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Sample records for recombination repair pathway

  1. A recombination execution checkpoint regulates the choice of homologous recombination pathway during DNA double-strand break repair

    PubMed Central

    Jain, Suvi; Sugawara, Neal; Lydeard, John; Vaze, Moreshwar; Tanguy Le Gac, Nicolas; Haber, James E.

    2009-01-01

    A DNA double-strand break (DSB) is repaired by gene conversion (GC) if both ends of the DSB share homology with an intact DNA sequence. However, if homology is limited to only one of the DSB ends, repair occurs by break-induced replication (BIR). It is not known how the homology status of the DSB ends is first assessed and what other parameters govern the choice between these repair pathways. Our data suggest that a “recombination execution checkpoint” (REC) regulates the choice of the homologous recombination pathway employed to repair a given DSB. This choice is made prior to the initiation of DNA synthesis, and is dependent on the relative position and orientation of the homologous sequences used for repair. The RecQ family helicase Sgs1 plays a key role in regulating the choice of the recombination pathway. Surprisingly, break repair and gap repair are fundamentally different processes, both kinetically and genetically, as Pol32 is required only for gap repair. We propose that the REC may have evolved to preserve genome integrity by promoting conservative repair, especially when a DSB occurs within a repeated sequence. PMID:19204116

  2. The Knowns Unknowns: Exploring the Homologous Recombination Repair Pathway in Toxoplasma gondii

    PubMed Central

    Fenoy, Ignacio M.; Bogado, Silvina S.; Contreras, Susana M.; Gottifredi, Vanesa; Angel, Sergio O.

    2016-01-01

    Toxoplasma gondii is an apicomplexan parasite of medical and veterinary importance which causes toxoplasmosis in humans. Great effort is currently being devoted toward the identification of novel drugs capable of targeting such illness. In this context, we believe that the thorough understanding of the life cycle of this model parasite will facilitate the identification of new druggable targets in T. gondii. It is important to exploit the available knowledge of pathways which could modulate the sensitivity of the parasite to DNA damaging agents. The homologous recombination repair (HRR) pathway may be of particular interest in this regard as its inactivation sensitizes other cellular models such as human cancer to targeted therapy. Herein we discuss the information available on T. gondii's HRR pathway from the perspective of its conservation with respect to yeast and humans. Special attention was devoted to BRCT domain-containing and end-resection associated proteins in T. gondii as in other experimental models such proteins have crucial roles in early/late steps or HRR and in the pathway choice for double strand break resolution. We conclude that T. gondii HRR pathway is a source of several lines of investigation that allow to to comprehend the extent of diversification of HRR in T. gondii. Such an effort will serve to determine if HRR could represent a potential targer for the treatment of toxoplasmosis. PMID:27199954

  3. Single nucleotide polymorphisms of nucleotide excision repair and homologous recombination repair pathways and their role in the risk of osteosarcoma

    PubMed Central

    Jin, Guojun; Wang, Min; Chen, Weida; Shi, Wei; Yin, Jiapeng; Gang, Wang

    2015-01-01

    Objective: To evaluate the influence of polymorphisms in nucleotide excision repair (NER) and homologous recombination repair (HRR) pathways on the development of osteosarcoma patients. Methods: Genotypes of ERCC1 rs11615 and rs3212986, ERCC2 rs1799793 and rs13181, NBN rs709816 and rs1805794, RAD51 rs1801320, rs1801321 and rs12593359, and XRCC3 rs861539 were conducted by Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) assay. Results: Total 148 osteosarcoma patients and 296 control subjects were collected from Taizhou First People’s Hospital. Conditional logistic regression analyses found that individuals carrying with GA+AA genotype of ERCC2 rs1799793 and GC+CC genotype of NBN rs1805794 were significantly associated with increased risk of osteosarcoma, and the ORs(95%CI) were 1.58(1.03-2.41) and 2.66(1.73-4.08), respectively. We found that GA+AA genotype of ERCC2 rs1799793 or GC+CC genotype of NBN rs1805794 were associated with an increased risk of osteosarcoma in females, with ORs(95%CI) of 2.42(1.20-4.87) and 2.01(1.07-4.23), respectively. Conclusion: Our results suggest that ERCC2 rs1799793 and NBN rs1805794 polymorphisms were associated with an increased risk for osteosarcoma, which suggests that NER and HRR pathways modulate the risk of developing osteosarcoma. PMID:26101473

  4. Selective utilization of nonhomologous end-joining and homologous recombination DNA repair pathways during nervous system development.

    PubMed

    Orii, Kenji E; Lee, Youngsoo; Kondo, Naomi; McKinnon, Peter J

    2006-06-27

    The repair of DNA double-strand breaks (DSBs) occurs via nonhomologous end-joining (NHEJ) or homologous recombination (HR). These mechanistically distinct pathways are critical for maintenance of genomic integrity and organismal survival. Although inactivation of either pathway leads to embryonic lethality, here we show selective requirements for each DNA DSB repair pathway at different stages of mammalian nervous system development. DNA damage-induced apoptosis resulting from inactivation of HR (Xrcc2 deficiency) only occurred in proliferating neural precursor cells, whereas disruption of NHEJ (DNA ligase IV deficiency) mainly affected differentiating cells at later developmental stages. Therefore, these data suggest that NHEJ is dispensable for a substantial portion of early development because DSB repair during this period utilizes HR. Moreover, DNA damage-induced apoptosis required the ataxia telangiectasia mutated (Atm) kinase after disruption of NHEJ, but not HR, during neurogenesis. However, embryonic lethality arising from disruption of either repair pathway was rescued by loss of p53 and resulted in specific tumor types reflective of the particular DSB repair pathway inactivated. Thus, these data reveal distinct tissue- and cell-type requirements for each DNA DSB repair pathway during neural development and provide insights for understanding the contributions of DNA DSB responses to disease.

  5. Genetic variations in the homologous recombination repair pathway genes modify risk of glioma.

    PubMed

    Zhang, Haishi; Liu, Yanhong; Zhou, Keke; Zhou, Chengcheng; Zhou, Renke; Cheng, Chunxia; Wei, Qingyi; Lu, Daru; Zhou, Liangfu

    2016-01-01

    Accumulative epidemiological evidence suggests that single nucleotide polymorphisms (SNPs) in genes involved in homologous recombination (HR) DNA repair pathway play an important role in glioma susceptibility. However, the effects of such SNPs on glioma risk remain unclear. We used a used a candidate pathway-based approach to elucidate the relationship between glioma risk and 12 putative functional SNPs in genes involved in the HR pathway. Genotyping was conducted on 771 histologically-confirmed glioma patients and 752 cancer-free controls from the Chinese Han population. Odds ratios (OR) were calculated both for each SNP individually and for grouped analyses, examining the effects of the numbers of adverse alleles on glioma risk, and evaluated their potential gene-gene interactions using the multifactor dimensionality reduction (MDR). In the single-locus analysis, two variants, the NBS1 rs1805794 (OR 1.42, 95% CI 1.15-1.76, P = 0.001), and RAD54L rs1048771 (OR 1.61, 95% CI 1.17-2.22, P = 0.002) were significantly associated with glioma risk. When we examined the joint effects of the risk-conferring alleles of these three SNPs, we found a significant trend indicating that the risk increases as the number of adverse alleles increase (P = 0.005). Moreover, the MDR analysis suggested a significant three-locus interaction model involving NBS1 rs1805794, MRE11 rs10831234, and ATM rs227062. These results suggested that these variants of the genes involved in the HR pathway may contribute to glioma susceptibility.

  6. Triple-helix formation induces recombination in mammalian cells via a nucleotide excision repair-dependent pathway.

    PubMed

    Faruqi, A F; Datta, H J; Carroll, D; Seidman, M M; Glazer, P M

    2000-02-01

    The ability to stimulate recombination in a site-specific manner in mammalian cells may provide a useful tool for gene knockout and a valuable strategy for gene therapy. We previously demonstrated that psoralen adducts targeted by triple-helix-forming oligonucleotides (TFOs) could induce recombination between tandem repeats of a supF reporter gene in a simian virus 40 vector in monkey COS cells. Based on work showing that triple helices, even in the absence of associated psoralen adducts, are able to provoke DNA repair and cause mutations, we asked whether intermolecular triplexes could stimulate recombination. Here, we report that triple-helix formation itself is capable of promoting recombination and that this effect is dependent on a functional nucleotide excision repair (NER) pathway. Transfection of COS cells carrying the dual supF vector with a purine-rich TFO, AG30, designed to bind as a third strand to a region between the two mutant supF genes yielded recombinants at a frequency of 0.37%, fivefold above background, whereas a scrambled sequence control oligomer was ineffective. In human cells deficient in the NER factor XPA, the ability of AG30 to induce recombination was eliminated, but it was restored in a corrected subline expressing the XPA cDNA. In comparison, the ability of triplex-directed psoralen cross-links to induce recombination was only partially reduced in XPA-deficient cells, suggesting that NER is not the only pathway that can metabolize targeted psoralen photoadducts into recombinagenic intermediates. Interestingly, the triplex-induced recombination was unaffected in cells deficient in DNA mismatch repair, challenging our previous model of a heteroduplex intermediate and supporting a model based on end joining. This work demonstrates that oligonucleotide-mediated triplex formation can be recombinagenic, providing the basis for a potential strategy to direct genome modification by using high-affinity DNA binding ligands.

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

    PubMed

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

    2014-10-17

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

  8. Ubiquitylation of Rad51d Mediated by E3 Ligase Rnf138 Promotes the Homologous Recombination Repair Pathway.

    PubMed

    Han, Deqiang; Liang, Junbo; Lu, Yalan; Xu, Longchang; Miao, Shiying; Lu, Lin-Yu; Song, Wei; Wang, Linfang

    2016-01-01

    Ubiquitylation has an important role as a signal transducer that regulates protein function, subcellular localization, or stability during the DNA damage response. In this study, we show that Ring domain E3 ubiquitin ligases RNF138 is recruited to DNA damage site quickly. And the recruitment is mediated through its Zinc finger domains. We further confirm that RNF138 is phosphorylated by ATM at Ser124. However, the phosphorylation was dispensable for recruitment to the DNA damage site. Our findings also indicate that RAD51 assembly at DSB sites following irradiation is dramatically affected in RNF138-deficient cells. Hence, RNF138 is likely involved in regulating homologous recombination repair pathway. Consistently, efficiency of homologous recombination decreased observably in RNF138-depleted cells. In addition, RNF138-deficient cell is hypersensitive to DNA damage insults, such as IR and MMS. And the comet assay confirmed that RNF138 directly participated in DNA damage repair. Moreover, we find that RAD51D directly interacted with RNF138. And the recruitment of RAD51D to DNA damage site is delayed and unstable in RNF138-depleted cells. Taken together, these results suggest that RNF138 promotes the homologous recombination repair pathway.

  9. Ubiquitylation of Rad51d Mediated by E3 Ligase Rnf138 Promotes the Homologous Recombination Repair Pathway

    PubMed Central

    Han, Deqiang; Liang, Junbo; Lu, Yalan; Xu, Longchang; Miao, Shiying; Lu, Lin-Yu; Song, Wei; Wang, Linfang

    2016-01-01

    Ubiquitylation has an important role as a signal transducer that regulates protein function, subcellular localization, or stability during the DNA damage response. In this study, we show that Ring domain E3 ubiquitin ligases RNF138 is recruited to DNA damage site quickly. And the recruitment is mediated through its Zinc finger domains. We further confirm that RNF138 is phosphorylated by ATM at Ser124. However, the phosphorylation was dispensable for recruitment to the DNA damage site. Our findings also indicate that RAD51 assembly at DSB sites following irradiation is dramatically affected in RNF138-deficient cells. Hence, RNF138 is likely involved in regulating homologous recombination repair pathway. Consistently, efficiency of homologous recombination decreased observably in RNF138-depleted cells. In addition, RNF138-deficient cell is hypersensitive to DNA damage insults, such as IR and MMS. And the comet assay confirmed that RNF138 directly participated in DNA damage repair. Moreover, we find that RAD51D directly interacted with RNF138. And the recruitment of RAD51D to DNA damage site is delayed and unstable in RNF138-depleted cells. Taken together, these results suggest that RNF138 promotes the homologous recombination repair pathway. PMID:27195665

  10. Homologous recombination and non-homologous end-joining repair pathways in bovine embryos with different developmental competence

    SciTech Connect

    Henrique Barreta, Marcos; Garziera Gasperin, Bernardo; Braga Rissi, Vitor; Cesaro, Matheus Pedrotti de; Ferreira, Rogerio; Oliveira, Joao Francisco de; Goncalves, Paulo Bayard Dias; Bordignon, Vilceu

    2012-10-01

    This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes were expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.

  11. Crosstalk between Translesion Synthesis, Fanconi Anemia Network, and Homologous Recombination Repair Pathways in Interstrand DNA Crosslink Repair and Development of Chemoresistance

    PubMed Central

    Haynes, Brittany; Saadat, Nadia; Myung, Brian; Shekhar, Malathy PV

    2014-01-01

    Bifunctional alkylating and platinum based drugs are chemotherapeutic agents used to treat cancer. These agents induce DNA adducts via formation of intrastrand or interstrand (ICL) DNA crosslinks, and DNA lesions of the ICL type are particularly toxic as they block DNA replication and/or DNA transcription. However, the therapeutic efficacies of these drugs are frequently limited due to the cancer cell’s enhanced ability to repair and tolerate these toxic DNA lesions. This ability to tolerate and survive the DNA damage is accomplished by a set of specialized low fidelity DNA polymerases called translesion synthesis (TLS) polymerases since high fidelity DNA polymerases are unable to replicate the damaged DNA template. TLS is a crucial initial step in ICL repair as it synthesizes DNA across the lesion thus preparing the damaged DNA template for repair by the homologous recombination (HR) pathway and Fanconi anemia (FA) network, processes critical for ICL repair. Here we review the molecular features and functional roles of TLS polymerases, discuss the collaborative interactions and cross-regulation of the TLS DNA damage tolerance pathway, the FA network and the BRCA-dependent HRR pathway, and the impact of TLS hyperactivation on development of chemoresistance. Finally, since TLS hyperactivation results from overexpression of Rad6/Rad18 ubiquitinating enzymes (fundamental components of the TLS pathway), increased PCNA ubiquitination, and/or increased recruitment of TLS polymerases, the potential benefits of selectively targeting critical components of the TLS pathway for enhancing anti-cancer therapeutic efficacy and curtailing chemotherapy-induced mutagenesis are also discussed. PMID:25795124

  12. Sulforaphane induces DNA double strand breaks predominantly repaired by homologous recombination pathway in human cancer cells

    SciTech Connect

    Sekine-Suzuki, Emiko; Yu, Dong; Kubota, Nobuo; Okayasu, Ryuichi; Anzai, Kazunori

    2008-12-12

    Cytotoxicity and DNA double strand breaks (DSBs) were studied in HeLa cells treated with sulforaphane (SFN), a well-known chemo-preventive agent. Cell survival was impaired by SFN in a concentration and treatment time-dependent manner. Both constant field gel electrophoresis (CFGE) and {gamma}-H2AX assay unambiguously indicated formation of DSBs by SFN, reflecting the cell survival data. These DSBs were predominantly processed by homologous recombination repair (HRR), judging from the SFN concentration-dependent manner of Rad51 foci formation. On the other hand, the phosphorylation of DNA-PKcs, a key non-homologous end joining (NHEJ) protein, was not observed by SFN treatment, suggesting that NHEJ may not be involved in DSBs induced by this chemical. G2/M arrest by SFN, a typical response for cells exposed to ionizing radiation was also observed. Our new data indicate the clear induction of DSBs by SFN and a useful anti-tumor aspect of SFN through the induction of DNA DSBs.

  13. Sulforaphane induces DNA double strand breaks predominantly repaired by homologous recombination pathway in human cancer cells.

    PubMed

    Sekine-Suzuki, Emiko; Yu, Dong; Kubota, Nobuo; Okayasu, Ryuichi; Anzai, Kazunori

    2008-12-12

    Cytotoxicity and DNA double strand breaks (DSBs) were studied in HeLa cells treated with sulforaphane (SFN), a well-known chemo-preventive agent. Cell survival was impaired by SFN in a concentration and treatment time-dependent manner. Both constant field gel electrophoresis (CFGE) and gamma-H2AX assay unambiguously indicated formation of DSBs by SFN, reflecting the cell survival data. These DSBs were predominantly processed by homologous recombination repair (HRR), judging from the SFN concentration-dependent manner of Rad51 foci formation. On the other hand, the phosphorylation of DNA-PKcs, a key non-homologous end joining (NHEJ) protein, was not observed by SFN treatment, suggesting that NHEJ may not be involved in DSBs induced by this chemical. G2/M arrest by SFN, a typical response for cells exposed to ionizing radiation was also observed. Our new data indicate the clear induction of DSBs by SFN and a useful anti-tumor aspect of SFN through the induction of DNA DSBs.

  14. BRCA1 Directs the Repair Pathway to Homologous Recombination by Promoting 53BP1 Dephosphorylation.

    PubMed

    Isono, Mayu; Niimi, Atsuko; Oike, Takahiro; Hagiwara, Yoshihiko; Sato, Hiro; Sekine, Ryota; Yoshida, Yukari; Isobe, Shin-Ya; Obuse, Chikashi; Nishi, Ryotaro; Petricci, Elena; Nakada, Shinichiro; Nakano, Takashi; Shibata, Atsushi

    2017-01-10

    BRCA1 promotes homologous recombination (HR) by activating DNA-end resection. By contrast, 53BP1 forms a barrier that inhibits DNA-end resection. Here, we show that BRCA1 promotes DNA-end resection by relieving the 53BP1-dependent barrier. We show that 53BP1 is phosphorylated by ATM in S/G2 phase, promoting RIF1 recruitment, which inhibits resection. 53BP1 is promptly dephosphorylated and RIF1 released, despite remaining unrepaired DNA double-strand breaks (DSBs). When resection is impaired by CtIP/MRE11 endonuclease inhibition, 53BP1 phosphorylation and RIF1 are sustained due to ongoing ATM signaling. BRCA1 depletion also sustains 53BP1 phosphorylation and RIF1 recruitment. We identify the phosphatase PP4C as having a major role in 53BP1 dephosphorylation and RIF1 release. BRCA1 or PP4C depletion impairs 53BP1 repositioning, EXO1 recruitment, and HR progression. 53BP1 or RIF1 depletion restores resection, RAD51 loading, and HR in PP4C-depleted cells. Our findings suggest that BRCA1 promotes PP4C-dependent 53BP1 dephosphorylation and RIF1 release, directing repair toward HR.

