/or staphylococcal V8 protease. H241 and 2C10 H chains each yielded a major labeled peptide fragment, but the peptides from the two antibodies were different. These experiments measured only some of the antibody-DNA interactions, probably with bases in the major groove of the DNA. They indicated that two MRL-1pr/1pr IgG anti-native DNA antibodies differ in their H and L chain contacts with DNA and provide an approach to identifying affinity-labeled binding sites in the antibodies.

  10. The DNA base excision repair protein Ape1/Ref-1 as a therapeutic and chemopreventive target.

    Science.gov (United States)

    Fishel, Melissa L; Kelley, Mark R

    2007-01-01

    With our growing understanding of the pathways involved in cell proliferation and signaling, targeted therapies, in the treatment of cancer are entering the clinical arena. New and emerging targets are proteins involved in DNA repair pathways. Inhibition of various proteins in the DNA repair pathways sensitizes cancer cells to DNA damaging agents such as chemotherapy and/or radiation. We study the apurinic endonuclease 1/redox factor-1 (Ape1/Ref-1) and believe that its crucial function in DNA repair and reduction-oxidation or redox signaling make it an excellent target for sensitizing tumor cells to chemotherapy. Ape1/Ref-1 is an essential enzyme in the base excision repair (BER) pathway which is responsible for the repair of DNA caused by oxidative and alkylation damage. As importantly, Ape1/Ref-1 also functions as a redox factor maintaining transcription factors in an active reduced state. Ape1/Ref-1 stimulates the DNA binding activity of numerous transcription factors that are involved in cancer promotion and progression such as AP-1 (Fos/Jun), NFkappaB, HIF-1alpha, CREB, p53 and others. We will discuss what is known regarding the pharmacological targeting of the DNA repair activity, as well as the redox activity of Ape1/Ref-1, and explore the budding clinical utility of inhibition of either of these functions in cancer treatment. A brief discussion of the effect of polymorphisms in its DNA sequence is included because of Ape1/Ref-1's importance to maintenance and integrity of the genome. Experimental modification of Ape1/Ref-1 activity changes the response of cells and of organisms to DNA damaging agents, suggesting that Ape1/Ref-1 may also be a productive target of chemoprevention. In this review, we will provide an overview of Ape1/Ref-1's activities and explore the potential of this protein as a target in cancer treatment as well as its role in chemoprevention.

  11. Nrf2 facilitates repair of radiation induced DNA damage through homologous recombination repair pathway in a ROS independent manner in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Jayakumar, Sundarraj; Pal, Debojyoti; Sandur, Santosh K., E-mail: sskumar@barc.gov.in

    2015-09-15

    Highlights: • Nrf2 inhibition in A549 cells led to attenuated DNA repair and radiosensitization. • Influence of Nrf2 on DNA repair is not linked to its antioxidant function. • Nrf2 influences DNA repair through homologous recombination (HR) repair pathway. • Many genes involved in HR pathway show ARE sequences in their upstream region. - Abstract: Nrf2 is a redox sensitive transcription factor that is involved in the co-ordinated transcription of genes involved in redox homeostasis. But the role of Nrf2 in DNA repair is not investigated in detail. We have employed A549 and MCF7 cells to study the role of Nrf2 on DNA repair by inhibiting Nrf2 using all-trans retinoic acid (ATRA) or by knock down approach prior to radiation exposure (4 Gy). DNA damage and repair analysis was studied by γH2AX foci formation and comet assay. Results suggested that the inhibition of Nrf2 in A549 or MCF7 cells led to significant slowdown in DNA repair as compared to respective radiation controls. The persistence of residual DNA damage even in the presence of free radical scavenger N-acetyl cysteine, suggested that the influence of Nrf2 on DNA repair was not linked to its antioxidant functions. Further, its influence on non-homologous end joining repair pathway was studied by inhibiting both Nrf2 and DNA-PK together. This led to synergistic reduction of survival fraction, indicating that Nrf2 may not be influencing the NHEJ pathway. To investigate the role of homologous recombination repair (HR) pathway, RAD51 foci formation was monitored. There was a significant reduction in the foci formation in cells treated with ATRA or shRNA against Nrf2 as compared to their respective radiation controls. Further, Nrf2 inhibition led to significant reduction in mRNA levels of RAD51. BLAST analysis was also performed on upstream regions of DNA repair genes to identify antioxidant response element and found that many repair genes that are involved in HR pathway may be regulated by Nrf2

  12. Comprehensive analysis of DNA repair gene variants and risk of meningioma

    DEFF Research Database (Denmark)

    Bethke, L.; Murray, A.; Webb, E.

    2008-01-01

    of meningioma and exposure to ionizing radiation is also well known and led us to examine whether variants in DNA repair genes contribute to disease susceptibility. METHODS: We analyzed 1127 tagging single-nucleotide polymorphisms (SNPs) that were selected to capture most of the common variation in 136 DNA...