  15. The RecRO pathway of DNA recombinational repair in Helicobacter pylori and its role in bacterial survival in the host

    PubMed Central

    Wang, Ge; Lo, Leja F.; Maier, Robert J.

    2011-01-01

    Two pathways for DNA recombination, AddAB (RecBCD-like) and RecRO, were identified in Helicobacter pylori, a pathogenic bacterium that colonizes human stomachs resulting in a series of gastric diseases. In this study, we examined the physiological roles of H. pylori RecRO pathway in DNA recombinational repair. We characterized H. pylori single mutants in recR and in recO, genes in the putative gap repair recombination pathway, and an addA recO double mutant that is thus deficient in both pathways that initiate DNA recombinational repair. The recR or recO single mutants showed the same level of sensitivity to mitomycin C as the parent strain, suggesting that the RecRO pathway is not responsible for the repair of DNA double strand breaks. However, H. pylori recR and recO mutants are highly sensitive to oxidative stress and separately to acid stress, two major stress conditions that H. pylori encounters in its physiological niche. The complementation of the recR mutant restored the sensitivity to oxidative and acid stress to the wild type level. By measuring DNA transformation frequencies, the recR and recO single mutants were shown to have no effect on inter-genomic recombination, whereas the addA recO double mutant had a greatly (~12-fold) reduced transformation frequency. On the other hand, the RecRO pathway was shown to play a significant role in intra-genomic recombination with direct repeat sequences. Whereas the recA strain had a deletion frequency 35-fold lower than that of background level, inactivation of recR resulted in a 4-fold decrease in deletion frequency. In a mouse infection model, the three mutant strains displayed a greatly reduced ability to colonize the host stomachs. The geometric means of colonization number for the wild type, recR, recO, and addA recO strains were 6 × 105, 1.6 × 104, 1.4 × 104 and 4 × 103 CFU/g stomach, respectively. H. pylori RecRO-mediated DNA recombinational repair (intra-genomic recombination) is thus involved in

  16. Yeast MPH1 gene functions in an error-free DNA damage bypass pathway that requires genes from Homologous recombination, but not from postreplicative repair.

    PubMed Central

    Schürer, K Anke; Rudolph, Christian; Ulrich, Helle D; Kramer, Wilfried

    2004-01-01

    The MPH1 gene from Saccharomyces cerevisiae, encoding a member of the DEAH family of proteins, had been identified by virtue of the spontaneous mutator phenotype of respective deletion mutants. Genetic analysis suggested that MPH1 functions in a previously uncharacterized DNA repair pathway that protects the cells from damage-induced mutations. We have now analyzed genetic interactions of mph1 with a variety of mutants from different repair systems with respect to spontaneous mutation rates and sensitivities to different DNA-damaging agents. The dependence of the mph1 mutator phenotype on REV3 and REV1 and the synergy with mutations in base and nucleotide excision repair suggest an involvement of MPH1 in error-free bypass of lesions. However, although we observed an unexpected partial suppression of the mph1 mutator phenotype by rad5, genetic interactions with other mutations in postreplicative repair imply that MPH1 does not belong to this pathway. Instead, mutations from the homologous recombination pathway were found to be epistatic to mph1 with respect to both spontaneous mutation rates and damage sensitivities. Determination of spontaneous mitotic recombination rates demonstrated that mph1 mutants are not deficient in homologous recombination. On the contrary, in an sgs1 background we found a pronounced hyperrecombination phenotype. Thus, we propose that MPH1 is involved in a branch of homologous recombination that is specifically dedicated to error-free bypass. PMID:15126389

  17. Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis.

    PubMed

    Maassen, Nicole; Freese, Stefan; Schruff, Barbara; Passoth, Volkmar; Klinner, Ulrich

    2008-08-01

    Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.

  18. Alternative end-joining and classical nonhomologous end-joining pathways repair different types of double-strand breaks during class-switch recombination.

    PubMed

    Cortizas, Elena M; Zahn, Astrid; Hajjar, Maurice E; Patenaude, Anne-Marie; Di Noia, Javier M; Verdun, Ramiro E

    2013-12-01

    Classical nonhomologous end-joining (C-NHEJ) and alternative end-joining (A-EJ) are the main DNA double-strand break (DSB) repair pathways when a sister chromatid is not available. However, it is not clear how one pathway is chosen over the other to process a given DSB. To address this question, we studied in mouse splenic B cells and CH12F3 cells how C-NHEJ and A-EJ repair DSBs initiated by the activation-induced deaminase during IgH (Igh) class-switch recombination (CSR). We show in this study that lowering the deamination density at the Igh locus increases DSB resolution by microhomology-mediated repair while decreasing C-NHEJ activity. This process occurs without affecting 53BP1 and γH2AX levels during CSR. Mechanistically, lowering deamination density increases exonuclease I recruitment and single-stranded DNA at the Igh locus and promotes C-terminal binding protein interacting protein and MSH2-dependent DSB repair during CSR. Indeed, reducing activation-induced deaminase levels increases CSR efficiency in C-NHEJ-defective cells, suggesting enhanced use of an A-EJ pathway. Our results establish a mechanism by which C-NHEJ and this C-terminal binding protein interacting protein/MSH2-dependent pathway that relies on microhomology can act concurrently but independently to repair different types of DSBs and reveal that the density of DNA lesions influences the choice of DSB repair pathway during CSR.

  19. The requirement for recombination factors differs considerably between different pathways of homologous double-strand break repair in somatic plant cells.

    PubMed

    Roth, Nadine; Klimesch, Jacqueline; Dukowic-Schulze, Stefanie; Pacher, Michael; Mannuss, Anja; Puchta, Holger

    2012-12-01

    In recent years, multiple factors involved in DNA double-strand break (DSB) repair have been characterised in Arabidopsis thaliana. Using homologous sequences in somatic cells, DSBs are mainly repaired by two different pathways: synthesis-dependent strand annealing (SDSA) and single-strand annealing (SSA). By applying recombination substrates in which recombination is initiated by the induction of a site-specific DSB by the homing endonuclease I-SceI, we were able to characterise the involvement of different factors in both pathways. The nucleases MRE11 and COM1, both involved in DSB end processing, were not required for either SDSA or SSA in our assay system. Both SDSA and SSA were even more efficient without MRE11, in accordance with the fact that a loss of MRE11 might negatively affect the efficiency of non-homologous end joining. Loss of the classical recombinase RAD51 or its two paralogues RAD51C and XRCC3, as well as the SWI2/SNF2 remodelling factor RAD54, resulted in a drastic deficiency in SDSA but had hardly any influence on SSA, confirming that a strand exchange reaction is only required for SDSA. The helicase FANCM, which is postulated to be involved in the stabilisation of recombination intermediates, is surprisingly not only needed for SDSA but to a lesser extent also for SSA. Both SSA and SDSA were affected only weakly when the SMC6B protein, implicated in sister chromatid recombination, was absent, indicating that SSA and SDSA are in most cases intrachromatid recombination reactions.

  20. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells.

    PubMed Central

    Takata, M; Sasaki, M S; Sonoda, E; Morrison, C; Hashimoto, M; Utsumi, H; Yamaguchi-Iwai, Y; Shinohara, A; Takeda, S

    1998-01-01

    Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54(-/-), KU70(-/-) and RAD54(-/-)/KU70(-/-), generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing gamma-radiation-induced DSBs during G1-early S phase while recombinational repair is preferentially used in late S-G2 phase. RAD54(-/-)/KU70(-/-) cells were profoundly more sensitive to gamma-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54(-/-) and RAD54(-/-)/KU70(-/-) cells, with RAD54(-/-)/KU70(-/-) cells exhibiting significantly higher levels of chromosomal aberrations than RAD54(-/-) cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle. PMID:9736627

  1. Cross-talk between nucleotide excision and homologous recombination DNA repair pathways in the mechanism of action of antitumor trabectedin.

    PubMed

    Herrero, Ana B; Martín-Castellanos, Cristina; Marco, Esther; Gago, Federico; Moreno, Sergio

    2006-08-15

    Trabectedin (Yondelis) is a potent antitumor drug that has the unique characteristic of killing cells by poisoning the DNA nucleotide excision repair (NER) machinery. The basis for the NER-dependent toxicity has not yet been elucidated but it has been proposed as the major determinant for the drug's cytotoxicity. To study the in vivo mode of action of trabectedin and to explore the role of NER in its cytotoxicity, we used the fission yeast Schizosaccharomyces pombe as a model system. Treatment of S. pombe wild-type cells with trabectedin led to cell cycle delay and activation of the DNA damage checkpoint, indicating that the drug causes DNA damage in vivo. DNA damage induced by the drug is mostly caused by the NER protein, Rad13 (the fission yeast orthologue to human XPG), and is mainly repaired by homologous recombination. By constructing different rad13 mutants, we show that the DNA damage induced by trabectedin depends on a 46-amino acid region of Rad13 that is homologous to a DNA-binding region of human nuclease FEN-1. More specifically, an arginine residue in Rad13 (Arg961), conserved in FEN1 (Arg314), was found to be crucial for the drug's cytotoxicity. These results lead us to propose a model for the action of trabectedin in eukaryotic cells in which the formation of a Rad13/DNA-trabectedin ternary complex, stabilized by Arg961, results in cell death.

  2. Impact of two DNA repair pathways, homologous recombination and non-homologous end joining, on bacterial spore inactivation under simulated martian environmental conditions

    NASA Astrophysics Data System (ADS)

    Moeller, Ralf; Schuerger, Andrew C.; Reitz, Günther; Nicholson, Wayne L.

    2011-09-01

    Spores of Bacillus subtilis were used as a model system to study the impact of the two major DNA double-strand break (DSB) repair mechanisms [homologous recombination (HR) and non-homologous end-joining (NHEJ)] on the survivability of air-dried mono- and multilayers of bacterial spores under a simulated martian environment; i.e., an environment with low temperature (-10 °C), pure CO 2 atmosphere (99.99% CO 2), 200-1100 nm UV-VIS-NIR radiation, and 0.69 kPa pressure. Spores in multilayers exhibited low inactivation rates compared to monolayers, mainly due to shadowing effects of overlying spores. Simulated martian UV irradiation reduced dramatically spore viability, whereas when shielded from martian UV radiation, spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to simulated martian environmental conditions than were wild-type spores. In addition, NHEJ-deficient spores were consistently more sensitive than HR-deficient spores to simulated Mars environmental conditions, suggesting that DSBs were an important type of DNA damage. The results indicated that both HR and NHEJ provide an efficient set of DNA repair pathways ensuring spore survival after exposure to simulated martian environmental conditions.

  3. Replication-Associated Recombinational Repair: Lessons from Budding Yeast

    PubMed Central

    Bonner, Jaclyn N.; Zhao, Xiaolan

    2016-01-01

    Recombinational repair processes multiple types of DNA lesions. Though best understood in the repair of DNA breaks, recombinational repair is intimately linked to other situations encountered during replication. As DNA strands are decorated with many types of blocks that impede the replication machinery, a great number of genomic regions cannot be duplicated without the help of recombinational repair. This replication-associated recombinational repair employs both the core recombination proteins used for DNA break repair and the specialized factors that couple replication with repair. Studies from multiple organisms have provided insights into the roles of these specialized factors, with the findings in budding yeast being advanced through use of powerful genetics and methods for detecting DNA replication and repair intermediates. In this review, we summarize recent progress made in this organism, ranging from our understanding of the classical template switch mechanisms to gap filling and replication fork regression pathways. As many of the protein factors and biological principles uncovered in budding yeast are conserved in higher eukaryotes, these findings are crucial for stimulating studies in more complex organisms. PMID:27548223

  4. Alternative end-joining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: Implications for the formation of chromosome translocations.

    PubMed

    Iliakis, George; Murmann, Tamara; Soni, Aashish

    2015-11-01

    DNA double strand breaks (DSB) are the most deleterious lesions for the integrity of the genome, as their misrepair can lead to the formation of chromosome translocations. Cells have evolved two main repair pathways to suppress the formation of these genotoxic lesions: homology-dependent, error-free homologous recombination repair (HRR), and potentially error-prone, classical, DNA-PK-dependent non-homologous end-joining (c-NHEJ). The most salient feature of c-NHEJ, speed, will largely suppress chromosome translocation formation, while sequence alterations at the junction remain possible. It is now widely accepted that when c-NHEJ is inactivated, globally or locally, an alternative form of end-joining (alt-EJ) removes DSBs. Alt-EJ operates with speed and fidelity markedly lower than c-NHEJ, causing thus with higher probability chromosome translocations, and generating more extensive sequence alterations at the junction. Our working hypothesis is that alt-EJ operates as a backup to c-NHEJ. Recent results show that alt-EJ can also backup abrogated HRR in G2 phase cells, again at the cost of elevated formation of chromosome translocations. These observations raise alt-EJ to a global rescuing mechanism operating on ends that have lost their chromatin context in ways that compromise processing by HRR or c-NHEJ. While responsible for eliminating from the genome highly cytotoxic DNA ends, alt-EJ provides this function at the price of increased translocation formation. Here, we analyze recent literature on the mechanisms of chromosome translocation formation and propose a functional hierarchy among DSB processing pathways that makes alt-EJ the global backup pathway. We discuss possible ramifications of this model in cellular DSB management and pathway choice, and analyze its implications in radiation carcinogenesis and the design of novel therapeutic approaches. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Interaction between Mismatch Repair and Genetic Recombination in Saccharomyces Cerevisiae

    PubMed Central

    Alani, E.; Reenan, RAG.; Kolodner, R. D.

    1994-01-01

    The yeast Saccharomyces cerevisiae encodes a set of genes that show strong amino acid sequence similarity to MutS and MutL, proteins required for mismatch repair in Escherichia coli. We examined the role of MSH2 and PMS1, yeast homologs of mutS and mutL, respectively, in the repair of base pair mismatches formed during meiotic recombination. By using specifically marked HIS4 and ARG4 alleles, we showed that msh2 mutants displayed a severe defect in the repair of all base pair mismatches as well as 1-, 2- and 4-bp insertion/deletion mispairs. The msh2 and pms1 phenotypes were indistinguishable, suggesting that the wild-type gene products act in the same repair pathway. A comparison of gene conversion events in wild-type and msh2 mutants indicated that mismatch repair plays an important role in genetic recombination. (1) Tetrad analysis at five different loci revealed that, in msh2 mutants, the majority of aberrant segregants displayed a sectored phenotype, consistent with a failure to repair mismatches created during heteroduplex formation. In wild type, base pair mismatches were almost exclusively repaired toward conversion rather than restoration. (2) In msh2 strains 10-19% of the aberrant tetrads were Ab4:4. (3) Polarity gradients at HIS4 and ARG4 were nearly abolished in msh2 mutants. The frequency of gene conversion at the 3' end of these genes was increased and was nearly the frequency observed at the 5' end. (4) Co-conversion studies were consistent with mismatch repair acting to regulate heteroduplex DNA tract length. We favor a model proposing that recombination events occur through the formation and resolution of heteroduplex intermediates and that mismatch repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA. PMID:8056309

  6. Nuclear position dictates DNA repair pathway choice

    PubMed Central

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

    2014-01-01

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

  7. Mismatch repair during homologous and homeologous recombination.

    PubMed

    Spies, Maria; Fishel, Richard

    2015-03-02

    Homologous recombination (HR) and mismatch repair (MMR) are inextricably linked. HR pairs homologous chromosomes before meiosis I and is ultimately responsible for generating genetic diversity during sexual reproduction. HR is initiated in meiosis by numerous programmed DNA double-strand breaks (DSBs; several hundred in mammals). A characteristic feature of HR is the exchange of DNA strands, which results in the formation of heteroduplex DNA. Mismatched nucleotides arise in heteroduplex DNA because the participating parental chromosomes contain nonidentical sequences. These mismatched nucleotides may be processed by MMR, resulting in nonreciprocal exchange of genetic information (gene conversion). MMR and HR also play prominent roles in mitotic cells during genome duplication; MMR rectifies polymerase misincorporation errors, whereas HR contributes to replication fork maintenance, as well as the repair of spontaneous DSBs and genotoxic lesions that affect both DNA strands. MMR suppresses HR when the heteroduplex DNA contains excessive mismatched nucleotides, termed homeologous recombination. The regulation of homeologous recombination by MMR ensures the accuracy of DSB repair and significantly contributes to species barriers during sexual reproduction. This review discusses the history, genetics, biochemistry, biophysics, and the current state of studies on the role of MMR in homologous and homeologous recombination from bacteria to humans.

  8. Mismatch Repair during Homologous and Homeologous Recombination

    PubMed Central

    Spies, Maria; Fishel, Richard

    2015-01-01

    Homologous recombination (HR) and mismatch repair (MMR) are inextricably linked. HR pairs homologous chromosomes before meiosis I and is ultimately responsible for generating genetic diversity during sexual reproduction. HR is initiated in meiosis by numerous programmed DNA double-strand breaks (DSBs; several hundred in mammals). A characteristic feature of HR is the exchange of DNA strands, which results in the formation of heteroduplex DNA. Mismatched nucleotides arise in heteroduplex DNA because the participating parental chromosomes contain nonidentical sequences. These mismatched nucleotides may be processed by MMR, resulting in nonreciprocal exchange of genetic information (gene conversion). MMR and HR also play prominent roles in mitotic cells during genome duplication; MMR rectifies polymerase misincorporation errors, whereas HR contributes to replication fork maintenance, as well as the repair of spontaneous DSBs and genotoxic lesions that affect both DNA strands. MMR suppresses HR when the heteroduplex DNA contains excessive mismatched nucleotides, termed homeologous recombination. The regulation of homeologous recombination by MMR ensures the accuracy of DSB repair and significantly contributes to species barriers during sexual reproduction. This review discusses the history, genetics, biochemistry, biophysics, and the current state of studies on the role of MMR in homologous and homeologous recombination from bacteria to humans. PMID:25731766

  9. Human DNA repair and recombination genes

    SciTech Connect

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

    1988-09-01

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

  10. Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

    PubMed Central

    Kuzminov, Andrei

    1999-01-01

    Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation. PMID:10585965

  11. Mismatch repair proteins: key regulators of genetic recombination.

    PubMed

    Surtees, J A; Argueso, J L; Alani, E

    2004-01-01

    Mismatch repair (MMR) systems are central to maintaining genome stability in prokaryotes and eukaryotes. MMR proteins play a fundamental role in avoiding mutations, primarily by removing misincorporation errors that occur during DNA replication. MMR proteins also act during genetic recombination in steps that include repairing mismatches in heteroduplex DNA, modulating meiotic crossover control, removing 3' non-homologous tails during double-strand break repair, and preventing recombination between divergent sequences. In this review we will, first, discuss roles for MMR proteins in repairing mismatches that occur during recombination, particularly during meiosis. We will also explore how studying this process has helped to refine models of double-strand break repair, and particularly to our understanding of gene conversion gradients. Second, we will examine the role of MMR proteins in repressing homeologous recombination, i.e. recombination between divergent sequences. We will also compare the requirements for MMR proteins in preventing homeologous recombination to the requirements for these proteins in mismatch repair.