  13. Congenital DNA repair deficiency results in protection against renal ischemia reperfusion injury in mice

    NARCIS (Netherlands)

    D. Susa (Denis); J.R. Mitchell (James); M. Verweij (Marielle); H.W.M. van de Ven (Marieke); H.P. Roest (Henk); S. van den Engel (Sandra); I.M. Bajema (Ingeborg); K. Mangundap (Kirsten); J.N.M. IJzermans (Jan); J.H.J. Hoeijmakers (Jan); R.W.F. de Bruin (Ron)

    2009-01-01

    textabstractCockayne syndrome and other segmental progerias with inborn defects in DNA repair mechanisms are thought to be due in part to hypersensitivity to endogenous oxidative DNA damage. The accelerated aging-like symptoms of this disorder include dysmyelination within the central nervous system

  14. Congenital DNA repair deficiency results in protection against renal ischemia reperfusion injury in mice

    NARCIS (Netherlands)

    D. Susa (Denis); J.R. Mitchell (James); M. Verweij (Marielle); H.W.M. van de Ven (Marieke); H.P. Roest (Henk); S. van den Engel (Sandra); I.M. Bajema (Ingeborg); K. Mangundap (Kirsten); J.N.M. IJzermans (Jan); J.H.J. Hoeijmakers (Jan); R.W.F. de Bruin (Ron)

    2009-01-01

    textabstractCockayne syndrome and other segmental progerias with inborn defects in DNA repair mechanisms are thought to be due in part to hypersensitivity to endogenous oxidative DNA damage. The accelerated aging-like symptoms of this disorder include dysmyelination within the central nervous system

  15. Acetylation regulates WRN catalytic activities and affects base excision DNA repair

    DEFF Research Database (Denmark)

    Muftuoglu, Meltem; Kusumoto, Rika; Speina, Elzbieta

    2008-01-01

    The Werner protein (WRN), defective in the premature aging disorder Werner syndrome, participates in a number of DNA metabolic processes, and we have been interested in the possible regulation of its function in DNA repair by post-translational modifications. Acetylation mediated by histone...

  16. Role of Human and Mouse Rad54 in DNA Recombination and Repair

    NARCIS (Netherlands)

    J. Essers (Jeroen)

    1999-01-01

    textabstractDNA double-strand breaks (DSBs) which can be induced by endogenously produced radicals or by ionizing radiation are among the most genotoxic DNA lesions. Repair of DSBs is of cardinal importance for the prevention of chromosomal fragmentation, translocations, and deletions. The genetic i

  17. Differential contributions of mammalian Rad54 paralogs to recombination, DNA damage repair, and meiosis.

    NARCIS (Netherlands)

    J. Wesoly (Joanna); S. Agarwal (Sheba); S. Sigurdsson (Stefan); W. Bussen (Wendy); S. Komen (Stephen); J. Qin (Jian); H. van Steeg (Harry); J. van Benthem (Jan); E. Wassenaar (Evelyne); W.M. Baarends (Willy); M. Ghazvini (Mehrnaz); A. Tafel (Agnieszka); H. Heath (Helen); N.J. Galjart (Niels); J. Essers (Jeroen); J.A. Grootegoed (Anton); N. Arnheim (Norman); O.Y. Bezzubova (Olga); J-M. Buerstedde; P. Sung (Patrick); R. Kanaar (Roland)

    2006-01-01

    textabstractHomologous recombination is a versatile DNA damage repair pathway requiring Rad51 and Rad54. Here we show that a mammalian Rad54 paralog, Rad54B, displays physical and functional interactions with Rad51 and DNA that are similar to those of Rad54. While ablation of Rad54 in mouse embryoni

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

    NARCIS (Netherlands)

    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

  19. Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks

    DEFF Research Database (Denmark)

    Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia

    2016-01-01

    Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depl...

  20. NAD(+) Replenishment Improves Lifespan and Healthspan in Ataxia Telangiectasia Models via Mitophagy and DNA Repair

    DEFF Research Database (Denmark)

    Fang, Evandro Fei; Kassahun, Henok; Croteau, Deborah L

    2016-01-01

    function, delay memory loss, and extend lifespan in both animal models. Mechanistically, treatments that increase intracellular NAD(+) also stimulate neuronal DNA repair and improve mitochondrial quality via mitophagy. This work links two major theories on aging, DNA damage accumulation, and mitochondrial...

  1. Immunohistochemical analysis of oxidative stress and DNA repair proteins in normal mammary and breast cancer tissues

    Directory of Open Access Journals (Sweden)

    Nardulli Ann M

    2010-01-01

    Full Text Available Abstract Background During the course of normal cellular metabolism, oxygen is consumed and reactive oxygen species (ROS are produced. If not effectively dissipated, ROS can accumulate and damage resident proteins, lipids, and DNA. Enzymes involved in redox regulation and DNA repair dissipate ROS and repair the resulting damage in order to preserve a functional cellular environment. Because increased ROS accumulation and/or unrepaired DNA damage can lead to initiation and progression of cancer and we had identified a number of oxidative stress and DNA repair proteins that influence estrogen responsiveness of MCF-7 breast cancer cells, it seemed possible that these proteins might be differentially expressed in normal mammary tissue, benign hyperplasia (BH, ductal carcinoma in situ (DCIS and invasive breast cancer (IBC. Methods Immunohistochemistry was used to examine the expression of a number of oxidative stress proteins, DNA repair proteins, and damage markers in 60 human mammary tissues which were classified as BH, DCIS or IBC. The relative mean intensity was determined for each tissue section and ANOVA was used to detect statistical differences in the relative expression of BH, DCIS and IBC compared to normal mammary tissue. Results We found that a number of these proteins were overexpressed and that the cellular localization was altered in human breast cancer tissue. Conclusions Our studies suggest that oxidative stress and DNA repair proteins not only protect normal cells from the damaging effects of ROS, but may also promote survival of mammary tumor cells.

  2. DNA Repair and Photoprotection: Mechanisms of Overcoming Environmental Ultraviolet Radiation Exposure in Halophilic Archaea

    Directory of Open Access Journals (Sweden)

    Daniel L. Jones

    2017-09-01

    Full Text Available Halophilic archaea push the limits of life at several extremes. In particular, they are noted for their biochemical strategies in dealing with osmotic stress, low water activity and cycles of desiccation in their hypersaline environments. Another feature common to their habitats is intense ultraviolet (UV radiation, which is a challenge that microorganisms must overcome. The consequences of high UV exposure include DNA lesions arising directly from bond rearrangement of adjacent bipyrimidines, or indirectly from oxidative damage, which may ultimately result in mutation and cell death. As such, these microorganisms have evolved a number of strategies to navigate the threat of DNA damage, which we differentiate into two categories: DNA repair and photoprotection. Photoprotection encompasses damage avoidance strategies that serve as a “first line of defense,” and in halophilic archaea include pigmentation by carotenoids, mechanisms of oxidative damage avoidance, polyploidy, and genomic signatures that make DNA less susceptible to photodamage. Photolesions that do arise are addressed by a number of DNA repair mechanisms that halophilic archaea efficiently utilize, which include photoreactivation, nucleotide excision repair, base excision repair, and homologous recombination. This review seeks to place DNA damage, repair, and photoprotection in the context of halophilic archaea and the solar radiation of their hypersaline environments. We also provide new insight into the breadth of strategies and how they may work together to produce remarkable UV-resistance for these microorganisms.

  3. Mitochondrial DNA repair and association with aging--an update

    DEFF Research Database (Denmark)

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

    2010-01-01

    Mitochondrial DNA is constantly exposed to oxidative injury. Due to its location close to the main site of reactive oxygen species, the inner mitochondrial membrane, mtDNA is more susceptible than nuclear DNA to oxidative damage. The accumulation of DNA damage is thought to play a critical role...

  4. Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus

    Science.gov (United States)

    Bothmer, Anne; Phadke, Tanushree; Barrera, Luis A.; Margulies, Carrie M; Lee, Christina S.; Buquicchio, Frank; Moss, Sean; Abdulkerim, Hayat S.; Selleck, William; Jayaram, Hariharan; Myer, Vic E.; Cotta-Ramusino, Cecilia

    2017-01-01

    The CRISPR–Cas9 system provides a versatile toolkit for genome engineering that can introduce various DNA lesions at specific genomic locations. However, a better understanding of the nature of these lesions and the repair pathways engaged is critical to realizing the full potential of this technology. Here we characterize the different lesions arising from each Cas9 variant and the resulting repair pathway engagement. We demonstrate that the presence and polarity of the overhang structure is a critical determinant of double-strand break repair pathway choice. Similarly, single nicks deriving from different Cas9 variants differentially activate repair: D10A but not N863A-induced nicks are repaired by homologous recombination. Finally, we demonstrate that homologous recombination is required for repairing lesions using double-stranded, but not single-stranded DNA as a template. This detailed characterization of repair pathway choice in response to CRISPR–Cas9 enables a more deterministic approach for designing research and therapeutic genome engineering strategies. PMID:28067217

  5. Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner

    NARCIS (Netherlands)

    Moser, Jill; Kool, Hanneke; Giakzidis, Ioannis; Caldecott, Keith; Mullenders, Leon H. F.; Fousteri, Maria I.

    2007-01-01

    Impaired gap filling and sealing of chromosomal DNA in nucleotide excision repair (NER) leads to genome instability. XRCC1-DNA ligase III alpha (XRCC1-Lig3) plays a central role in the repair of DNA single-strand breaks but has never been implicated in NER. Here we show that XRCC1-Lig3 is indispensa

  6. Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner

    NARCIS (Netherlands)

    Moser, Jill; Kool, Hanneke; Giakzidis, Ioannis; Caldecott, Keith; Mullenders, Leon H. F.; Fousteri, Maria I.