  12. Signaling Pathways in Cartilage Repair

    PubMed Central

    Mariani, Erminia; Pulsatelli, Lia; Facchini, Andrea

    2014-01-01

    In adult healthy cartilage, chondrocytes are in a quiescent phase characterized by a fine balance between anabolic and catabolic activities. In ageing, degenerative joint diseases and traumatic injuries of cartilage, a loss of homeostatic conditions and an up-regulation of catabolic pathways occur. Since cartilage differentiation and maintenance of homeostasis are finely tuned by a complex network of signaling molecules and biophysical factors, shedding light on these mechanisms appears to be extremely relevant for both the identification of pathogenic key factors, as specific therapeutic targets, and the development of biological approaches for cartilage regeneration. This review will focus on the main signaling pathways that can activate cellular and molecular processes, regulating the functional behavior of cartilage in both physiological and pathological conditions. These networks may be relevant in the crosstalk among joint compartments and increased knowledge in this field may lead to the development of more effective strategies for inducing cartilage repair. PMID:24837833

  13. On the mutagenicity of homologous recombination and double-strand break repair in bacteriophage.

    PubMed

    Shcherbakov, Victor P; Plugina, Lidiya; Shcherbakova, Tamara; Sizova, Svetlana; Kudryashova, Elena

    2011-01-02

    The double-strand break (DSB) repair via homologous recombination is generally construed as a high-fidelity process. However, some molecular genetic observations show that the recombination and the recombinational DSB repair may be mutagenic and even highly mutagenic. Here we developed an effective and precise method for studying the fidelity of DSB repair in vivo by combining DSBs produced site-specifically by the SegC endonuclease with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. The method is based on the comparison of the rate of reversion of rII mutation in the presence and in the absence of a DSB repair event initiated in the proximity of the mutation. We observed that DSB repair may moderately (up to 6-fold) increase the apparent reversion frequency, the effect of being dependent on the mutation structure. We also studied the effect of the T4 recombinase deficiency (amber mutation in the uvsX gene) on the fidelity of DSB repair. We observed that DSBs are still repaired via homologous recombination in the uvsX mutants, and the apparent fidelity of this repair is higher than that seen in the wild-type background. The mutator effect of the DSB repair may look unexpected given that most of the normal DNA synthesis in bacteriophage T4 is performed via a recombination-dependent replication (RDR) pathway, which is thought to be indistinguishable from DSB repair. There are three possible explanations for the observed mutagenicity of DSB repair: (1) the origin-dependent (early) DNA replication may be more accurate than the RDR; (2) the step of replication initiation may be more mutagenic than the process of elongation; and (3) the apparent mutagenicity may just reflect some non-randomness in the pool of replicating DNA, i.e., preferential replication of the sequences already involved in replication. We discuss the DSB repair pathway in the absence of UvsX recombinase.

  14. Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae

    PubMed Central

    Pâques, Frédéric; Haber, James E.

    1999-01-01

    The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination. PMID:10357855

  15. Impairment of the non-homologous end joining and homologous recombination pathways of DNA double strand break repair: Impact on spontaneous and radiation-induced mammary and intestinal tumour risk in Apc min/+ mice.

    PubMed

    Haines, Jackie W; Coster, Margaret; Bouffler, Simon D

    2015-11-01

    Female Apc(min/+) mice carrying the BALB/c variant of Prkdc or heterozygous knockout for Xrcc2, were sham- or 2 Gy X-irradiated as adults to compare the effect of mild impairments of double-strand break (DSB) repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR) respectively on spontaneous and radiation-induced mammary and intestinal tumorigenesis. Mice with impaired NHEJ showed no difference in incidence of spontaneous mammary tumours, compared with matched controls, (2.46 fold, P=0.121) and significantly less following irradiation (radiation-induced excess; 0.35 fold, P=0.008). In contrast mice with impaired HR presented with significantly less spontaneous mammary tumours than matched controls (0.33 fold, P=0.027) and significantly more following irradiation (radiation-induced excess; 3.3 fold, P=0.016). Spontaneous and radiation-induced intestinal adenoma multiplicity in the same groups were significantly greater than matched controls for mice with impaired NHEJ (sham; 1.29 fold, P<0.001, radiation-induced excess; 2.55 fold, P<0.001) and mice with impaired HR showed no significant differences (sham; 0.92 fold, P=0.166, radiation-induced excess; 1.16, P=0.274). Genetic insertion events were common in spontaneous tumours from NHEJ impaired mice compared with matched controls. γH2AX foci analysis suggests a significantly faster rate of DSB repair (MANOVA P<0.001) in intestinal than mammary tissue; apoptosis was also higher in irradiated intestine. To conclude, results suggest that pathway of choice for repair of spontaneous and radiation-induced DSBs is influenced by tissue type. NHEJ appears to play a greater role in DSB repair in intestinal tissue since impairment by functional change of Prkdc significantly increases the rate of mis-repair in intestinal but not mammary tissue. HR appears to play a greater role in DSB repair in adult mammary tissue since impaired HR results in significant changes in mammary but not in the intestinal

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

    PubMed

    Weiner, Allon; Zauberman, Nathan; Minsky, Abraham

    2009-10-01

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

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

    PubMed

    Rupp, W Dean

    2013-12-13

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

  18. DNA double-strand break repair in Penaeus monodon is predominantly dependent on homologous recombination.

    PubMed

    Srivastava, Shikha; Dahal, Sumedha; Naidu, Sharanya J; Anand, Deepika; Gopalakrishnan, Vidya; Kooloth Valappil, Rajendran; Raghavan, Sathees C

    2017-01-24

    DNA double-strand breaks (DSBs) are mostly repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) in higher eukaryotes. In contrast, HR-mediated DSB repair is the major double-strand break repair pathway in lower order organisms such as bacteria and yeast. Penaeus monodon, commonly known as black tiger shrimp, is one of the economically important crustaceans facing large-scale mortality due to exposure to infectious diseases. The animals can also get exposed to chemical mutagens under the culture conditions as well as in wild. Although DSB repair mechanisms have been described in mammals and some invertebrates, its mechanism is unknown in the shrimp species. In the present study, we show that HR-mediated DSB repair is the predominant mode of repair in P. monodon Robust repair was observed at a temperature of 30 °C, when 2 µg of cell-free extract derived from hepatopancreas was used for the study. Although HR occurred through both reciprocal recombination and gene conversion, the latter was predominant when the bacterial colonies containing recombinants were evaluated. Unlike mammals, NHEJ-mediated DSB repair was undetectable in P. monodon However, we could detect evidence for an alternative mode of NHEJ that uses microhomology, termed as microhomology-mediated end joining (MMEJ). Interestingly, unlike HR, MMEJ was predominant at lower temperatures. Therefore, the results suggest that, while HR is major DSB repair pathway in shrimp, MMEJ also plays a role in ensuring the continuity and stability of the genome.

  19. Multifactorial Resistance of Bacillus subtilis Spores to High-Energy Proton Radiation: Role of Spore Structural Components and the Homologous Recombination and Non-Homologous End Joining DNA Repair Pathways

    PubMed Central

    Reitz, Günther; Li, Zuofeng; Klein, Stuart; Nicholson, Wayne L.

    2012-01-01

    Abstract The space environment contains high-energy charged particles (e.g., protons, neutrons, electrons, α-particles, heavy ions) emitted by the Sun and galactic sources or trapped in the radiation belts. Protons constitute the majority (87%) of high-energy charged particles. Spores of Bacillus species are one of the model systems used for astro- and radiobiological studies. In this study, spores of different Bacillus subtilis strains were used to study the effects of high energetic proton irradiation on spore survival. Spores of the wild-type B. subtilis strain [mutants deficient in the homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways and mutants deficient in various spore structural components such as dipicolinic acid (DPA), α/β-type small, acid-soluble spore protein (SASP) formation, spore coats, pigmentation, or spore core water content] were irradiated as air-dried multilayers on spacecraft-qualified aluminum coupons with 218 MeV protons [with a linear energy transfer (LET) of 0.4 keV/μm] to various final doses up to 2500 Gy. Spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to proton radiation than wild-type spores, indicating that both HR and NHEJ DNA repair pathways are needed for spore survival. Spores lacking DPA, α/β-type SASP, or with increased core water content were also significantly more sensitive to proton radiation, whereas the resistance of spores lacking pigmentation or spore coats was essentially identical to that of the wild-type spores. Our results indicate that α/β-type SASP, core water content, and DPA play an important role in spore resistance to high-energy proton irradiation, suggesting their essential function as radioprotectants of the spore interior. Key Words: Bacillus—Spores—DNA repair—Protection—High-energy proton radiation. Astrobiology 12, 1069–1077. PMID:23088412

  20. Multifactorial resistance of Bacillus subtilis spores to high-energy proton radiation: role of spore structural components and the homologous recombination and non-homologous end joining DNA repair pathways.

    PubMed

    Moeller, Ralf; Reitz, Günther; Li, Zuofeng; Klein, Stuart; Nicholson, Wayne L

    2012-11-01

    The space environment contains high-energy charged particles (e.g., protons, neutrons, electrons, α-particles, heavy ions) emitted by the Sun and galactic sources or trapped in the radiation belts. Protons constitute the majority (87%) of high-energy charged particles. Spores of Bacillus species are one of the model systems used for astro- and radiobiological studies. In this study, spores of different Bacillus subtilis strains were used to study the effects of high energetic proton irradiation on spore survival. Spores of the wild-type B. subtilis strain [mutants deficient in the homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways and mutants deficient in various spore structural components such as dipicolinic acid (DPA), α/β-type small, acid-soluble spore protein (SASP) formation, spore coats, pigmentation, or spore core water content] were irradiated as air-dried multilayers on spacecraft-qualified aluminum coupons with 218 MeV protons [with a linear energy transfer (LET) of 0.4 keV/μm] to various final doses up to 2500 Gy. Spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to proton radiation than wild-type spores, indicating that both HR and NHEJ DNA repair pathways are needed for spore survival. Spores lacking DPA, α/β-type SASP, or with increased core water content were also significantly more sensitive to proton radiation, whereas the resistance of spores lacking pigmentation or spore coats was essentially identical to that of the wild-type spores. Our results indicate that α/β-type SASP, core water content, and DPA play an important role in spore resistance to high-energy proton irradiation, suggesting their essential function as radioprotectants of the spore interior.

  1. DNA repair pathways in human multiple myeloma

    PubMed Central

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

    2013-01-01

    Every day, cells are faced with thousands of DNA lesions, which have to be repaired to preserve cell survival and function. DNA repair is more or less accurate and could result in genomic instability and cancer. We review here the current knowledge of the links between molecular features, treatment, and DNA repair in multiple myeloma (MM), a disease characterized by the accumulation of malignant plasma cells producing a monoclonal immunoglobulin. Genetic instability and abnormalities are two hallmarks of MM cells and aberrant DNA repair pathways are involved in disease onset, primary translocations in MM cells, and MM progression. Two major drugs currently used to treat MM, the alkylating agent Melphalan and the proteasome inhibitor Bortezomib act directly on DNA repair pathways, which are involved in response to treatment and resistance. A better knowledge of DNA repair pathways in MM could help to target them, thus improving disease treatment. PMID:23966156

  2. Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates.

    PubMed

    Guo, Xiaoge; Jinks-Robertson, Sue

    2013-12-01

    Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the collateral

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

    PubMed

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

    2011-05-15

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

  4. Inhibition of spontaneous mutagenesis by vanillin and cinnamaldehyde in Escherichia coli: Dependence on recombinational repair.

    PubMed

    Shaughnessy, Daniel T; Schaaper, Roel M; Umbach, David M; DeMarini, David M

    2006-12-01

    Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that effectively inhibit both induced and spontaneous mutations. We have shown previously that VAN and CIN reduced the spontaneous mutant frequency in Salmonella TA104 (hisG428, rfa, DeltauvrB, pKM101) by approximately 50% and that both compounds significantly reduced mutations at GC sites but not at AT sites. Previous studies have suggested that VAN and CIN may reduce mutations in bacterial model systems by modulating DNA repair pathways, particularly by enhancing recombinational repair. To further explore the basis for inhibition of spontaneous mutation by VAN and CIN, we have determined the effects of these compounds on survival and mutant frequency in five Escherichia coli strains derived from the wild-type strain NR9102 with different DNA repair backgrounds. At nontoxic doses, both VAN and CIN significantly reduced mutant frequency in the wild-type strain NR9102, in the nucleotide excision repair-deficient strain NR11634 (uvrB), and in the recombination-proficient but SOS-deficient strain NR11475 (recA430). In contrast, in the recombination-deficient and SOS-deficient strain NR11317 (recA56), both VAN and CIN not only failed to inhibit the spontaneous mutant frequency but actually increased the mutant frequency. In the mismatch repair-defective strain NR9319 (mutL), only CIN was antimutagenic. Our results show that the antimutagenicity of VAN and CIN against spontaneous mutation required the RecA recombination function but was independent of the SOS and nucleotide excision repair pathways. Thus, we propose the counterintuitive notion that these antimutagens actually produce a type of DNA damage that elicits recombinational repair (but not mismatch, SOS, or nucleotide excision repair), which then repairs not only the damage induced by VAN and CIN but also other DNA damage-resulting in an antimutagenic effect on spontaneous mutation.

  5. Inhibition of spontaneous mutagenesis by vanillin and cinnamaldehyde in Escherichia coli: Dependence on recombinational repair

    PubMed Central

    Shaughnessy, Daniel T.; Schaaper, Roel M.; Umbach, David M.; DeMarini, David M.

    2007-01-01

    Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that effectively inhibit both induced and spontaneous mutations. We have shown previously that VAN and CIN reduced the spontaneous mutant frequency in Salmonella TA104 (hisG428, rfa, ΔuvrB, pKM101) by approximately 50% and that both compounds significantly reduced mutations at GC sites but not at AT sites. Previous studies have suggested that VAN and CIN may reduce mutations in bacterial model systems by modulating DNA repair pathways, particularly by enhancing recombinational repair. To further explore the basis for inhibition of spontaneous mutation by VAN and CIN, we have determined the effects of these compounds on survival and mutant frequency in five Escherichia coli strains derived from the wild-type strain NR9102 with different DNA repair backgrounds. At nontoxic doses, both VAN and CIN significantly reduced mutant frequency in the wild-type strain NR9102, in the nucleotide excision repair-deficient strain NR11634 (uvrB), and in the recombination-proficient but SOS-deficient strain NR11475 (recA430). In contrast, in the recombination-deficient and SOS-deficient strain NR11317 (recA56), both VAN and CIN not only failed to inhibit the spontaneous mutant frequency but actually increased the mutant frequency. In the mismatch repair-defective strain NR9319 (mutL), only CIN was antimutagenic. Our results show that the antimutagenicity of VAN and CIN against spontaneous mutation required the RecA recombination function but was independent of the SOS and nucleotide excision repair pathways. Thus, we propose the counterintuitive notion that these antimutagens actually produce a type of DNA damage that elicits recombinational repair (but not mismatch, SOS, or nucleotide excision repair), which then repairs not only the damage induced by VAN and CIN but also other DNA damage—resulting in an antimutagenic effect on spontaneous mutation. PMID:16999979

  6. An alternative eukaryotic DNA excision repair pathway.

    PubMed Central

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

    1995-01-01

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

  7. DNA helicases in recombination and repair: construction of a delta uvrD delta helD delta recQ mutant deficient in recombination and repair.

    PubMed Central

    Mendonca, V M; Klepin, H D; Matson, S W

    1995-01-01

    DNA helicases play pivotal roles in homologous recombination and recombinational DNA repair. They are involved in both the generation of recombinogenic single-stranded DNA ends and branch migration of synapsed Holliday junctions. Escherichia coli helicases II (uvrD), IV (helD), and RecQ (recQ) have all been implicated in the presynaptic stage of recombination in the RecF pathway. To probe for functional redundancy among these helicases, mutant strains containing single, double, and triple deletions in the helD, uvrD, and recQ genes were constructed and examined for conjugational recombination efficiency and DNA repair proficiency. We were unable to construct a strain harboring a delta recQ delta uvrD double deletion in a recBC sbcB(C) background (RecF pathway), suggesting that a delta recQ deletion mutation was lethal to the cell in a recBC sbcB(C) delta D background. However, we were able to construct a triple delta recQ delta uvrD Delta helD mutant in the recBC sbcB(C) background. This may be due to the increased mutator frequency in delta uvrD mutants which may have resulted in the fortuitous accumulation of a suppressor mutation(s). The triple helicase mutant recBC sbcB(C) delta uvrD delta recQ delta helD severely deficient in Hfr-mediated conjugational recombination and in the repair of methylmethane sulfonate-induced DNA damage. This suggests that the presence of at least one helicase--helicase II, RecQ helicase, or helicase IV--is essential for homologous recombination and recombinational DNA repair in a recBC sbcB(C) background. The triple helicase mutant was recombination and repair proficient in a rec+ background. Genetic analysis of the various double mutants unmasked additional functional redundancies with regard to conjugational recombination and DNA repair, suggesting that mechanisms of recombination depend both on the DNA substrates and on the genotype of the cell. PMID:7868608

  8. The effect of caffeine on repair in Chlamydomonas reinhardtii. I. Enhancement of recombination repair.

    PubMed

    Rosen, H; Rehn, M M; Johnson, B A

    1980-05-01

    The effect of caffeine on repair was studied in the green alga Chlamydomonas reinhardtii. Treatment of UV-irradiated wild-type (UVS+) cells with a sublethal level of caffeine caused a significant increase in survival compared to untreated UV-irradiated cells. Caffeine did not affect survival in the repair-deficient strain UVSE1, which is deficient in repair of UV-induced damage carried out by enzymes associated with recombination during meiosis. A significant increase in survival in the presence of caffeine was observed in the repair-deficient strain UVSE4 in which recombination during meiosis is not affected. Treatment of zygotes homozygous for UVS+, UVSE1, or UVSE4 with sublethal levels of caffeine caused marked increases in recombination frequency in UVS+ and UVSE4 zygotes and no increase in recombination in UVSE1 zygotes. These results indicate that caffeine increases recombination in normal strains. Increased opportunity for recombination caused by caffeine would not result in increased recombination frequency in the UVSE1 strain, assuming limited-recombination enzyme activity in this strain. The observed increase in survival following UV-irradiation in the presence of caffeine in strains having normal recombination would therefore be associated with a caffeine-induced increase in opportunities for recombination repair.