    2007-01-01

    Impaired gap filling and sealing of chromosomal DNA in nucleotide excision repair (NER) leads to genome instability. XRCC1-DNA ligase III alpha (XRCC1-Lig3) plays a central role in the repair of DNA single-strand breaks but has never been implicated in NER. Here we show that XRCC1-Lig3 is

  7. Inhibition of DNA repair by Pentoxifylline and related methylxanthine derivatives.

    Science.gov (United States)

    Böhm, Lothar; Roos, Wynand Paul; Serafin, Antonio Mendes

    2003-11-15

    The methylxanthine drug Pentoxifylline is reviewed for new properties which have emerged only relatively recently and for which clinical applications can be expected. After a summary on the established systemic effects of Pentoxifylline on the microcirculation and reduction of tumour anoxia, the role of the drug in the treatment of vasoocclusive disorders, cerebral ischemia, infectious diseases, septic shock and acute respiratory distress, the review focuses on another level of drug action which is based on in vitro observations in a variety of cell lines. Pentoxifylline and the related drug Caffeine are known radiosensitizers especially in p53 mutant cells. The explanation that the drug abrogates the G2 block and shortens repair in G2 by promoting early entry into mitosis is not anymore tenable because enhancement of radiotoxicity requires presence of the drug during irradiation and fails when the drug is added after irradiation at the G2 maximum. Repair assays by measurement of recovery ratios and by delayed plating experiments indeed strongly suggested a role in repair which is now confirmed for Pentoxifylline by constant field gel electrophoresis (CFGE) measurements and for Pentoxifylline and for Caffeine by use of a variety of repair mutants. The picture now emerging shows that Caffeine and Pentoxifylline inhibit homologous recombination by targeting members of the PIK kinase family (ATM and ATR) which facilitate repair in G2. Pentoxifylline induced repair inhibition between irradiation dose fractions to counter interfraction repair has been successfully applied in a model for stereotactic surgery. Another realistic avenue of application of Pentoxifylline in tumour therapy comes from experiments which show that repair events in G2 can be targeted directly by addition of cytotoxic drugs and Pentoxifylline at the G2 maximum. Under these conditions massive dose enhancement factors of up to 80 have been observed suggesting that it may be possible to realise

  8. Rad54 and Mus81 cooperation promotes DNA damage repair and restrains chromosome missegregation

    DEFF Research Database (Denmark)

    Ghamrasni, S El; Cardoso, R; Li, L;

    2016-01-01

    Rad54 and Mus81 mammalian proteins physically interact and are important for the homologous recombination DNA repair pathway; however, their functional interactions in vivo are poorly defined. Here, we show that combinatorial loss of Rad54 and Mus81 results in hypersensitivity to DNA......-damaging agents, defects on both the homologous recombination and non-homologous DNA end joining repair pathways and reduced fertility. We also observed that while Mus81 deficiency diminished the cleavage of common fragile sites, very strikingly, Rad54 loss impaired this cleavage to even a greater extent....... The inefficient repair of DNA double-strand breaks (DSBs) in Rad54(-/-)Mus81(-/-) cells was accompanied by elevated levels of chromosome missegregation and cell death. Perhaps as a consequence, tumor incidence in Rad54(-/-)Mus81(-/-) mice remained comparable to that in Mus81(-/-) mice. Our study highlights...

  9. DNA damage response and repair data with pharmacological modulators of Tousled

    Directory of Open Access Journals (Sweden)

    Prakash Srinivasan Timiri Shanmugam

    2016-06-01

    Full Text Available Human Tousled kinase 1 (TLK1 plays an important role in chromatin remodeling, replication, and DNA damage response and repair. TLK1 activity is immediately, but transiently, downregulated after genotoxic insult, and its recovery is important for exit from checkpoint arrest and cell survival after radiation. The data in this article compliments research presented in the paper titled, “Tousled kinase activator, gallic acid, promotes DNA repair and suppresses radiation cytotoxicity in salivary gland cells” [1]. The identification of small molecule activators and inhibitors of TLK1 provided an opportunity to pharmacologically alter the protein׳s activity to elucidate its role in DNA damage response pathways. TLK1 effectors, gallic acid (GA and thioridazine (THD activate and inhibit the kinase, respectively, and the data report on the impact of these compounds and the significance of TLK1 to DNA break repair and the survival of human salivary acinar cells.

  10. ppGpp couples transcription to DNA repair in E. coli.

    Science.gov (United States)

    Kamarthapu, Venu; Epshtein, Vitaly; Benjamin, Bradley; Proshkin, Sergey; Mironov, Alexander; Cashel, Michael; Nudler, Evgeny

    2016-05-20

    The small molecule alarmone (p)ppGpp mediates bacterial adaptation to nutrient deprivation by altering the initiation properties of RNA polymerase (RNAP). ppGpp is generated in Escherichia coli by two related enzymes, RelA and SpoT. We show that ppGpp is robustly, but transiently, induced in response to DNA damage and is required for efficient nucleotide excision DNA repair (NER). This explains why relA-spoT-deficient cells are sensitive to diverse genotoxic agents and ultraviolet radiation, whereas ppGpp induction renders them more resistant to such challenges. The mechanism of DNA protection by ppGpp involves promotion of UvrD-mediated RNAP backtracking. By rendering RNAP backtracking-prone, ppGpp couples transcription to DNA repair and prompts transitions between repair and recovery states.