  9. Exploiting Synthetic Lethal Relationships: Chemical Inhibition of Recombinational Repair as a Strategy to Selectively Target Tumor Cells

    DTIC Science & Technology

    2011-03-01

    14. ABSTRACT Homologous recombination is a key cellular pathway to repair or tolerate complex DNA damage such as DNA double-stranded breaks...interstrand DNA crosslinks, or single-stranded DNA gaps. In addition, homologous recombination is required for the recovery of stalled or broken...interstrand crosslinkers) agents as principal modalities in anti-tumor therapy. The importance of the homologous recombination pathway for breast cancer

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

    PubMed

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

    2014-11-20

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-12-15

    PARP inhibitors (PARPis) are being used in patients with BRCA1/2 mutations. However, doubly deficient BRCA1(-/-)53BP1(-/-) cells or tumors become resistant to PARPis. Since 53BP1 or its known downstream effectors, PTIP and RIF1 (RAP1-interacting factor 1 homolog), lack enzymatic activities directly implicated in DNA repair, we decided to further explore the 53BP1-dependent pathway. In this study, we uncovered a nuclease, Artemis, as a PTIP-binding protein. Loss of Artemis restores PARPi resistance in BRCA1-deficient cells. Collectively, our data demonstrate that Artemis is the major downstream effector of the 53BP1 pathway, which prevents end resection and promotes nonhomologous end-joining and therefore directly competes with the homologous recombination repair pathway. © 2014 Wang et al.; Published by Cold Spring Harbor Laboratory Press.

  13. Endonucleases involved in repair and recombination of DNA

    SciTech Connect

    Linn, S.M.

    1988-01-01

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

  14. Extensive Interallelic Polymorphisms Drive Meiotic Recombination into a Crossover Pathway

    PubMed Central

    Dooner, Hugo K.

    2002-01-01

    Recombinants isolated from most meiotic intragenic recombination experiments in maize, but not in yeast, are borne principally on crossover chromosomes. This excess of crossovers is not explained readily by the canonical double-strand break repair model of recombination, proposed to account for a large body of yeast data, which predicts that crossovers (COs) and noncrossovers (NCOs) should be recovered equally. An attempt has been made here to identify general rules governing the recovery of the CO and NCO classes of intragenic recombinants in maize. Recombination was analyzed in bz heterozygotes between a variety of mutations derived from the same or different progenitor alleles. The mutations include point mutations, transposon insertions, and transposon excision footprints. Consequently, the differences between the bz heteroalleles ranged from just two nucleotides to many nucleotides, indels, and insertions. In this article, allelic pairs differing at only two positions are referred to as dimorphic to distinguish them from polymorphic pairs, which differ at multiple positions. The present study has revealed the following effects at these bz heteroalleles: (1) recombination between polymorphic heteroalleles produces mostly CO chromosomes; (2) recombination between dimorphic heteroalleles produces both CO and NCO chromosomes, in ratios apparently dependent on the nature of the heteroalleles; and (3) in dimorphic heterozygotes, the two NCO classes are recovered in approximately equal numbers when the two mutations are point mutations but not when one or both mutations are insertions. These observations are discussed in light of a recent version of the double-strand break repair model of recombination that postulates separate pathways for the formation of CO and NCO products. PMID:12034905

  15. Restriction-Stimulated Homologous Recombination of Plasmids by the Rece Pathway of Escherichia Coli

    PubMed Central

    Nussbaum, A.; Shalit, M.; Cohen, A.

    1992-01-01

    To test the double-strand break (DSB) repair model in recombination by the RecE pathway of Escherichia coli, we constructed chimeric phages that allow restriction-mediated release of linear plasmid substrates of the bioluminescence recombination assay in infected EcoRI(+) cells. Kinetics of DSB repair and expression of recombination products were followed by Southern hybridization and by the bioluminescence recombination assay, respectively. Plasmid recombinants were analyzed with restriction endonucleases. Our results indicate that a DSB can induce more than one type of RecE-mediated recombination. A DSB within the homology induced intermolecular recombination that followed the rules of the DSB repair model: (1) Recombination was enhanced by in vivo restriction. (2) Repair of the break depended on homologous sequences on the resident plasmid. (3) Break-repair was frequently associated with conversion of alleles that were cis to the break. (4) Conversion frequency decreased as the distance from the break increased. (5) Some clones contained a mixture of plasmid recombinants as expected by replication of a heteroduplex in the primary recombinant. The rules of the DSB repair model were not followed when recombination was induced by a DSB outside the homology. Both the cut and the uncut substrates were recipients in conversion events. Recombination events were associated with deletions that spanned the break site, but these deletions did not reach the homology. We propose that a break outside the homology may stimulate a RecE-mediated recombination pathway that does not involve direct participation of DNA ends in the homologous pairing reaction. PMID:1732167

  16. DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

    PubMed Central

    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

  17. Single-stranded oligonucleotide-mediated gene repair in mammalian cells has a mechanism distinct from homologous recombination repair.

    PubMed

    Wang, Zai; Zhou, Zhong-Jun; Liu, De-Pei; Huang, Jian-Dong

    2006-11-24

    Single-stranded DNA oligonucleotide (SSO)-mediated gene repair has great potentials for gene therapy and functional genomic studies. However, its underlying mechanism remains unclear. Previous studies from other groups have suggested that DNA damage response via the ATM/ATR pathway may be involved in this process. In this study, we measured the effect of two ATM/ATR inhibitors caffeine and pentoxifylline on the correction efficiency in SSO-mediated gene repair. We also checked their effect on double-stranded break (DSB)-induced homologous recombination repair (HRR) as a control, which is well known to be dependent on the ATM/ATR pathway. We found these inhibitors could completely inhibit DSB-induced HRR, but could only partially inhibit SSO-mediated process, indicating SSO-mediated gene repair is not dependent on the ATM/ATR pathway. Furthermore, we found that thymidine treatment promotes SSO-mediated gene repair, but inhibits DSB-induced HRR. Collectively, our results demonstrate that SSO-mediated and DSB-induced gene repairs have distinct mechanisms.

  18. Aging impairs double-strand break repair by homologous recombination in Drosophila germ cells.

    PubMed

    Delabaere, Laetitia; Ertl, Henry A; Massey, Dashiell J; Hofley, Carolyn M; Sohail, Faraz; Bienenstock, Elisa J; Sebastian, Hans; Chiolo, Irene; LaRocque, Jeannine R

    2017-04-01

    Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double-strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error-free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I-SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR-white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, we propose that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  19. DNA double-strand break repair in Penaeus monodon is predominantly dependent on homologous recombination.

    PubMed

    Srivastava, Shikha; Dahal, Sumedha; Naidu, Sharanya J; Anand, Deepika; Gopalakrishnan, Vidya; Kooloth Valappil, Rajendran; Raghavan, Sathees C

    2017-04-01

    DNA double-strand breaks (DSBs) are mostly repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) in higher eukaryotes. In contrast, HR-mediated DSB repair is the major double-strand break repair pathway in lower order organisms such as bacteria and yeast. Penaeus monodon, commonly known as black tiger shrimp, is one of the economically important crustaceans facing large-scale mortality due to exposure to infectious diseases. The animals can also get exposed to chemical mutagens under the culture conditions as well as in wild. Although DSB repair mechanisms have been described in mammals and some invertebrates, its mechanism is unknown in the shrimp species. In the present study, we show that HR-mediated DSB repair is the predominant mode of repair in P. monodon. Robust repair was observed at a temperature of 30 °C, when 2 µg of cell-free extract derived from hepatopancreas was used for the study. Although HR occurred through both reciprocal recombination and gene conversion, the latter was predominant when the bacterial colonies containing recombinants were evaluated. Unlike mammals, NHEJ-mediated DSB repair was undetectable in P. monodon. However, we could detect evidence for an alternative mode of NHEJ that uses microhomology, termed as microhomology-mediated end joining (MMEJ). Interestingly, unlike HR, MMEJ was predominant at lower temperatures. Therefore, the results suggest that, while HR is major DSB repair pathway in shrimp, MMEJ also plays a role in ensuring the continuity and stability of the genome. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  20. A Life Investigating Pathways That Repair Broken Chromosomes.

    PubMed

    Haber, James E

    2016-11-23

    Double-strand breaks (DSBs) pose a severe challenge to genome integrity; consequently, cells have developed efficient mechanisms to repair DSBs through several pathways of homologous recombination and other nonhomologous end-joining processes. Much of our understanding of these pathways has come from the analysis of site-specific DSBs created by the HO endonuclease in the budding yeast Saccharomyces cerevisiae. I was fortunate to get in on the ground floor of analyzing the fate of synchronously induced DSBs through the study of what I coined "in vivo biochemistry." I have had the remarkable good fortune to profit from the development of new techniques that have permitted an ever more detailed dissection of these repair mechanisms, which are described here.

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

    PubMed Central

    Coleman, Kate E.; Huang, Tony T.

    2016-01-01

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

  2. Characterization of Bacillus subtilis recombinational pathways.

    PubMed Central

    Alonso, J C; Lüder, G; Tailor, R H

    1991-01-01

    Recombination in Bacillus subtilis requires the products of numerous rec loci. To dissect the various mechanisms which may be involved in genetic recombination, we constructed a series of isogenic strains containing more than one mutant rec allele. On the basis of their impairment in genetic exchange, the various loci (represented by specific rec alleles) were classified into different epistatic groups. Group alpha consists of rec genes represented by recB, recD, recF, recG, recL, and recR mutations, while group beta comprises the addA and addB mutations. Group gamma consists of the recH and recP mutations. These results suggest that B. subtilis has multiple pathways for genetic recombination and that the products of the genes within the alpha, beta, and gamma epistatic groups are involved in these alternative recombination pathways. The RecA protein is required in all three pathways of intermolecular recombination. PMID:1905712

  3. The Functions of BRCA2 in Homologous Recombinational Repair

    DTIC Science & Technology

    2004-07-01

    February 2004. 2. A manuscript related to this project entitled "Human Rad51C deficiency destabilizes XRCC3 , impairs recombination and radiosensitizes...Cell Biol 15: 1968-1973 15. Brenneman MA et al. (2000) XRCC3 is required for efficient repair of chromosome breaks by homologous recombination. Mutat...JBiol Chem 278: 2469-2478 APPENDICES 1. Lio, Y-C, Schild, D, Brenneman, MA, Redpath, JL, and Chen, DJ (2004) Human RadS1C deficiency destabilizes XRCC3

  4. The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination.

    PubMed

    Burgess, Rebecca C; Rahman, Sadia; Lisby, Michael; Rothstein, Rodney; Zhao, Xiaolan

    2007-09-01

    Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to replication centers. In addition, like SUMO E2 mutants, slx8Delta mutants exhibited clonal lethality, which was due to the overamplification of 2 microm, an extrachromosomal plasmid. Interestingly, in both SUMO E2 and slx8Delta mutants, clonal lethality was rescued by deleting genes required for Rad51-independent recombination but not those involved in Rad51-dependent events. These results suggest that sumoylation negatively regulates Rad51-independent recombination, and indeed, the Slx5-Slx8 complex affected the sumoylation of several enzymes involved in early steps of Rad51-independent recombination. We propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2010-07-09

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

  8. Fhit and CHK1 have opposing effects on homologous recombination repair.

    PubMed

    Hu, Baocheng; Wang, Hongyan; Wang, Xiang; Lu, Hua-Rui; Huang, Cuifen; Powell, Simon N; Huebner, Kay; Wang, Ya

    2005-10-01

    Fragile histidine triad (FHIT) gene deletion or promoter methylation and reduced Fhit protein expression occur in approximately 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified. We previously reported that compared with Fhit+/+ cells, Fhit-/- cells with an overactivated ATR/CHK1 pathway show increased mutation frequency and resistance to DNA damage-induced killing, indicating that Fhit and the CHK1 pathway have opposing roles in cells responding to DNA damage. In this study, we show that cells, with or without Fhit expression, have similar DNA double-strand break induction levels and similar rejoining rates following ionizing radiation, indicating that the effect of Fhit on cell radiosensitivity is independent of nonhomologous end-joining. By combining I-SceI-induced-DNA double-strand break system and small interfering RNA approach, we also show that knocking down Fhit increases the efficiency of homologous recombination repair of cells, but knocking down Chk1 decreases the efficiency of homologous recombination repair, associated with the sensitivity to ionizing radiation-induced killing. Taken together, the results show that the role of Fhit in affecting the sensitivity of cells to ionizing radiation-induced killing is through the CHK1 pathway linked to homologous recombination repair. These results also illustrate the importance of balanced checkpoint activation in genomic stability and suggest a connection between the radioresistance and mutagenesis, carcinogenesis, as well as tumor progression in Fhit-deficient cells or tissue.

  9. Induction of direct repeat recombination by psoralen-DNA adducts in Saccharomyces cerevisiae: defects in DNA repair increase gene copy number variation.

    PubMed

    Saffran, Wilma A; Ahmed, Anam; Binyaminov, Olga; Gonzalez, Cynthia; Gupta, Amita; Fajardo, Manuel A; Kishun, Devindra; Nandram, Ashana; Reyes, Kenneth; Scalercio, Karina; Senior, Charles W

    2014-09-01

    Psoralen photoreaction produces covalent monoadducts and interstrand crosslinks in DNA. The interstrand DNA crosslinks are complex double strand lesions that require the involvement of multiple pathways for repair. Homologous recombination, which can carry out error-free repair, is a major pathway for crosslink repair; however, some recombination pathways can also produce DNA rearrangements. Psoralen photoreaction-induced recombination in yeast was measured using direct repeat substrates that can detect gene conversions, a form of conservative recombination, as well as deletions and triplications, which generate gene copy number changes. In repair-proficient cells the major products of recombination were gene conversions, along with substantial fractions of deletions. Deficiencies in DNA repair pathways increased non-conservative recombination products. Homologous recombination-deficient rad51, rad54, and rad57 strains had low levels of crosslink-induced recombination, and most products were deletions produced by single strand annealing. Nucleotide excision repair-deficient rad1 and rad2 yeast had increased levels of triplications, and rad1 cells had lower crosslink-induced recombination. Deficiencies in post-replication repair increased crosslink-induced recombination and gene copy number changes. Loss of REV3 function, in the error-prone branch, and of RAD5 and UBC13, in the error-free branch, produced moderate increases in deletions and triplications; rad18 cells, deficient in both post-replication repair sub-pathways, exhibited hyperrecombination, with primarily non-conservative products. Proper functioning of all the DNA repair pathways tested was required to maintain genomic stability and avoid gene copy number variation in response to interstrand crosslinks. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Aberrant recombination and repair during immunoglobulin class switching in BRCA1-deficient human B cells.

    PubMed

    Björkman, Andrea; Qvist, Per; Du, Likun; Bartish, Margarita; Zaravinos, Apostolos; Georgiou, Konstantinos; Børglum, Anders D; Gatti, Richard A; Törngren, Therese; Pan-Hammarström, Qiang

    2015-02-17

    Breast cancer type 1 susceptibility protein (BRCA1) has a multitude of functions that contribute to genome integrity and tumor suppression. Its participation in the repair of DNA double-strand breaks (DSBs) during homologous recombination (HR) is well recognized, whereas its involvement in the second major DSB repair pathway, nonhomologous end-joining (NHEJ), remains controversial. Here we have studied the role of BRCA1 in the repair of DSBs in switch (S) regions during immunoglobulin class switch recombination, a physiological, deletion/recombination process that relies on the classical NHEJ machinery. A shift to the use of microhomology-based, alternative end-joining (A-EJ) and increased frequencies of intra-S region deletions as well as insertions of inverted S sequences were observed at the recombination junctions amplified from BRCA1-deficient human B cells. Furthermore, increased use of long microhomologies was found at recombination junctions derived from E3 ubiquitin-protein ligase RNF168-deficient, Fanconi anemia group J protein (FACJ, BRIP1)-deficient, or DNA endonuclease RBBP8 (CtIP)-compromised cells, whereas an increased frequency of S-region inversions was observed in breast cancer type 2 susceptibility protein (BRCA2)-deficient cells. Thus, BRCA1, together with its interaction partners, seems to play an important role in repairing DSBs generated during class switch recombination by promoting the classical NHEJ pathway. This may not only provide a general mechanism underlying BRCA1's function in maintaining genome stability and tumor suppression but may also point to a previously unrecognized role of BRCA1 in B-cell lymphomagenesis.

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

    PubMed Central

    2017-01-01

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

  12. The mismatch repair system reduces meiotic homeologous recombination and stimulates recombination-dependent chromosome loss.

    PubMed Central

    Chambers, S R; Hunter, N; Louis, E J; Borts, R H

    1996-01-01

    Efficient genetic recombination requires near-perfect homology between participating molecules. Sequence divergence reduces the frequency of recombination, a process that is dependent on the activity of the mismatch repair system. The effects of chromosomal divergence in diploids of Saccharomyces cerevisiae in which one copy of chromosome III is derived from a closely related species, Saccharomyces paradoxus, have been examined. Meiotic recombination between the diverged chromosomes is decreased by 25-fold. Spore viability is reduced with an observable increase in the number of tetrads with only two or three viable spores. Asci with only two viable spores are disomic for chromosome III, consistent with meiosis I nondisjunction of the homeologs. Asci with three viable spores are highly enriched for recombinants relative to tetrads with four viable spores. In 96% of the class with three viable spores, only one spore possesses a recombinant chromosome III, suggesting that the recombination process itself contributes to meiotic death. This phenomenon is dependent on the activities of the mismatch repair genes PMS1 and MSH2. A model of mismatch-stimulated chromosome loss is proposed to account for this observation. As expected, crossing over is increased in pms1 and msh2 mutants. Furthermore, genetic exchange in pms1 msh2 double mutants is affected to a greater extent than in either mutant alone, suggesting that the two proteins act independently to inhibit homeologous recombination. All mismatch repair-deficient strains exhibited reductions in the rate of chromosome III nondisjunction. PMID:8887641

  13. Mdt1 Facilitates Efficient Repair of Blocked DNA Double-Strand Breaks and Recombinational Maintenance of Telomeres▿

    PubMed Central

    Pike, Brietta L.; Heierhorst, Jörg

    2007-01-01

    DNA recombination plays critical roles in DNA repair and alternative telomere maintenance. Here we show that absence of the SQ/TQ cluster domain-containing protein Mdt1 (Ybl051c) renders Saccharomyces cerevisiae particularly hypersensitive to bleomycin, a drug that causes 3′-phospho-glycolate-blocked DNA double-strand breaks (DSBs). mdt1Δ also hypersensitizes partially recombination-defective cells to camptothecin-induced 3′-phospho-tyrosyl protein-blocked DSBs. Remarkably, whereas mdt1Δ cells are unable to restore broken chromosomes after bleomycin treatment, they efficiently repair “clean” endonuclease-generated DSBs. Epistasis analyses indicate that MDT1 acts in the repair of bleomycin-induced DSBs by regulating the efficiency of the homologous recombination pathway as well as telomere-related functions of the KU complex. Moreover, mdt1Δ leads to severe synthetic growth defects with a deletion of the recombination facilitator and telomere-positioning factor gene CTF18 already in the absence of exogenous DNA damage. Importantly, mdt1Δ causes a dramatic shift from the usually prevalent type II to the less-efficient type I pathway of recombinational telomere maintenance in the absence of telomerase in liquid senescence assays. As telomeres resemble protein-blocked DSBs, the results indicate that Mdt1 acts in a novel blocked-end-specific recombination pathway that is required for the efficiency of both drug-induced DSB repair and telomerase-independent telomere maintenance. PMID:17636027

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

    PubMed

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

    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.

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

    PubMed Central

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

    2015-01-01

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

  16. Roles of RECQ helicases in recombination based DNA repair, genomic stability and aging

    PubMed Central

    Singh, Dharmendra Kumar; Ahn, Byungchan

    2009-01-01

    The maintenance of the stability of genetic material is an essential feature of every living organism. Organisms across all kingdoms have evolved diverse and highly efficient repair mechanisms to protect the genome from deleterious consequences of various genotoxic factors that might tend to destabilize the integrity of the genome in each generation. One such group of proteins that is actively involved in genome surveillance is the RecQ helicase family. These proteins are highly conserved DNA helicases, which have diverse roles in multiple DNA metabolic processes such as DNA replication, recombination and DNA repair. In humans, five RecQ helicases have been identified and three of them namely, WRN, BLM and RecQL4 have been linked to genetic diseases characterized by genome instability, premature aging and cancer predisposition. This helicase family plays important roles in various DNA repair pathways including protecting the genome from illegitimate recombination during chromosome segregation in mitosis and assuring genome stability. This review mainly focuses on various roles of human RecQ helicases in the process of recombination-based DNA repair to maintain genome stability and physiological consequences of their defects in the development of cancer and premature aging. PMID:19083132

  17. Roles of RECQ helicases in recombination based DNA repair, genomic stability and aging.

    PubMed

    Singh, Dharmendra Kumar; Ahn, Byungchan; Bohr, Vilhelm A

    2009-06-01

    The maintenance of the stability of genetic material is an essential feature of every living organism. Organisms across all kingdoms have evolved diverse and highly efficient repair mechanisms to protect the genome from deleterious consequences of various genotoxic factors that might tend to destabilize the integrity of the genome in each generation. One such group of proteins that is actively involved in genome surveillance is the RecQ helicase family. These proteins are highly conserved DNA helicases, which have diverse roles in multiple DNA metabolic processes such as DNA replication, recombination and DNA repair. In humans, five RecQ helicases have been identified and three of them namely, WRN, BLM and RecQL4 have been linked to genetic diseases characterized by genome instability, premature aging and cancer predisposition. This helicase family plays important roles in various DNA repair pathways including protecting the genome from illegitimate recombination during chromosome segregation in mitosis and assuring genome stability. This review mainly focuses on various roles of human RecQ helicases in the process of recombination-based DNA repair to maintain genome stability and physiological consequences of their defects in the development of cancer and premature aging.

  18. Repairing a double-strand chromosome break by homologous recombination: revisiting Robin Holliday's model.

    PubMed Central

    Haber, James E; Ira, Gregorz; Malkova, Anna; Sugawara, Neal

    2004-01-01

    Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast Saccharomyces cerevisiae, one can follow recombination by physically monitoring DNA after the synchronous induction of a double-strand break (DSB) in both wild-type and mutant cells. A particularly well-studied system has been the switching of yeast mating-type (MAT) genes, where a DSB can be induced synchronously by expression of the site-specific HO endonuclease. Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis-dependent strand annealing pathway leading to noncrossovers and a two-end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break-induced replication. Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking. PMID:15065659

  19. Repairing a double-strand chromosome break by homologous recombination: revisiting Robin Holliday's model.

    PubMed

    Haber, James E; Ira, Gregorz; Malkova, Anna; Sugawara, Neal

    2004-01-29

    Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast Saccharomyces cerevisiae, one can follow recombination by physically monitoring DNA after the synchronous induction of a double-strand break (DSB) in both wild-type and mutant cells. A particularly well-studied system has been the switching of yeast mating-type (MAT) genes, where a DSB can be induced synchronously by expression of the site-specific HO endonuclease. Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis-dependent strand annealing pathway leading to noncrossovers and a two-end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break-induced replication. Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking.

  20. A non-canonical mismatch repair pathway in prokaryotes

    PubMed Central

    Castañeda-García, A.; Prieto, A. I.; Rodríguez-Beltrán, J.; Alonso, N.; Cantillon, D.; Costas, C.; Pérez-Lago, L.; Zegeye, E. D.; Herranz, M.; Plociński, P.; Tonjum, T.; García de Viedma, D.; Paget, M.; Waddell, S. J.; Rojas, A. M.; Doherty, A. J.; Blázquez, J.

    2017-01-01

    Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS–MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS–MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature. PMID:28128207

  1. A non-canonical mismatch repair pathway in prokaryotes.

    PubMed

    Castañeda-García, A; Prieto, A I; Rodríguez-Beltrán, J; Alonso, N; Cantillon, D; Costas, C; Pérez-Lago, L; Zegeye, E D; Herranz, M; Plociński, P; Tonjum, T; García de Viedma, D; Paget, M; Waddell, S J; Rojas, A M; Doherty, A J; Blázquez, J

    2017-01-27

    Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS-MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS-MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature.

  2. DNA Repair Pathway Choice Is Influenced by the Health of Drosophila melanogaster

    PubMed Central

    Wang, Alethea D.; Agrawal, Aneil F.

    2012-01-01

    In nature, individuals vary tremendously in condition and this may be an important source of variation in mutation rate. Condition is likely to affect cell state and thereby impact the amount of DNA damage sustained and/or the way it is repaired. Here, we focus on DNA repair. If low-condition individuals are less capable of devoting the same level of resources to accurate repair, they may suffer higher mutation rates. However, repair decisions are also governed by various aspects of cell physiology, which may render the prediction that “higher-condition individuals use better repair mechanisms” too simplistic. We use a larval diet manipulation in Drosophila melanogaster to create high- and low-condition individuals and then contrast their relative usage of three repair pathways [homologous recombination (HR), single-strand annealing (SSA), and nonhomologous end joining (NHEJ)] that differ in their mechanistic requirements and their mutational consequences. We find that low-condition flies are more likely than high-condition flies to use the most conservative of these three repair pathways, suggesting that physiological constraints on repair pathway usage may be more important than energetic costs. We also show that the repair differences between high- and low-condition flies resemble those between young and old flies, suggesting the underlying mechanisms may be similar. Finally, we observe that the effect of larval diet on adult repair increases as flies age, indicating that developmental differences early in life can have long-lasting consequences. PMID:22813892

  3. Homologous recombination contributes to the repair of zinc-finger-nuclease induced double strand breaks in pig primary cells and facilitates recombination with exogenous DNA.

    PubMed

    Klymiuk, Nikolai; Fezert, Pauline; Wünsch, Annegret; Kurome, Mayuko; Kessler, Barbara; Wolf, Eckhard

    2014-05-10

    Site-specific nucleases have become powerful tools for genome editing by the introduction of end-joining-mediated mutations, but it is unclear to which extent induced double strand breaks will also facilitate homologous recombination with exogenous DNA. This question is, however, of particular importance for somatic cells, which have to be modified for the generation of large animal models, but, on the other hand, have also been described to be reluctant to recombination-based DNA repair. Here, we examined zinc-finger nucleases for their potential to introduce modifications in pig somatic cells via end-joining or recombination. We found that co-transfection with nuclease-encoding plasmids resulted in a dramatic boost of recombination with different targeting vectors, suggesting a much more prominent role of this repair pathway in somatic cells than was previously thought. Although recombination with any of the vectors even occurred on both alleles of the target gene, we found also evidence for distinct properties of the used vectors regarding their preference for mono-allelic or bi-allelic modification. Thus, we show that the combined usage of site-specific nucleases and targeting vectors does not only promote homologous recombination in somatic cells but might also resemble a promising tool for detailed examination of DNA repair pathways.

  4. The Mechanism of Double-Strand DNA Break Repair by the Nonhomologous DNA End Joining Pathway

    PubMed Central

    Lieber, Michael R.

    2011-01-01

    Double-strand DNA breaks are common events in eukaryotic cells, and there are two major pathways for repairing them: homologous recombination and nonhomologous DNA end joining (NHEJ). The diverse causes of DSBs result in a diverse chemistry of DNA ends that must be repaired. Across NHEJ evolution, the enzymes of the NHEJ pathway exhibit a remarkable degree of structural tolerance in the range of DNA end substrate configurations upon which they can act. In vertebrate cells, the nuclease, polymerases and ligase of NHEJ are the most mechanistically flexible and multifunctional enzymes in each of their classes. Unlike repair pathways for more defined lesions, NHEJ repair enzymes act iteratively, act in any order, and can function independently of one another at each of the two DNA ends being joined. NHEJ is critical not only for the repair of pathologic DSBs as in chromosomal translocations, but also for the repair of physiologic DSBs created during V(D)J recombination and class switch recombination. Therefore, patients lacking normal NHEJ are not only sensitive to ionizing radiation, but also severely immunodeficient. PMID:20192759

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

    PubMed

    Walden, Helen; Deans, Andrew J

    2014-01-01

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

  6. Recombinant methods for screening human DNA excision repair proficiency

    SciTech Connect

    Athas, W.F.

    1988-01-01

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

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

    PubMed

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

    2017-03-20

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

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

    PubMed Central

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

    2017-01-01

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

  9. An Overview of the Molecular Mechanisms of Recombinational DNA Repair.

    PubMed

    Kowalczykowski, Stephen C

    2015-11-02

    Recombinational DNA repair is a universal aspect of DNA metabolism and is essential for genomic integrity. It is a template-directed process that uses a second chromosomal copy (sister, daughter, or homolog) to ensure proper repair of broken chromosomes. The key steps of recombination are conserved from phage through human, and an overview of those steps is provided in this review. The first step is resection by helicases and nucleases to produce single-stranded DNA (ssDNA) that defines the homologous locus. The ssDNA is a scaffold for assembly of the RecA/RAD51 filament, which promotes the homology search. On finding homology, the nucleoprotein filament catalyzes exchange of DNA strands to form a joint molecule. Recombination is controlled by regulating the fate of both RecA/RAD51 filaments and DNA pairing intermediates. Finally, intermediates that mature into Holliday structures are disjoined by either nucleolytic resolution or topological dissolution. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.

  10. Presynaptic Filament Dynamics in Homologous Recombination and DNA Repair

    PubMed Central

    Liu, Jie; Ehmsen, Kirk T.; Heyer, Wolf-Dietrich; Morrical, Scott W.

    2014-01-01

    Homologous Recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA. Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we review the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments: some intrinsic such as recombinase ATP binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examine dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examine the biochemical properties of recombination proteins from four model systems (T4 phage, E. coli, S. cerevisiae, and H. sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We propose that the presynaptic filament has evolved to rely on multiple external factors for increased multi-level regulation of HR processes in genomes with greater structural and sequence complexity. PMID:21599536

  11. Personalised pathway analysis reveals association between DNA repair pathway dysregulation and chromosomal instability in sporadic breast cancer.

    PubMed

    Liu, Chao; Srihari, Sriganesh; Lal, Samir; Gautier, Benoît; Simpson, Peter T; Khanna, Kum Kum; Ragan, Mark A; Lê Cao, Kim-Anh

    2016-01-01

    The Homologous Recombination (HR) pathway is crucial for the repair of DNA double-strand breaks (DSBs) generated during DNA replication. Defects in HR repair have been linked to the initiation and development of a wide variety of human malignancies, and exploited in chemical, radiological and targeted therapies. In this study, we performed a personalised pathway analysis independently for four large sporadic breast cancer cohorts to investigate the status of HR pathway dysregulation in individual sporadic breast tumours, its association with HR repair deficiency and its impact on tumour characteristics. Specifically, we first manually curated a list of HR genes according to our recent review on this pathway (Liu et al., 2014), and then applied a personalised pathway analysis method named Pathifier (Drier et al., 2013) on the expression levels of the curated genes to obtain an HR score quantifying HR pathway dysregulation in individual tumours. Based on the score, we observed a great diversity in HR dysregulation between and within gene expression-based breast cancer subtypes, and by using two published HR-defect signatures, we found HR pathway dysregulation reflects HR repair deficiency. Furthermore, we identified a novel association between HR pathway dysregulation and chromosomal instability (CIN) in sporadic breast cancer. Although CIN has long been considered as a hallmark of most solid tumours, with recent extensive studies highlighting its importance in tumour evolution and drug resistance, the molecular basis of CIN in sporadic cancers remains poorly understood. Our results imply that HR pathway dysregulation might contribute to CIN in sporadic breast cancer.

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

    PubMed

    Neher, Tracy M; Turchi, John J

    2011-06-15

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

  13. Competitive repair by naturally dispersed repetitive DNA during non-allelic homologous recombination

    SciTech Connect

    Hoang, Margaret L.; Tan, Frederick J.; Lai, David C.; Celniker, Sue E.; Hoskins, Roger A.; Dunham, Maitreya J.; Zheng, Yixian; Koshland, Douglas

    2010-08-27

    Genome rearrangements often result from non-allelic homologous recombination (NAHR) between repetitive DNA elements dispersed throughout the genome. Here we systematically analyze NAHR between Ty retrotransposons using a genome-wide approach that exploits unique features of Saccharomyces cerevisiae purebred and Saccharomyces cerevisiae/Saccharomyces bayanus hybrid diploids. We find that DNA double-strand breaks (DSBs) induce NAHR-dependent rearrangements using Ty elements located 12 to 48 kilobases distal to the break site. This break-distal recombination (BDR) occurs frequently, even when allelic recombination can repair the break using the homolog. Robust BDR-dependent NAHR demonstrates that sequences very distal to DSBs can effectively compete with proximal sequences for repair of the break. In addition, our analysis of NAHR partner choice between Ty repeats shows that intrachromosomal Ty partners are preferred despite the abundance of potential interchromosomal Ty partners that share higher sequence identity. This competitive advantage of intrachromosomal Tys results from the relative efficiencies of different NAHR repair pathways. Finally, NAHR generates deleterious rearrangements more frequently when DSBs occur outside rather than within a Ty repeat. These findings yield insights into mechanisms of repeat-mediated genome rearrangements associated with evolution and cancer.

  14. Nonhomologous end-joining repair plays a more important role than homologous recombination repair in defining radiosensitivity after exposure to high-LET radiation.

    PubMed

    Takahashi, Akihisa; Kubo, Makoto; Ma, Hongyu; Nakagawa, Akiko; Yoshida, Yukari; Isono, Mayu; Kanai, Tatsuaki; Ohno, Tatsuya; Furusawa, Yoshiya; Funayama, Tomoo; Kobayashi, Yasuhiko; Nakano, Takashi

    2014-09-01

    DNA double-strand breaks (DSBs) induced by ionizing radiation pose a major threat to cell survival. The cell can respond to the presence of DSBs through two major repair pathways: homologous recombination (HR) and nonhomologous end joining (NHEJ). Higher levels of cell death are induced by high-linear energy transfer (LET) radiation when compared to low-LET radiation, even at the same physical doses, due to less effective and efficient DNA repair. To clarify whether high-LET radiation inhibits all repair pathways or specifically one repair pathway, studies were designed to examine the effects of radiation with different LET values on DNA DSB repair and radiosensitivity. Embryonic fibroblasts bearing repair gene (NHEJ-related Lig4 and/or HR-related Rad54) knockouts (KO) were used and their responses were compared to wild-type cells. The cells were exposed to X rays, spread-out Bragg peak (SOBP) carbon ion beams as well as with carbon, iron, neon and argon ions. Cell survival was measured with colony-forming assays. The sensitization enhancement ratio (SER) values were calculated using the 10% survival dose of wild-type cells and repair-deficient cells. Cellular radiosensitivity was listed in descending order: double-KO cells > Lig4-KO cells > Rad54-KO cells > wild-type cells. Although Rad54-KO cells had an almost constant SER value, Lig4-KO cells showed a high-SER value when compared to Rad54-KO cells, even with increasing LET values. These results suggest that with carbon-ion therapy, targeting NHEJ repair yields higher radiosensitivity than targeting homologous recombination repair.

  15. The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair.

    PubMed

    Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg; Dziegielewski, Jaroslaw; Syljuåsen, Randi G; Lundin, Cecilia; Bartek, Jiri; Helleday, Thomas

    2005-02-01

    The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.