  11. Fission yeast Pxd1 promotes proper DNA repair by activating Rad16XPF and inhibiting Dna2.

    Directory of Open Access Journals (Sweden)

    Jia-Min Zhang

    2014-09-01

    Full Text Available 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.

  12. Gypenosides causes DNA damage and inhibits expression of DNA repair genes of human oral cancer SAS cells.

    Science.gov (United States)

    Lu, Kung-Wen; Chen, Jung-Chou; Lai, Tung-Yuan; Yang, Jai-Sing; Weng, Shu-Wen; Ma, Yi-Shih; Tang, Nou-Ying; Lu, Pei-Jung; Weng, Jing-Ru; Chung, Jing-Gung

    2010-01-01

    Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino, a Chinese medical plant. Recently, Gyp has been shown to induce cell cycle arrest and apoptosis in many human cancer cell lines. However, there is no available information to address the effects of Gyp on DNA damage and DNA repair-associated gene expression in human oral cancer cells. Therefore, we investigated whether Gyp induced DNA damage and DNA repair gene expression in human oral cancer SAS cells. The results from flow cytometric assay indicated that Gyp-induced cytotoxic effects led to a decrease in the percentage of viable SAS cells. The results from comet assay revealed that the incubation of SAS cells with Gyp led to a longer DNA migration smear (comet tail) when compared with control and this effect was dose-dependent. The results from real-time PCR analysis indicated that treatment of SAS cells with 180 mug/ml of Gyp for 24 h led to a decrease in 14-3-3sigma, DNA-dependent serine/threonine protein kinase (DNAPK), p53, ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and breast cancer gene 1 (BRCA1) mRNA expression. These observations may explain the cell death caused by Gyp in SAS cells. Taken together, Gyp induced DNA damage and inhibited DNA repair-associated gene expressions in human oral cancer SAS cells in vitro.

  13. The Heterochromatic Barrier to DNA Double Strand Break Repair: How to Get the Entry Visa

    Directory of Open Access Journals (Sweden)

    Aaron A. Goodarzi

    2012-09-01

    Full Text Available Over recent decades, a deep understanding of pathways that repair DNA double strand breaks (DSB has been gained from biochemical, structural, biophysical and cellular studies. DNA non-homologous end-joining (NHEJ and homologous recombination (HR represent the two major DSB repair pathways, and both processes are now well understood. Recent work has demonstrated that the chromatin environment at a DSB significantly impacts upon DSB repair and that, moreover, dramatic modifications arise in the chromatin surrounding a DSB. Chromatin is broadly divided into open, transcriptionally active, euchromatin (EC and highly compacted, transcriptionally inert, heterochromatin (HC, although these represent extremes of a spectrum. The HC superstructure restricts both DSB repair and damage response signaling. Moreover, DSBs within HC (HC-DSBs are rapidly relocalized to the EC-HC interface. The damage response protein kinase, ataxia telangiectasia mutated (ATM, is required for HC-DSB repair but is dispensable for the relocalization of HC-DSBs. It has been proposed that ATM signaling enhances HC relaxation in the DSB vicinity and that this is a prerequisite for HC-DSB repair. Hence, ATM is essential for repair of HC-DSBs. Here, we discuss how HC impacts upon the response to DSBs and how ATM overcomes the barrier that HC poses to repair.

  14. Protecting DNA from errors and damage: an overview of DNA repair mechanisms in plants compared to mammals.

    Science.gov (United States)

    Spampinato, Claudia P

    2017-05-01

    The genome integrity of all organisms is constantly threatened by replication errors and DNA damage arising from endogenous and exogenous sources. Such base pair anomalies must be accurately repaired to prevent mutagenesis and/or lethality. Thus, it is not surprising that cells have evolved multiple and partially overlapping DNA repair pathways to correct specific types of DNA errors and lesions. Great progress in unraveling these repair mechanisms at the molecular level has been made by several talented researchers, among them Tomas Lindahl, Aziz Sancar, and Paul Modrich, all three Nobel laureates in Chemistry for 2015. Much of this knowledge comes from studies performed in bacteria, yeast, and mammals and has impacted research in plant systems. Two plant features should be mentioned. Plants differ from higher eukaryotes in that they lack a reserve germline and cannot avoid environmental stresses. Therefore, plants have evolved different strategies to sustain genome fidelity through generations and continuous exposure to genotoxic stresses. These strategies include the presence of unique or multiple paralogous genes with partially overlapping DNA repair activities. Yet, in spite (or because) of these differences, plants, especially Arabidopsis thaliana, can be used as a model organism for functional studies. Some advantages of this model system are worth mentioning: short life cycle, availability of both homozygous and heterozygous lines for many genes, plant transformation techniques, tissue culture methods and reporter systems for gene expression and function studies. Here, I provide a current understanding of DNA repair genes in plants, with a special focus on A. thaliana. It is expected that this review will be a valuable resource for future functional studies in the DNA repair field, both in plants and animals.

  15. Molecular Mechanisms of Ultraviolet Radiation-Induced DNA Damage and Repair

    Directory of Open Access Journals (Sweden)

    Rajesh P. Rastogi

    2010-01-01

    Full Text Available DNA is one of the prime molecules, and its stability is of utmost importance for proper functioning and existence of all living systems. Genotoxic chemicals and radiations exert adverse effects on genome stability. Ultraviolet radiation (UVR (mainly UV-B: 280–315 nm is one of the powerful agents that can alter the normal state of life by inducing a variety of mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs, 6-4 photoproducts (6-4PPs, and their Dewar valence isomers as well as DNA strand breaks by interfering the genome integrity. To counteract these lesions, organisms have developed a number of highly conserved repair mechanisms such as photoreactivation, base excision repair (BER, nucleotide excision repair (NER, and mismatch repair (MMR. Additionally, double-strand break repair (by homologous recombination and nonhomologous end joining, SOS response, cell-cycle checkpoints, and programmed cell death (apoptosis are also operative in various organisms with the expense of specific gene products. This review deals with UV-induced alterations in DNA and its maintenance by various repair mechanisms.

  16. DNA repair gene polymorphisms in relation to chromosome aberration frequencies in retired radiation workers

    Energy Technology Data Exchange (ETDEWEB)

    Wilding, Craig S. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom)]. E-mail: craig.wilding@westlakes.ac.uk; Relton, Caroline L. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom); Paediatric and Lifecourse Epidemiology Research Group, School of Clinical Medical Sciences (Child Health), Newcastle University, Sir James Spence Institute, Royal Victoria Infirmary, Newcastle-upon-Tyne NE1 4LP (United Kingdom); Rees, Gwen S. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom); Tarone, Robert E. [International Epidemiology Institute, 1455 Research Boulevard, Suite 550, Rockville, MD 20850 (United States); Whitehouse, Caroline A. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom); Tawn, E. Janet [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom)

    2005-02-15

    Polymorphic variation in DNA repair genes was examined in a group of retired workers from the British Nuclear Fuels plc facility at Sellafield in relation to previously determined translocation frequencies in peripheral blood lymphocytes. Variation at seven polymorphisms in four genes involved in the base excision repair (XRCC1 R194W, R399Q and a [AC]{sub n} microsatellite in the 3' UTR) and double strand break repair (XRCC3 T241M and a [AC]{sub n} microsatellite in intron 3 of XRCC3, XRCC4 I134T, and a GACTAn microsatellite located 120kb 5' of XRCC5) pathways was determined for 291 retired radiation workers who had received cumulative occupational external radiation doses of between 0 and 1873mSv. When the interaction between radiation dose and each DNA repair gene polymorphism was examined in relation to translocation frequency there was no evidence for any of the polymorphisms studied influencing the response to occupational exposure. A positive interaction observed between genotype (individuals with at least one allele >=20 repeat units) at a microsatellite locus in the XRCC3 gene and smoking status should be interpreted cautiously because interactions were investigated for seven polymorphisms and two exposures. Nonetheless, further research is warranted to examine whether this DNA repair gene variant might be associated with a sub-optimal repair response to smoking-induced DNA damage and hence an increased frequency of translocations.

  17. A cell-free system for studying a priming factor involved in repair of bleomycin-damaged DNA.

    Directory of Open Access Journals (Sweden)

    Seki,Shuji

    1989-04-01

    Full Text Available A simple cell-free system for studying a priming factor involved in the repair of bleomycin-damaged DNA was established. The template-primer used for the repair DNA synthesis was prepared by treating the closed circular, superhelical form of pUC19 plasmid DNA with 2.2 microM bleomycin and 20 microM ferrous ions. Single-strand breaks were introduced into pUC19 DNA by the bleomycin treatment, and the DNA was consequently converted largely into the open circular form. A system for repair of this bleomycin-damaged DNA was constructed with a priming factor, DNA polymerase (DNA polymerase beta or Klenow fragment of DNA polymerase I, ATP, T4 DNA ligase and four deoxynucleoside triphosphates. After incubation, the conformation of the DNA was analyzed by agarose gel electrophoresis and electron microscopy. The open circular DNA was largely converted to the closed circular DNA, indicating that the single-strand breaks of DNA were repaired. When the priming factor was omitted, DNA repair did not occur. The present system seemed to be applicable to the study of priming factors involved in the repair of DNA with single-strand breaks caused not only by bleomycin but also by ionizing radiation or active oxygen.