  16. The Drosophila Meiotic Recombination Gene Mei-9 Encodes a Homologue of the Yeast Excision Repair Protein Rad1

    PubMed Central

    Sekelsky, J. J.; McKim, K. S.; Chin, G. M.; Hawley, R. S.

    1995-01-01

    Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cerevisiae, many genes required to repair DNA double-strand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cerevisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cerevisiae excision repair protein Rad1, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cerevisiae, in that it requires an NER protein. The biochemical properties of the Rad1 protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion. PMID:8647398

  17. The Saccharomyces cerevisiae DNA recombination and repair functions of the RAD52 epistasis group inhibit Ty1 transposition.

    PubMed Central

    Rattray, A J; Shafer, B K; Garfinkel, D J

    2000-01-01

    RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or delta) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA. PMID:10655210

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

    PubMed

    Kakarougkas, A; Jeggo, P A

    2014-03-01

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

  19. Exploiting the homologous recombination DNA repair network for targeted cancer therapy.

    PubMed

    Peng, Guang; Lin, Shiaw-Yih

    2011-02-10

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

  20. How cancer cells hijack DNA double-strand break repair pathways to gain genomic instability.

    PubMed

    Jeggo, Penny A; Löbrich, Markus

    2015-10-01

    DNA DSBs (double-strand breaks) are a significant threat to the viability of a normal cell, since they can result in loss of genetic material if mitosis or replication is attempted in their presence. Consequently, evolutionary pressure has resulted in multiple pathways and responses to enable DSBs to be repaired efficiently and faithfully. Cancer cells, which are under pressure to gain genomic instability, have a striking ability to avoid the elegant mechanisms by which normal cells maintain genomic stability. Current models suggest that, in normal cells, DSB repair occurs in a hierarchical manner that promotes rapid and efficient rejoining first, with the utilization of additional steps or pathways of diminished accuracy if rejoining is unsuccessful or delayed. In the present review, we evaluate the fidelity of DSB repair pathways and discuss how cancer cells promote the utilization of less accurate processes. Homologous recombination serves to promote accuracy and stability during replication, providing a battlefield for cancer to gain instability. Non-homologous end-joining, a major DSB repair pathway in mammalian cells, usually operates with high fidelity and only switches to less faithful modes if timely repair fails. The transition step is finely tuned and provides another point of attack during tumour progression. In addition to DSB repair, a DSB signalling response activates processes such as cell cycle checkpoint arrest, which enhance the possibility of accurate DSB repair. We consider the ways by which cancers modify and hijack these processes to gain genomic instability.

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

    PubMed

    Nowosielska, Anetta

    2007-01-01

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

  2. Eliminating both canonical and short-patch mismatch repair in Drosophila melanogaster suggests a new meiotic recombination model.

    PubMed

    Crown, K Nicole; McMahan, Susan; Sekelsky, Jeff

    2014-09-01

    In most meiotic systems, recombination is essential to form connections between homologs that ensure their accurate segregation from one another. Meiotic recombination is initiated by DNA double-strand breaks that are repaired using the homologous chromosome as a template. Studies of recombination in budding yeast have led to a model in which most early repair intermediates are disassembled to produce noncrossovers. Selected repair events are stabilized so they can proceed to form double-Holliday junction (dHJ) intermediates, which are subsequently resolved into crossovers. This model is supported in yeast by physical isolation of recombination intermediates, but the extent to which it pertains to animals is unknown. We sought to test this model in Drosophila melanogaster by analyzing patterns of heteroduplex DNA (hDNA) in recombination products. Previous attempts to do this have relied on knocking out the canonical mismatch repair (MMR) pathway, but in both yeast and Drosophila the resulting recombination products are complex and difficult to interpret. We show that, in Drosophila, this complexity results from a secondary, short-patch MMR pathway that requires nucleotide excision repair. Knocking out both canonical and short-patch MMR reveals hDNA patterns that reveal that many noncrossovers arise after both ends of the break have engaged with the homolog. Patterns of hDNA in crossovers could be explained by biased resolution of a dHJ; however, considering the noncrossover and crossover results together suggests a model in which a two-end engagement intermediate with unligated HJs can be disassembled by a helicase to a produce noncrossover or nicked by a nuclease to produce a crossover. While some aspects of this model are similar to the model from budding yeast, production of both noncrossovers and crossovers from a single, late intermediate is a fundamental difference that has important implications for crossover control.

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

    PubMed

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

    2017-03-15

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

  4. The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair of radiation damage and recombination.

    PubMed Central

    Kooistra, R; Vreeken, K; Zonneveld, J B; de Jong, A; Eeken, J C; Osgood, C J; Buerstedde, J M; Lohman, P H; Pastink, A

    1997-01-01

    The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRADS4-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution. PMID:9315669

  5. Frequency of intrachromosomal homologous recombination induced by UV radiation in normally repairing and excision repair-deficient human cells

    SciTech Connect

    Tsujimura, T.; Maher, V.M.; McCormick, J.J. ); Godwin, A.R.; Liskay, R.M. )

    1990-02-01

    To investigate the role of DNA damage and nucleotide excision repair in intrachromosomal homologous recombination, a plasmid containing duplicated copies of the gene coding for hygromycin resistance was introduced into the genome of a repair-proficient human cell line, KMST-6, and two repair-deficient lines, XP2OS(SV) from xeroderma pigmentosum complementation group A and XP2YO(SV) from complementation group F. Neither hygromycin-resistance gene codes for a functional enzyme because each contains an insertion/deletion mutation at a unique site, but recombination between the two defective genes can yield hygromycin-resistant cells. The rates of spontaneous recombination in normal and xeroderma pigmentosum cell strains containing the recombination substrate were found to be similar. The frequency of UV-induced recombination was determined for three of these cell strains. At low doses, the group A cell strain and the group F cell strain showed a significant increase in frequency of recombinants. The repair-proficient cell strain required 10-to 20-fold higher doses of UV to exhibit comparable increases in frequency of recombinants. These results suggest that unexcised DNA damage, rather than the excision repair process per se, stimulates such recombination.

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

    PubMed

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

    2014-05-01

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

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

    PubMed

    Aparicio, Tomas; Baer, Richard; Gautier, Jean

    2014-07-01

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

  8. Role of the RecBCD Recombination Pathway in Salmonella Virulence

    PubMed Central

    Cano, David A.; Pucciarelli, M. Graciela; García-del Portillo, Francisco; Casadesús, Josep

    2002-01-01

    Mutants of Salmonella enterica lacking the RecBC function are avirulent in mice and unable to grow inside macrophages (N. A. Buchmeier, C. J. Lipps, M. Y. H. So, and F. Heffron, Mol. Microbiol. 7:933–936, 1993). The virulence-related defects of RecBC− mutants are not suppressed by sbcB and sbcCD mutations, indicating that activation of the RecF recombination pathway cannot replace the virulence-related function(s) of RecBCD. Functions of the RecF pathway such as RecJ and RecF are not required for virulence. Since the RecBCD pathway, but not the RecF pathway, is known to participate in the repair of double-strand breaks produced during DNA replication, we propose that systemic infection by S. enterica may require RecBCD-mediated recombinational repair to prime DNA replication inside phagocytes. Mutants lacking both RecD and RecJ are also attenuated in mice and are unable to proliferate in macrophages, suggesting that exonucleases V and IX provide alternative functions for RecBCD-mediated recombinational repair during Salmonella infection. PMID:11751841

  9. Potential role for the BLM helicase in recombinational repair via a conserved interaction with RAD51.

    PubMed

    Wu, L; Davies, S L; Levitt, N C; Hickson, I D

    2001-06-01

    Bloom's syndrome (BS) is an autosomal recessive disorder that predisposes individuals to a wide range of cancers. The gene mutated in BS, BLM, encodes a member of the RecQ family of DNA helicases. The precise role played by these enzymes in the cell remains to be determined. However, genome-wide hyper-recombination is a feature of many RecQ helicase-deficient cells. In eukaryotes, a central step in homologous recombination is catalyzed by the RAD51 protein. In response to agents that induce DNA double-strand breaks, RAD51 accumulates in nuclear foci that are thought to correspond to sites of recombinational repair. Here, we report that purified BLM and human RAD51 interact in vitro and in vivo, and that residues in the N- and C-terminal domains of BLM can independently mediate this interaction. Consistent with these observations, BLM localizes to a subset of RAD51 nuclear foci in normal human cells. Moreover, the number of BLM foci and the extent to which BLM and RAD51 foci co-localize increase in response to ionizing radiation. Nevertheless, the formation of RAD51 foci does not require functional BLM. Indeed, in untreated BS cells, an abnormally high proportion of the cells contain RAD51 nuclear foci. Exogenous expression of BLM markedly reduces the fraction of cells containing RAD51 foci. The interaction between BLM and RAD51 appears to have been evolutionarily conserved since the C-terminal domain of Sgs1, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Rad51. Furthermore, genetic analysis reveals that the SGS1 and RAD51 genes are epistatic indicating that they operate in a common pathway. Potential roles for BLM in the RAD51 recombinational repair pathway are discussed.

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

    PubMed Central

    Boulton, S J; Jackson, S P

    1996-01-01

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

  11. PARP-mediated repair, homologous recombination, and back-up non-homologous end joining-like repair of single-strand nicks.

    PubMed

    Metzger, Michael J; Stoddard, Barry L; Monnat, Raymond J

    2013-07-01

    Double-strand breaks (DSBs) in chromosomal DNA can induce both homologous recombination (HR) and non-homologous end-joining (NHEJ). Recently we showed that single-strand nicks induce HR with a significant reduction in toxicity and mutagenic effects associated with NHEJ. To further investigate the differences and similarities of DSB- and nick-induced repair, we used an integrated reporter system in human cells to measure HR and NHEJ produced by the homing endonuclease I-AniI and a designed 'nickase' variant that nicks the same target site, focusing on the PARP and HR repair pathways. PARP inhibitors, which block single-strand break repair, increased the rate of nick-induced HR up to 1.7-fold but did not affect DSB-induced HR or mutNHEJ. Additionally, expression of the PALB2 WD40 domain in trans acted as a dominant-negative inhibitor of both DSB- and nick-induced HR, sensitized cells to PARP inhibition, and revealed an alternative mutagenic repair pathway for nicks. Thus, while both DSB- and nick-induced HR use a common pathway, their substrates are differentially processed by cellular factors. These results also suggest that the synthetic lethality of PARP and BRCA may be due to repair of nicks through an error prone, NHEJ-like mechanism that is active when both PARP and HR pathways are blocked.

  12. Induced DNA repair pathway in mammalian cells

    SciTech Connect

    Overberg, R.

    1985-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  14. Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells.

    PubMed

    Takahashi, Akihisa; Mori, Eiichiro; Nakagawa, Yosuke; Kajihara, Atsuhisa; Kirita, Tadaaki; Pittman, Douglas L; Hasegawa, Masatoshi; Ohnishi, Takeo

    2016-11-13

    Heat shock induces DNA double-strand breaks (DSBs), but the precise mechanism of repairing heat-induced damage is unclear. Here, we investigated the DNA repair pathways involved in cell death induced by heat shock. B02, a specific inhibitor of human RAD51 (homologous recombination; HR), and NU7026, a specific inhibitor of DNA-PK (non-homologous end-joining; NHEJ), were used for survival assays of human cancer cell lines with different p53-gene status. Mouse embryonic fibroblasts (MEFs) lacking Lig4 (NHEJ) and/or Rad54 (HR) were used for survival assays and a phosphorylated histone H2AX at Ser139 (γH2AX) assay. MEFs lacking Rad51d (HR) were used for survival assays. SPD8 cells were used to measure HR frequency after heat shock. Human cancer cells were more sensitive to heat shock in the presence of B02 despite their p53-gene status, and the effect of B02 on heat sensitivity was specific to the G2 phase. Rad54-deficient MEFs were sensitive to heat shock and showed prolonged γH2AX signals following heat shock. Rad51d-deficient MEFs were also sensitive to heat shock. Moreover, heat shock-stimulated cells had increased HR. The HR pathway plays an important role in the survival of mammalian cells against death induced by heat shock via the repair of heat-induced DNA DSBs.

  15. Phosphoglycerate mutase 1 regulates dNTP pool and promotes homologous recombination repair in cancer cells

    PubMed Central

    Qu, Jia; Sun, Wenyi; Zhong, Jie; Lv, Hao; Zhu, Mingrui; Xu, Jun; Jin, Nan; Tan, Minjia; Geng, Meiyu

    2017-01-01

    Glycolytic enzymes are known to play pivotal roles in cancer cell survival, yet their molecular mechanisms remain poorly understood. Phosphoglycerate mutase 1 (PGAM1) is an important glycolytic enzyme that coordinates glycolysis, pentose phosphate pathway, and serine biosynthesis in cancer cells. Herein, we report that PGAM1 is required for homologous recombination (HR) repair of DNA double-strand breaks (DSBs) caused by DNA-damaging agents. Mechanistically, PGAM1 facilitates DSB end resection by regulating the stability of CTBP-interacting protein (CtIP). Knockdown of PGAM1 in cancer cells accelerates CtIP degradation through deprivation of the intracellular deoxyribonucleotide triphosphate pool and associated activation of the p53/p73 pathway. Enzymatic inhibition of PGAM1 decreases CtIP protein levels, impairs HR repair, and hence sensitizes BRCA1/2-proficient breast cancer to poly(ADP-ribose) polymerase (PARP) inhibitors. Together, this study identifies a metabolically dependent function of PGAM1 in promoting HR repair and reveals a potential therapeutic opportunity for PGAM1 inhibitors in combination with PARP inhibitors. PMID:28122957

  16. Harmine suppresses homologous recombination repair and inhibits proliferation of hepatoma cells

    PubMed Central

    Zhang, Lei; Zhang, Fan; Zhang, Wenjun; Chen, Lu; Gao, Neng; Men, Yulong; Xu, Xiaojun; Jiang, Ying

    2015-01-01

    To avoid cell cycle arrest or apoptosis, rapidly proliferating cancer cells have to promote DNA double strand break (DSB) repair to fix replication stress induced DSBs. Therefore, developing drugs blocking homologous recombination (HR) and nonhomologous end joining (NHEJ) – 2 major DSB repair pathways – holds great potential for cancer therapy. Over the last few decades, much attention has been paid to explore drugs targeting DSB repair pathways for cancer therapy. Here, using 2 well-established reporters for analyzing HR and NHEJ efficiency, we found that both HR and NHEJ are elevated in hepatoma cell lines Hep3B and HuH7 compared with normal liver cell lines Chang liver and QSG-7701. Our further study found that Harmine, a natural compound, negatively regulates HR but not NHEJ by interfering Rad51 recruitment, resulting in severe cytotoxicity in hepatoma cells. Furthermore, NHEJ inhibitor Nu7441 markedly sensitizes Hep3B cells to the anti-proliferative effects of Harmine. Taken together, our study suggested that Harmine holds great promise as an oncologic drug and combination of Harmine with a NHEJ inhibitor might be an effective strategy for anti-cancer treatment. PMID:26382920

  17. Homologous recombination-mediated double-strand break repair in mouse testicular extracts and comparison with different germ cell stages.

    PubMed

    Srivastava, Niloo; Raman, Mercy J

    2007-01-01

    Homologous recombination (HR) is established as a significant contributor to double-strand break (DSB) repair in mammalian somatic cells; however, its role in mammalian germ cells has not been characterized, although being conservative in nature it is anticipated to be the major pathway in germ cells. The germ cell system has inherent limitations by which intact cell approaches are not feasible. The present study, therefore, investigates HR-mediated DSB repair in mouse germ cell extracts by using an in vitro plasmid recombination assay based on functional rescue of a neomycin (neo) gene. A significantly high-fold increase in neo+ (Kan(R)) colonies following incubation of two plasmid substrates (neo delta1 and neo delta2) with testicular extracts demonstrated the extracts' ability to catalyze intermolecular recombination. A significant enhancement in recombinants upon linearization of one of the plasmids suggested the existence of an HR-mediated DSB repair activity. Comparison of the activity at sequential developmental stages, spermatogonia, spermatocytes and spermatids revealed its presence at all the stages; spermatocyte being the most proficient stage. Further, restriction analysis of recombinant plasmids indicated the predominance of gene conversion in enriched spermatocytes (mostly pachytenes), in contrast to gonial and spermatid extracts that showed higher reciprocal exchange. In conclusion, this study demonstrates HR repair activity at all stages of male germ cells, suggesting an important role of HR-mediated DSB repair during mammalian spermatogenesis. Further, the observed preference of gene conversion over reciprocal exchange at spermatocyte stage correlates with the close association of gene conversion with the meiotic recombination program.

  18. Sirtuin 6 (SIRT6) rescues the decline of homologous recombination repair during replicative senescence

    PubMed Central

    Mao, Zhiyong; Tian, Xiao; Van Meter, Michael; Ke, Zhonghe; Gorbunova, Vera; Seluanov, Andrei

    2012-01-01

    Genomic instability is a hallmark of aging tissues. Genomic instability may arise from the inefficient or aberrant function of DNA double-stranded break (DSB) repair. DSBs are repaired by homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). HR is a precise pathway, whereas NHEJ frequently leads to deletions or insertions at the repair site. Here, we used normal human fibroblasts with a chromosomally integrated HR reporter cassette to examine the changes in HR efficiency as cells progress to replicative senescence. We show that HR declines sharply with increasing replicative age, with an up to 38-fold decrease in efficiency in presenescent cells relative to young cells. This decline is not explained by a reduction of the number of cells in S/G2/M stage as presenescent cells are actively dividing. Expression of proteins involved in HR such as Rad51, Rad51C, Rad52, NBS1, and Sirtuin 6 (SIRT6) diminished with cellular senescence. Supplementation of Rad51, Rad51C, Rad52, and NBS1 proteins, either individually or in combination, did not rescue the senescence-related decline of HR. However, overexpression of SIRT6 in “middle-aged” and presenescent cells strongly stimulated HR repair, and this effect was dependent on mono-ADP ribosylation activity of poly(ADP-ribose) polymerase (PARP1). These results suggest that in aging cells, the precise HR pathway becomes repressed giving way to a more error-prone NHEJ pathway. These changes in the processing of DSBs may contribute to age-related genomic instability and a higher incidence of cancer with age. SIRT6 activation provides a potential therapeutic strategy to prevent the decline in genome maintenance. PMID:22753495

  19. The nucleotide excision repair system of Borrelia burgdorferi is the sole pathway involved in repair of DNA damage by UV light.

    PubMed

    Hardy, Pierre-Olivier; Chaconas, George

    2013-05-01

    To survive and avoid accumulation of mutations caused by DNA damage, the genomes of prokaryotes encode a variety of DNA repair pathways most well characterized in Escherichia coli. Some of these are required for the infectivity of various pathogens. In this study, the importance of 25 DNA repair/recombination genes for Borrelia burgdorferi survival to UV-induced DNA damage was assessed. In contrast to E. coli, where 15 of these genes have an effect on survival of UV irradiation, disruption of recombinational repair, transcription-coupled repair, methyl-directed mismatch correction, and repair of arrested replication fork pathways did not decrease survival of B. burgdorferi exposed to UV light. However, the disruption of the B. burgdorferi nucleotide excision repair (NER) pathway (uvrA, uvrB, uvrC, and uvrD) resulted in a 10- to 1,000-fold increase in sensitivity to UV light. A functional NER pathway was also shown to be required for B. burgdorferi resistance to nitrosative damage. Finally, disruption of uvrA, uvrC, and uvrD had only a minor effect upon murine infection by increasing the time required for dissemination.

  20. A role for XLF in DNA repair and recombination in human somatic cells.

    PubMed

    Fattah, Farjana Jahan; Kweon, Junghun; Wang, Yongbao; Lee, Eu Han; Kan, Yinan; Lichter, Natalie; Weisensel, Natalie; Hendrickson, Eric A

    2014-03-01

    Classic non-homologous end-joining (C-NHEJ) is required for the repair of radiation-induced DNA double-strand breaks (DSBs) in mammalian cells and plays a critical role in lymphoid V(D)J recombination. A core C-NHEJ component is the DNA ligase IV co-factor, Cernunnos/XLF (hereafter XLF). In patients, mutations in XLF cause predicted increases in radiosensitivity and deficits in immune function, but also cause other less well-understood pathologies including neural disorders. To characterize XLF function(s) in a defined genetic system, we used a recombinant adeno-associated virus-mediated gene targeting strategy to inactivate both copies of the XLF locus in the human HCT116 cell line. Analyses of XLF-null cells (which were viable) showed that they were highly sensitive to ionizing radiation and a radiomimetic DNA damaging agent, etoposide. XLF-null cells had profound DNA DSB repair defects as measured by in vivo plasmid end-joining assays and were also dramatically impaired in their ability to form either V(D)J coding or signal joints on extrachromosomal substrates. Thus, our somatic XLF-null cell line recapitulates many of the phenotypes expected from XLF patient cell lines. Subsequent structure:function experiments utilizing the expression of wild-type and mutant XLF cDNAs demonstrated that all of the phenotypes of an XLF deficiency could be rescued by the overexpression of a wild-type XLF cDNA. Unexpectedly, mutant forms of XLF bearing point mutations at amino acid positions L115 and L179, also completely complemented the null phenotype suggesting, in contrast to predictions to the contrary, that these mutations do not abrogate XLF function. Finally, we demonstrate that the absence of XLF causes a small, but significant, increase in homologous recombination, implicating XLF in DSB pathway choice regulation. We conclude that human XLF is a non-essential, but critical, C-NHEJ-repair factor.

  1. Activation of homologous recombination DNA repair in human skin fibroblasts continuously exposed to X-ray radiation.

    PubMed

    Osipov, Andreyan N; Grekhova, Anna; Pustovalova, Margarita; Ozerov, Ivan V; Eremin, Petr; Vorobyeva, Natalia; Lazareva, Natalia; Pulin, Andrey; Zhavoronkov, Alex; Roumiantsev, Sergey; Klokov, Dmitry; Eremin, Ilya

    2015-09-29

    Molecular and cellular responses to protracted ionizing radiation exposures are poorly understood. Using immunofluorescence microscopy, we studied the kinetics of DNA repair foci formation in normal human fibroblasts exposed to X-rays at a dose rate of 4.5 mGy/min for up to 6 h. We showed that both the number of γH2AX foci and their integral fluorescence intensity grew linearly with time of irradiation up to 2 h. A plateau was observed between 2 and 6 h of exposure, indicating a state of balance between formation and repair of DNA double-strand breaks. In contrast, the number and intensity of foci formed by homologous recombination protein RAD51 demonstrated a continuous increase during 6 h of irradiation. We further showed that the enhancement of the homologous recombination repair was not due to redistribution of cell cycle phases. Our results indicate that continuous irradiation of normal human cells triggers DNA repair responses that are different from those elicited after acute irradiation. The observed activation of the error-free homologous recombination DNA double-strand break repair pathway suggests compensatory adaptive mechanisms that may help alleviate long-term biological consequences and could potentially be utilized both in radiation protection and medical practices.

  2. Activation of homologous recombination DNA repair in human skin fibroblasts continuously exposed to X-ray radiation

    PubMed Central

    Osipov, Andreyan N.; Grekhova, Anna; Pustovalova, Margarita; Ozerov, Ivan V.; Eremin, Petr; Vorobyeva, Natalia; Lazareva, Natalia; Pulin, Andrey; Zhavoronkov, Alex; Roumiantsev, Sergey; Klokov, Dmitry; Eremin, Ilya

    2015-01-01

    Molecular and cellular responses to protracted ionizing radiation exposures are poorly understood. Using immunofluorescence microscopy, we studied the kinetics of DNA repair foci formation in normal human fibroblasts exposed to X-rays at a dose rate of 4.5 mGy/min for up to 6 h. We showed that both the number of γH2AX foci and their integral fluorescence intensity grew linearly with time of irradiation up to 2 h. A plateau was observed between 2 and 6 h of exposure, indicating a state of balance between formation and repair of DNA double-strand breaks. In contrast, the number and intensity of foci formed by homologous recombination protein RAD51 demonstrated a continuous increase during 6 h of irradiation. We further showed that the enhancement of the homologous recombination repair was not due to redistribution of cell cycle phases. Our results indicate that continuous irradiation of normal human cells triggers DNA repair responses that are different from those elicited after acute irradiation. The observed activation of the error-free homologous recombination DNA double-strand break repair pathway suggests compensatory adaptive mechanisms that may help alleviate long-term biological consequences and could potentially be utilized both in radiation protection and medical practices. PMID:26337087

  3. A Dominant Mutation in Human RAD51 Reveals Its Function in DNA Interstrand Crosslink Repair Independent of Homologous Recombination.

    PubMed

    Wang, Anderson T; Kim, Taeho; Wagner, John E; Conti, Brooke A; Lach, Francis P; Huang, Athena L; Molina, Henrik; Sanborn, Erica M; Zierhut, Heather; Cornes, Belinda K; Abhyankar, Avinash; Sougnez, Carrie; Gabriel, Stacey B; Auerbach, Arleen D; Kowalczykowski, Stephen C; Smogorzewska, Agata

    2015-08-06

    Repair of DNA interstrand crosslinks requires action of multiple DNA repair pathways, including homologous recombination. Here, we report a de novo heterozygous T131P mutation in RAD51/FANCR, the key recombinase essential for homologous recombination, in a patient with Fanconi anemia-like phenotype. In vitro, RAD51-T131P displays DNA-independent ATPase activity, no DNA pairing capacity, and a co-dominant-negative effect on RAD51 recombinase function. However, the patient cells are homologous recombination proficient due to the low ratio of mutant to wild-type RAD51 in cells. Instead, patient cells are sensitive to crosslinking agents and display hyperphosphorylation of Replication Protein A due to increased activity of DNA2 and WRN at the DNA interstrand crosslinks. Thus, proper RAD51 function is important during DNA interstrand crosslink repair outside of homologous recombination. Our study provides a molecular basis for how RAD51 and its associated factors may operate in a homologous recombination-independent manner to maintain genomic integrity. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. RAD6 Promotes Homologous Recombination Repair by Activating the Autophagy-Mediated Degradation of Heterochromatin Protein HP1

    PubMed Central

    Chen, Su; Wang, Chen; Sun, Luxi; Wang, Da-Liang; Chen, Lu; Huang, Zhuan; Yang, Qi; Gao, Jie; Yang, Xi-Bin; Chang, Jian-Feng; Chen, Ping; Lan, Li

    2014-01-01

    Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients. PMID:25384975

  5. DNA repair and recombination in higher plants: insights from comparative genomics of arabidopsis and rice

    PubMed Central

    2010-01-01

    Background The DNA repair and recombination (DRR) proteins protect organisms against genetic damage, caused by environmental agents and other genotoxic agents, by removal of DNA lesions or helping to abide them. Results We identified genes potentially involved in DRR mechanisms in Arabidopsis and rice using similarity searches and conserved domain analysis against proteins known to be involved in DRR in human, yeast and E. coli. As expected, many of DRR genes are very similar to those found in other eukaryotes. Beside these eukaryotes specific genes, several prokaryotes specific genes were also found to be well conserved in plants. In Arabidopsis, several functionally important DRR gene duplications are present, which do not occur in rice. Among DRR proteins, we found that proteins belonging to the nucleotide excision repair pathway were relatively more conserved than proteins needed for the other DRR pathways. Sub-cellular localization studies of DRR gene suggests that these proteins are mostly reside in nucleus while gene drain in between nucleus and cell organelles were also found in some cases. Conclusions The similarities and dissimilarities in between plants and other organisms' DRR pathways are discussed. The observed differences broaden our knowledge about DRR in the plants world, and raises the potential question of whether differentiated functions have evolved in some cases. These results, altogether, provide a useful framework for further experimental studies in these organisms. PMID:20646326

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

    PubMed Central

    Deng, W P; Nickoloff, J A

    1994-01-01

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

  7. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis.

    PubMed

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia; Altmannova, Veronika; Sebesta, Marek; Pacesa, Martin; Fugger, Kasper; Sorensen, Claus Storgaard; Lee, Marietta Y W T; Haracska, Lajos; Krejci, Lumir

    2016-04-20

    Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.

  8. Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

    PubMed Central

    Mosig, Gisela; Gewin, John; Luder, Andreas; Colowick, Nancy; Vo, Daniel

    2001-01-01

    Two major pathways of recombination-dependent DNA replication, “join-copy” and “join-cut-copy,” can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses. PMID:11459968

  9. Homologous recombination rescues ssDNA gaps generated by nucleotide excision repair and reduced translesion DNA synthesis in yeast G2 cells

    PubMed Central

    Ma, Wenjian; Westmoreland, James W.; Resnick, Michael A.

    2013-01-01

    Repair of DNA bulky lesions often involves multiple repair pathways such as nucleotide-excision repair, translesion DNA synthesis (TLS), and homologous recombination (HR). Although there is considerable information about individual pathways, little is known about the complex interactions or extent to which damage in single strands, such as the damage generated by UV, can result in double-strand breaks (DSBs) and/or generate HR. We investigated the consequences of UV-induced lesions in nonreplicating G2 cells of budding yeast. In contrast to WT cells, there was a dramatic increase in ssDNA gaps for cells deficient in the TLS polymerases η (Rad30) and ζ (Rev3). Surprisingly, repair in TLS-deficient G2 cells required HR repair genes RAD51 and RAD52, directly revealing a redundancy of TLS and HR functions in repair of ssDNAs. Using a physical assay that detects recombination between circular sister chromatids within a few hours after UV, we show an approximate three-fold increase in recombinants in the TLS mutants over that in WT cells. The recombination, which required RAD51 and RAD52, does not appear to be caused by DSBs, because a dose of ionizing radiation producing 20 times more DSBs was much less efficient than UV in producing recombinants. Thus, in addition to revealing TLS and HR functional redundancy, we establish that UV-induced recombination in TLS mutants is not attributable to DSBs. These findings suggest that ssDNA that might originate during the repair of closely opposed lesions or of ssDNA-containing lesions or from uncoupled replication may drive recombination directly in various species, including humans. PMID:23858457

  10. Knockout targeting of the Drosophila nap1 gene and examination of DNA repair tracts in the recombination products.

    PubMed Central

    Lankenau, Susanne; Barnickel, Thorsten; Marhold, Joachim; Lyko, Frank; Mechler, Bernard M; Lankenau, Dirk-Henner

    2003-01-01

    We used ends-in gene targeting to generate knockout mutations of the nucleosome assembly protein 1 (Nap1) gene in Drosophila melanogaster. Three independent targeted null-knockout mutations were produced. No wild-type NAP1 protein could be detected in protein extracts. Homozygous Nap1(KO) knockout flies were either embryonic lethal or poorly viable adult escapers. Three additional targeted recombination products were viable. To gain insight into the underlying molecular processes we examined conversion tracts in the recombination products. In nearly all cases the I-SceI endonuclease site of the donor vector was replaced by the wild-type Nap1 sequence. This indicated exonuclease processing at the site of the double-strand break (DSB), followed by replicative repair at donor-target junctions. The targeting products are best interpreted either by the classical DSB repair model or by the break-induced recombination (BIR) model. Synthesis-dependent strand annealing (SDSA), which is another important recombinational repair pathway in the germline, does not explain ends-in targeting products. We conclude that this example of gene targeting at the Nap1 locus provides added support for the efficiency of this method and its usefulness in targeting any arbitrary locus in the Drosophila genome. PMID:12618400

  11. Kin17 facilitates multiple double-strand break repair pathways that govern B cell class switching

    PubMed Central

    Le, Michael X.; Haddad, Dania; Ling, Alexanda K.; Li, Conglei; So, Clare C.; Chopra, Amit; Hu, Rui; Angulo, Jaime F.; Moffat, Jason; Martin, Alberto

    2016-01-01

    Class switch recombination (CSR) in B cells requires the timely repair of DNA double-stranded breaks (DSBs) that result from lesions produced by activation-induced cytidine deaminase (AID). Through a genome-wide RNAi screen, we identified Kin17 as a gene potentially involved in the maintenance of CSR in murine B cells. In this study, we confirm a critical role for Kin17 in CSR independent of AID activity. Furthermore, we make evident that DSBs generated by AID or ionizing radiation require Kin17 for efficient repair and resolution. Our report shows that reduced Kin17 results in an elevated deletion frequency following AID mutational activity in the switch region. In addition, deficiency in Kin17 affects the functionality of multiple DSB repair pathways, namely homologous recombination, non-homologous end-joining, and alternative end-joining. This report demonstrates the importance of Kin17 as a critical factor that acts prior to the repair phase of DSB repair and is of bona fide importance for CSR. PMID:27853268

  12. A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants.

    PubMed Central

    Merrill, B J; Holm, C

    1999-01-01

    To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants. PMID:10511542

  13. An alternative pathway of recombination of chromosomal fragments precedes recA-dependent recombination in the radioresistant bacterium Deinococcus radiodurans.

    PubMed

    Daly, M J; Minton, K W

    1996-08-01

    Deinococcus radiodurans R1 and other members of this genus are able to repair and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. The ability of R1 to repair completely > 100 double-strand breaks in its chromosome without lethality or mutagenesis is recA dependent. However, during the first 1.5 h after irradiation, recA+ and recA cells show similar increases in the average size of chromosomal fragments. In recA+ cells, DNA continues to enlarge to wild-type size within 29 h. However, in recA cells, no DNA repair is observed following the first 1.5 h postirradiation. This recA-independent effect was studied further, using two slightly different Escherichia coli plasmids forming adjacent duplication insertions in the chromosome, providing repetitive sequences suitable for circularization by non-recA-dependent pathways following irradiation. After exposure to 1.75 Mrad (17,500 Gy), circular derivatives of the integration units were detected in both recA+ and recA cells. These DNA circles were formed in the first 1.5 h postirradiation, several hours before the onset of detectable recA-dependent homologous recombination. By comparison, D. radiodurans strains containing the same E. coli plasmids as nonrepetitive direct insertions did not form circular derivatives of the integration units before or after irradiation in recA+ or recA cells. The circular derivatives of the tandemly integrated plasmids were formed before the onset of recA-dependent repair and have structures consistent with the hypothesis that DNA repair occurring immediately postirradiation is by a recA-independent single-strand annealing reaction and may be a preparatory step for further DNA repair in wild-type D. radiodurans.

  14. Multiple branches of the meiotic recombination pathway contribute independently to homolog pairing and stable juxtaposition during meiosis in budding yeast

    PubMed Central

    Peoples-Holst, Tamara L.; Burgess, Sean M.

    2005-01-01

    A unique aspect of meiosis is the segregation of homologous chromosomes at the meiosis I division. Homologs are physically connected prior to segregation by crossing over between nonsister chromatids. Crossovers arise from the repair of induced double-strand breaks (DSBs). In many organisms, more DSBs are formed than crossovers in a given nucleus. It has been previously suggested that repair of DSBs to noncrossover recombination products aids homolog alignment. Here we explore how two modes of the meiotic recombination pathway (crossover and noncrossover) and meiotic telomere reorganization contribute to the pairing and close juxtaposition of homologous chromosomes in budding yeast. We found that intermediates in the DSB repair pathway leading to both crossover and noncrossover recombination products contribute independently to close, stable homolog juxtaposition (CSHJ), a measurable state of homolog pairing. Analysis of the ndj1Δ mutant indicates that the effect of meiotic telomere reorganization on CSHJ is exerted through recombination intermediates at interstitial chromosomal loci, perhaps through the noncrossover branch of the DSB repair pathway. We suggest that transient, early DSB-initiated interactions, including those that give rise to noncrossovers, are important for homolog recognition and juxtaposition. PMID:15805472

  15. Transcription-associated recombination is independent of XRCC2 and mechanistically separate from homology-directed DNA double-strand break repair.

    PubMed

    Savolainen, Linda; Helleday, Thomas

    2009-02-01

    It has previously been shown that transcription greatly enhances recombination in mammalian cells. However, the proteins involved in catalysing this process and the recombination pathways involved in transcription-associated recombination (TAR) are still unknown. It is well established that both the BRCA2 protein and the RAD51 paralog protein XRCC2 are required for homologous recombination. Here, we show that the BRCA2 protein is also required for TAR, while the XRCC2 protein is not involved. Expression of the XRCC2 gene in XRCC2 mutated irs1 cells restores the defect in homologous recombination repair of an I-SceI-induced DNA double-strand break, while TAR is unaffected. Interestingly, the XRCC2-deficient irs1 cells are also proficient in recombination induced at slowed replication forks, suggesting that TAR is mechanistically linked with this recombination pathway. In conclusion, we show that TAR depends on BRCA2 but is independent of XRCC2, and that this recombination pathway is separate from that used to repair a two-ended DNA double-strand break.