  18. Association Between Polymorphisms of DNA Repair Gene XRCC1 and DNA Damage in Asbestos-Exposed Workers

    Institute of Scientific and Technical Information of China (English)

    XIAO-HONG ZHAO; GUANG JIA; YONG-QUAN LIU; SHAO-WEI LIU; LEI YAN; YU JIN; NIAN LIU

    2006-01-01

    Objective To compare the asbestos-induced DNA damage and repair capacities of DNA damage between 104 asbestos exposed workers and 101 control workers in Qingdao City of China and to investigate the possible association between polymorphisms in codon 399 of XRCC1 and susceptibility to asbestosis. Methods DNA damage levels in peripheral bloodlymphocytes were determined by comet assay, and XRCC 1 genetic polymorphisms of DNA samples from 51 asbestosis cases and 53 non-asbestosis workers with a similar asbestos exposure history were analyzed by PCR/RFLP. Results The basal comet scores (3.95±2.95) were significantly higher in asbestos-exposed workers than in control workers (0.10±0.28). After 1 h H2O2 stimulation, DNA damage of lymphocytes exhibited different increases. After a 4 h repair period, the comet scores were 50.98±19.53 in asbestos-exposed workers and 18.32±12.04 in controls. The residual DNA damage (RD) was significantly greater (P<0.01) in asbestos-exposed workers (35.62%) than in controls (27.75%). XRCC1 genetic polymorphism in 104 asbestos-exposed workers was not associated with increased risk of asbestosis. But compared with polymorphisms in the DNA repair gene XRCC1 (polymorphisms in codon 399) and the DNA damage induced by asbestos, the comet scores in asbestosis cases with Gln/Gln, Gln/Arg, and Arg/Arg were 40.26±18.94, 38.03±28.22, and 32.01±11.65, respectively, which were higher than those in non-asbestosis workers with the same genotypes (25.58±11.08, 37.08±14.74, and 29.38±10.15). There were significant differences in the comet scores between asbestosis cases and non-asbestosis workers with Gln/Gln by Student's t-test (P<0.05 or 0.01). The comet scores were higher in asbestosis workers with Gln/Gln than in those with Arg/Arg and in non-asbestosis workers exposed to asbestos, but without statistically significant difference. Conclusions Exposure to asbestos may be related to DNA damage or the capacity of cells to repair H2O2-induced

  19. Repair on the go: E. coli maintains a high proliferation rate while repairing a chronic DNA double-strand break.

    Directory of Open Access Journals (Sweden)

    Elise Darmon

    Full Text Available DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.

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

    Directory of Open Access Journals (Sweden)

    Victor D. Martinez

    2011-01-01

    Full Text Available 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.

  1. Islet expression of the DNA repair enzyme 8-oxoguanosine DNA glycosylase (Ogg1) in human type 2 diabetes

    OpenAIRE

    Yoon Kun-Ho; Wang-Rodriguez Jessica; Dib Sergio A.; Anachkov Kamen A; Tyrberg Björn; Levine Fred

    2002-01-01

    Abstract Background It has become increasingly clear that β-cell failure plays a critical role in the pathogenesis of type 2 diabetes. Free-radical mediated β-cell damage has been intensively studied in type 1 diabetes, but not in human type 2 diabetes. Therefore, we studied the protein expression of the DNA repair enzyme Ogg1 in pancreases from type 2 diabetics. Ogg1 was studied because it is the major enzyme involved in repairing 7,8-dihydro-8-oxoguanosine DNA adducts, a lesion previously o...

  2. DNA repair enhancement of aqueous extracts of Uncaria tomentosa in a human volunteer study.

    Science.gov (United States)

    Sheng, Y; Li, L; Holmgren, K; Pero, R W

    2001-07-01

    The Uncaria tomentosa water extracts (C-Med-100) have been shown to enhance DNA repair, mitogenic response and leukocyte recovery after chemotherapy-induced DNA damage in vivo. In this study, the effect of C-Med-100 supplement was evaluated in a human volunteer study. Twelve apparently healthy adults working in the same environment were randomly assigned into 3 groups with age and gender matched. One group was daily supplemented with a 250 mg tablet containing an aqueous extract of Uncaria tomentosa of C-Med-100, and another group with a 350 mg tablet, for 8 consecutive weeks. DNA repair after induction of DNA damage by a standard dose of hydrogen peroxide was measured 3 times before supplement and 3 times after the supplement for the last 3 weeks of the 8 week-supplement period. There were no drug-related toxic responses to C-Med-100 supplement when judged in terms of clinical symptoms, serum clinical chemistry, whole blood analysis and leukocyte differential counts. There was a statistically significant decrease of DNA damage and a concomitant increase of DNA repair in the supplement groups (250 and 350 mg/day) when compared with non-supplemented controls (p < 0.05). There was also an increased tendency of PHA induced lymphocyte proliferation in the treatment groups. Taken together, this trial has confirmed the earlier results obtained in the rat model when estimating DNA repair enhancement by C-Med-100.

  3. Early Steps in the DNA Base Excision Repair Pathway of a Fission Yeast Schizosaccharomyces pombe

    Directory of Open Access Journals (Sweden)

    Kyoichiro Kanamitsu

    2010-01-01

    Full Text Available DNA base excision repair (BER accounts for maintaining genomic integrity by removing damaged bases that are generated endogenously or induced by genotoxic agents. In this paper, we describe the roles of enzymes functioning in the early steps of BER in fission yeast. Although BER is an evolutionarily conserved process, some unique features of the yeast repair pathway were revealed by genetic and biochemical approaches. AP sites generated by monofunctional DNA glycosylases are incised mainly by AP lyase activity of Nth1p, a sole bifunctional glycosylase in yeast, to leave a blocked 3′ end. The major AP endonuclease Apn2p functions predominantly in removing the 3′ block. Finally, a DNA polymerase fills the gap, and a DNA ligase seals the nick (Nth1p-dependent or short patch BER. Apn1p backs up Apn2p. In long patch BER, Rad2p endonuclease removes flap DNA containing a lesion after DNA synthesis. A UV-specific endonuclease Uve1p engages in an alternative pathway by nicking DNA on the 5′ side of oxidative damage. Nucleotide excision repair and homologous recombination are involved in repair of BER intermediates including the AP site and single-strand break with the 3′ block. Other enzymes working in 3′ end processing are also discussed.

  4. DNA breakage caused by dimethyl mercury and its repair in a slime mould, Physarum polycephalum

    Energy Technology Data Exchange (ETDEWEB)

    Yatscoff, R.W.; Cummins, J.E.

    1975-10-02

    Methylmercury is known to produce a radiomimetic breakage of slime mould DNA. This DNA damage is characterized and the fact that the breakage is independent of DNA replication is established. Conclusive evidence is presented for the existence of a dimethyl mercury repair system and it is shown that strains of Physarum differing in geographical origin have widely different sensitivity to dimethyl mercury damage. 8 references, 2 figures.

  5. Fluorometric analysis of the formation and repair of DNA breaks in irradiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Ryabchenko, N.I.; Proskuryakov, S.Ya.; Ivannik, B.P.; Kutmin, A.I. (Akademiya Meditsinskikh Nauk SSSR, Obninsk. Nauchno-Issledovatel' skij Inst. Meditsinskoj Radiologii)

    A study was made of the dependence of the fluorescence of ethidium bromide upon NaOH concentration after staining of single- and double-strand DNA in cell lysates was demonstrated. The method of fluorometry was used to study the dose dependence of a change in the share of double-stranded DNA in the irradiated thymocytes and Ehrlich ascites carcinoma cells which permitted to determine the appearance and repair of DNA breaks in these cells.

  6. Effect of inhibitors of cellular metabolism on postradiation repair and degradation of DNA in rat thymocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ivannik, B.P.; Golubeva, R.V.; Proskuryakov, S.Ya.; Murzaev, V.I.; Ryabchenko, N.I.

    1979-10-01

    The viscosimetric method was used to determine the molecular weight of high polymer single-stranded DNA in alkaline nuclear lysates for the study of the effects of a number of inhibitors of synthesis of DNA (hydroxyurea), macroergic elements (2,4-dinitrophenol, EDTA) and DNAase (Na/sup +/ citrate, Ca/sup + +/ ions) on the process of repair and secondary post-radiation degradation of DNA of rat thymocytes exposed to radiation in a dosage of 3 kR.

  7. Excess single-stranded DNA inhibits meiotic double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Rebecca Johnson

    2007-11-01

    Full Text Available During meiosis, self-inflicted DNA double-strand breaks (DSBs are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE, in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects

  8. Genomically amplified Akt3 activates DNA repair pathway and promotes glioma progression.

    Science.gov (United States)

    Turner, Kristen M; Sun, Youting; Ji, Ping; Granberg, Kirsi J; Bernard, Brady; Hu, Limei; Cogdell, David E; Zhou, Xinhui; Yli-Harja, Olli; Nykter, Matti; Shmulevich, Ilya; Yung, W K Alfred; Fuller, Gregory N; Zhang, Wei

    2015-03-17

    Akt is a robust oncogene that plays key roles in the development and progression of many cancers, including glioma. We evaluated the differential propensities of the Akt isoforms toward progression in the well-characterized RCAS/Ntv-a mouse model of PDGFB-driven low grade glioma. A constitutively active myristoylated form of Akt1 did not induce high-grade glioma (HGG). In stark contrast, Akt2 and Akt3 showed strong progression potential with 78% and 97% of tumors diagnosed as HGG, respectively. We further revealed that significant variations in polarity and hydropathy values among the Akt isoforms in both the pleckstrin homology domain (P domain) and regulatory domain (R domain) were critical in mediating glioma progression. Gene expression profiles from representative Akt-derived tumors indicated dominant and distinct roles for Akt3, consisting primarily of DNA repair pathways. TCGA data from human GBM closely reflected the DNA repair function, as Akt3 was significantly correlated with a 76-gene signature DNA repair panel. Consistently, compared with Akt1 and Akt2 overexpression models, Akt3-expressing human GBM cells had enhanced activation of DNA repair proteins, leading to increased DNA repair and subsequent resistance to radiation and temozolomide. Given the wide range of Akt3-amplified cancers, Akt3 may represent a key resistance factor.

  9. The effect of aging on the DNA damage and repair capacity in 2BS cells undergoing oxidative stress.

    Science.gov (United States)

    Wang, Jin-Ling; Wang, Pei-Chang

    2012-01-01

    Aging is associated with a reduction in the DNA repair capacity under oxidative stress. However, whether the DNA damage and repair capacity can be a biomarker of aging remains controversial. In this study, we demonstrated two cause-and-effect relationships, the one is between the