  16. DNA replication meets genetic exchange: chromosomal damage and its repair by homologous recombination.

    PubMed

    Kuzminov, A

    2001-07-17

    Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair.

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

    PubMed

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

    2007-10-12

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

  18. Suberoylanilide hydroxamic acid (SAHA) enhances olaparib activity by targeting homologous recombination DNA repair in ovarian cancer.

    PubMed

    Konstantinopoulos, Panagiotis A; Wilson, Andrew J; Saskowski, Jeanette; Wass, Erica; Khabele, Dineo

    2014-06-01

    Approximately 50% of serous epithelial ovarian cancers (EOC) contain molecular defects in homologous recombination (HR) DNA repair pathways. Poly(ADP-ribose) polymerase inhibitors (PARPi) have efficacy in HR-deficient, but not in HR-proficient, EOC tumors as a single agent. Our goal was to determine whether the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), can sensitize HR-proficient ovarian cancer cells to the PARPi AZD-2281 (olaparib). Ovarian cancer cell lines (SKOV-3, OVCAR-8, NCI/ADR-Res, UWB1.289 BRCA1null and UWB1.289+BRCA1 wild-type) were treated with saline vehicle, olaparib, SAHA or olaparib/SAHA. Sulforhodamine B (SRB) assessed cytotoxicity and immunofluorescence and Western blot assays assessed markers of apoptosis (cleaved PARP) and DNA damage (pH2AX and RAD51). Drug effects were also tested in SKOV-3 xenografts in Nude mice. Affymetrix microarray experiments were performed in vehicle and SAHA-treated SKOV-3 cells. In a microarray analysis, SAHA induced coordinated down-regulation of HR pathway genes, including RAD51 and BRCA1. Nuclear co-expression of RAD51 and pH2AX, a marker of efficient HR repair, was reduced approximately 40% by SAHA treatment alone and combined with olaparib. SAHA combined with olaparib induced apoptosis and pH2AX expression to a greater extent than either drug alone. Olaparib reduced cell viability at increasing concentrations and SAHA enhanced these effects in 4 of 5 cell lines, including BRCA1 null and wild-type cells, in vitro and in SKOV-3 xenografts in vivo. These results provide preclinical rationale for targeting DNA damage response pathways by combining small molecule PARPi with HDACi as a mechanism for reducing HR efficiency in ovarian cancer. Copyright © 2014. Published by Elsevier Inc.

  19. Suberoylanilide Hydroxamic Acid (SAHA) enhances olaparib activity by targeting homologous recombination DNA repair in ovarian cancer

    PubMed Central

    Konstantinopoulos, Panagiotis A.; Wilson, Andrew J.; Saskowski, Jeanette; Wass, Erica; Khabele, Dineo

    2015-01-01

    Objectives Approximately 50% of serous epithelial ovarian cancers (EOC) contain molecular defects in homologous recombination (HR) DNA repair pathways. Poly(ADP-ribose) polymerase inhibitors (PARPi) have efficacy in HR-deficient, but not HR-proficient, EOC tumors as a single agent. Our goal was to determine whether the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), can sensitize HR-proficient ovarian cancer cells to the PARPi AZD-2281 (olaparib). Methods Ovarian cancer cell lines (SKOV-3, OVCAR-8, NCI/ADR-Res, UWB1.289 BRCA1null and UWB1.289 + BRCA1 wild-type) were treated with saline vehicle, olaparib, SAHA or olaparib/SAHA. Sulforhodamine B (SRB) assessed cytotoxicity and immunofluorescence and Western blot assays assessed markers of apoptosis (cleaved PARP) and DNA damage (pH2AX and RAD51). Drug effects were also tested in SKOV-3 xenografts in Nude mice. Affymetrix microarray experiments were performed in vehicle and SAHA-treated SKOV-3 cells. Results In a microarray analysis, SAHA induced coordinated down-regulation of HR pathway genes, including RAD51 and BRCA1. Nuclear co-expression of RAD51 and pH2AX, a marker of efficient HR repair, was reduced approximately 40% by SAHA treatment alone and combined with olaparib. SAHA combined with olaparib induced apoptosis and pH2AX expression to a greater extent than either drug alone. Olaparib reduced cell viability at increasing concentrations and SAHA enhanced these effects in 4 of 5 cell lines, including BRCA1 null and wild-type cells, in vitro and in SKOV-3 xenografts in vivo. Conclusions These results provide preclinical rationale for targeting DNA damage response pathways by combining small molecule PARPi with HDACi as a mechanism for reducing HR efficiency in ovarian cancer. PMID:24631446

  20. Prolonged particulate chromate exposure does not inhibit homologous recombination repair in North Atlantic right whale (Eubalaena glacialis) lung cells.

    PubMed

    Browning, Cynthia L; Wise, Catherine F; Wise, John Pierce

    2017-09-15

    Chromosome instability is a common feature of cancers that forms due to the misrepair of DNA double strand breaks. Homologous recombination (HR) repair is a high fidelity DNA repair pathway that utilizes a homologous DNA sequence to accurately repair such damage and protect the genome. Prolonged exposure (>72h) to the human lung carcinogen, particulate hexavalent chromium (Cr(VI)), inhibits HR repair, resulting in increased chromosome instability in human cells. Comparative studies have shown acute Cr(VI) exposure induces less chromosome damage in whale cells than human cells, suggesting investigating the effect of this carcinogen in other species may inform efforts to prevent Cr(VI)-induced chromosome instability. Thus, the goal of this study was to determine the effect of prolonged Cr(VI) exposure on HR repair and clastogenesis in North Atlantic right whale (Eubalaena glacialis) lung cells. We show particulate Cr(VI) induces HR repair activity after both acute (24h) and prolonged (120h) exposure in North Atlantic right whale cells. Although the RAD51 response was lower following prolonged Cr(VI) exposure compared to acute exposure, the response was sufficient for HR repair to occur. In accordance with active HR repair, no increase in Cr(VI)-induced clastogenesis was observed with increased exposure time. These results suggest prolonged Cr(VI) exposure affects HR repair and genomic stability differently in whale and human lung cells. Future investigation of the differences in how human and whale cells respond to chemical carcinogens may provide valuable insight into mechanisms of preventing chemical carcinogenesis. Copyright © 2017. Published by Elsevier Inc.

  1. Recombination between divergent sequences leads to cell death in a mismatch-repair-independent manner.

    PubMed

    Inbar, O; Kupiec, M

    2000-07-01

    Homologous recombination is an important DNA repair mechanism in vegetative cells. During the repair of double-strand breaks, genetic information is transferred between the interacting DNA sequences, thus creating a gene-conversion event. Gene conversion of a functional member of a gene family, which uses an inactive member (such as a pseudogene) as a template, might have deleterious consequences. It is therefore important for the cell to prevent recombination between divergent sequences. We have studied the repair of a double-strand break by recombination in a haploid yeast strain carrying 99% identical alleles located on different chromosomes. The fate of the broken chromosome was followed in the whole cell population without imposing selective constraints. Our results show that all the cells were able to repair the broken chromosome by gene conversion. During the repair, the cells arrest in the cell cycle with a "dumbbell" configuration characteristic of G2/M-arrested cells. Surprisingly, although all the cells repaired the broken chromosome, 60% of them were unable to resume growth and to form colonies after the repair was completed. The low level of cell recovery was due to the 1% divergence between the alleles, but was not dependent on the function of the mismatch-repair system. Cell death, however, could be prevented by the presence of an alternative source of perfect homology located on a different chromosome.

  2. Double-strand break repair and genetic recombination in topoisomerase and primase mutants of bacteriophage T4.

    PubMed

    Shcherbakov, Victor P; Kudryashova, Elena

    2014-09-01

    -branch migration step of the DSB repair pathway and partially deficient in HJ initiation. In apparent contradiction to their effects on the DSB-induced site-specific recombination, the topoisomerase and primase mutants demonstrated about 3-8-fold increase in the recombinant frequencies in the ordinary crosses, with the recombination running exclusively via patches. This implies that most of the spontaneous recombination events are not initiated by dsDNA ends in these mutants.

  3. Alkyltransferase-like proteins: Molecular switches between DNA repair pathways

    PubMed Central

    Tubbs, Julie L.; Tainer, John A.

    2011-01-01

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

  4. Electronic Pathways in Photoactivated Repair of UV Mutated DNA

    NASA Astrophysics Data System (ADS)

    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.

  5. CtIP-BRCA1 modulates the choice of DNA double-strand break repair pathway throughout the cell cycle

    PubMed Central

    Yun, Maximina H.; Hiom, Kevin

    2009-01-01

    The repair of DNA double-strand breaks (DSB) is tightly regulated during the cell cycle. In G1 phase, the absence of a sister chromatid means that repair of DSB occurs through non-homologous end-joining (NHEJ) or microhomology-mediated end-joining (MMEJ)1. These pathways often involve loss of DNA sequences at the break site and are therefore error-prone. In late S and G2 phases, even though DNA end-joining pathways remain functional2, there is an increase in repair of DSB by homologous recombination (HR), which is mostly error-free3,4. Consequently, the relative contribution of these different pathways to DSB repair in the cell cycle has a profound influence on the maintenance of genetic integrity. How then are DSB directed for repair by different, potentially competing, repair pathways? Here we identify a role for CtIP in this process in DT40. We establish that CtIP is not only required for repair of DSB by HR in S/G2 phase, but also for MMEJ in G1. The function of CtIP in HR, but not MMEJ, is dependent on the phosphorylation of serine residue 327 and recruitment of BRCA1. Cells expressing CtIP protein that cannot be phosphorylated at serine 327 are specifically defective in HR and exhibit decreased level of single-stranded DNA (ssDNA) after DNA damage, while MMEJ remains unaffected. Our data support a model in which phosphorylation of serine 327 of CtIP as cells enter S-phase and the recruitment of BRCA1 functions as a molecular switch to shift the balance of DSB repair from error-prone DNA end-joining to error-free homologous recombination (Supplementary Fig. 1). PMID:19357644

  6. Sgs1 and Mph1 Helicases Enforce the Recombination Execution Checkpoint During DNA Double-Strand Break Repair in Saccharomyces cerevisiae.

    PubMed

    Jain, Suvi; Sugawara, Neal; Mehta, Anuja; Ryu, Taehyun; Haber, James E

    2016-06-01

    We have previously shown that a recombination execution checkpoint (REC) regulates the choice of the homologous recombination pathway used to repair a given DNA double-strand break (DSB) based on the homology status of the DSB ends. If the two DSB ends are synapsed with closely-positioned and correctly-oriented homologous donors, repair proceeds rapidly by the gene conversion (GC) pathway. If, however, homology to only one of the ends is present, or if homologies to the two ends are situated far away from each other or in the wrong orientation, REC blocks the rapid initiation of new DNA synthesis from the synapsed end(s) and repair is carried out by the break-induced replication (BIR) machinery after a long pause. Here we report that the simultaneous deletion of two 3'→5' helicases, Sgs1 and Mph1, largely abolishes the REC-mediated lag normally observed during the repair of large gaps and BIR substrates, which now get repaired nearly as rapidly and efficiently as GC substrates. Deletion of SGS1 and MPH1 also produces a nearly additive increase in the efficiency of both BIR and long gap repair; this increase is epistatic to that seen upon Rad51 overexpression. However, Rad51 overexpression fails to mimic the acceleration in repair kinetics that is produced by sgs1Δ mph1Δ double deletion.

  7. Sgs1 and Mph1 Helicases Enforce the Recombination Execution Checkpoint During DNA Double-Strand Break Repair in Saccharomyces cerevisiae

    PubMed Central

    Jain, Suvi; Sugawara, Neal; Mehta, Anuja; Ryu, Taehyun; Haber, James E.

    2016-01-01

    We have previously shown that a recombination execution checkpoint (REC) regulates the choice of the homologous recombination pathway used to repair a given DNA double-strand break (DSB) based on the homology status of the DSB ends. If the two DSB ends are synapsed with closely-positioned and correctly-oriented homologous donors, repair proceeds rapidly by the gene conversion (GC) pathway. If, however, homology to only one of the ends is present, or if homologies to the two ends are situated far away from each other or in the wrong orientation, REC blocks the rapid initiation of new DNA synthesis from the synapsed end(s) and repair is carried out by the break-induced replication (BIR) machinery after a long pause. Here we report that the simultaneous deletion of two 3′→5′ helicases, Sgs1 and Mph1, largely abolishes the REC-mediated lag normally observed during the repair of large gaps and BIR substrates, which now get repaired nearly as rapidly and efficiently as GC substrates. Deletion of SGS1 and MPH1 also produces a nearly additive increase in the efficiency of both BIR and long gap repair; this increase is epistatic to that seen upon Rad51 overexpression. However, Rad51 overexpression fails to mimic the acceleration in repair kinetics that is produced by sgs1Δ mph1Δ double deletion. PMID:27075725

  8. Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways

    PubMed Central

    Arnal, Suzzette M.; Holub, Abigail J.; Salus, Sandra S.; Roth, David B.

    2010-01-01

    V(D)J recombination entails double-stranded DNA cleavage at the antigen receptor loci by the RAG1/2 proteins, which recognize conserved recombination signal sequences (RSSs) adjoining variable (V), diversity (D) and joining (J) gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ). Certain mutations in RAG1/2 destabilize the PCC, allowing DNA ends to access inappropriate repair pathways such as alternative NHEJ, an error-prone pathway implicated in chromosomal translocations. The PCC is thus thought to discourage aberrant rearrangements by controlling repair pathway choice. Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability. We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ. These data suggest that some non-consensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the PCC’s ability to restrict repair pathway choice. PMID:20139091

  9. Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways.

    PubMed

    Arnal, Suzzette M; Holub, Abigail J; Salus, Sandra S; Roth, David B

    2010-05-01

    V(D)J recombination entails double-stranded DNA cleavage at the antigen receptor loci by the RAG1/2 proteins, which recognize conserved recombination signal sequences (RSSs) adjoining variable (V), diversity (D) and joining (J) gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ). Certain mutations in RAG1/2 destabilize the PCC, allowing DNA ends to access inappropriate repair pathways such as alternative NHEJ, an error-prone pathway implicated in chromosomal translocations. The PCC is thus thought to discourage aberrant rearrangements by controlling repair pathway choice. Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability. We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ. These data suggest that some non-consensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the PCC's ability to restrict repair pathway choice.

  10. DNA damage tolerance by recombination: Molecular pathways and DNA structures.

    PubMed

    Branzei, Dana; Szakal, Barnabas

    2016-08-01

    Replication perturbations activate DNA damage tolerance (DDT) pathways, which are crucial to promote replication completion and to prevent fork breakage, a leading cause of genome instability. One mode of DDT uses translesion synthesis polymerases, which however can also introduce mutations. The other DDT mode involves recombination-mediated mechanisms, which are generally accurate. DDT occurs prevalently postreplicatively, but in certain situations homologous recombination is needed to restart forks. Fork reversal can function to stabilize stalled forks, but may also promote error-prone outcome when used for fork restart. Recent years have witnessed important advances in our understanding of the mechanisms and DNA structures that mediate recombination-mediated damage-bypass and highlighted principles that regulate DDT pathway choice locally and temporally. In this review we summarize the current knowledge and paradoxes on recombination-mediated DDT pathways and their workings, discuss how the intermediate DNA structures may influence genome integrity, and outline key open questions for future research. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

  11. PARP3 affects the relative contribution of homologous recombination and nonhomologous end-joining pathways

    PubMed Central

    Guirouilh Barbat, Josée; Bonnet, Marie-Elise; Illuzzi, Giuditta; Ronde, Philippe; Gauthier, Laurent R.; Magroun, Najat; Rajendran, Anbazhagan; Lopez, Bernard S.; Scully, Ralph; Boussin, François D.; Schreiber, Valérie; Dantzer, Françoise

    2014-01-01

    The repair of toxic double-strand breaks (DSB) is critical for the maintenance of genome integrity. The major mechanisms that cope with DSB are: homologous recombination (HR) and classical or alternative nonhomologous end joining (C-NHEJ versus A-EJ). Because these pathways compete for the repair of DSB, the choice of the appropriate repair pathway is pivotal. Among the mechanisms that influence this choice, deoxyribonucleic acid (DNA) end resection plays a critical role by driving cells to HR, while accurate C-NHEJ is suppressed. Furthermore, end resection promotes error-prone A-EJ. Increasing evidence define Poly(ADP-ribose) polymerase 3 (PARP3, also known as ARTD3) as an important player in cellular response to DSB. In this work, we reveal a specific feature of PARP3 that together with Ku80 limits DNA end resection and thereby helps in making the choice between HR and NHEJ pathways. PARP3 interacts with and PARylates Ku70/Ku80. The depletion of PARP3 impairs the recruitment of YFP-Ku80 to laser-induced DNA damage sites and induces an imbalance between BRCA1 and 53BP1. Both events result in compromised accurate C-NHEJ and a concomitant increase in DNA end resection. Nevertheless, HR is significantly reduced upon PARP3 silencing while the enhanced end resection causes mutagenic deletions during A-EJ. As a result, the absence of PARP3 confers hypersensitivity to anti-tumoral drugs generating DSB. PMID:24598253

  12. MOF phosphorylation by ATM regulates 53BP1-mediated DSB repair pathway choice

    PubMed Central

    Gupta, Arun; Hunt, Clayton R.; Hegdec, Muralidhar L.; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh1, Mayank; Ramnarain, Deepti B.; Hittelman, Walter N.; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K.; Ludwig, Thomas; Pandita, Raj K.; Tyler, Jessica K.; Pandita, Tej K.

    2014-01-01

    Cell cycle phase is a critical determinant of the choice between DNA damage repair by non-homologous end joining (NHEJ) or homologous recombination (HR). Here we report that DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF co-localizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S- and G2-phase but not G1-phase cells. Expression of MOF-T392A also reverses the reduction in DSB associated 53BP1 seen in wild type S/G2-phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair and decreased cell survival following irradiation. These data support a model whereby ATM mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2-phase. PMID:24953651

  13. MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

    PubMed

    Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

    2014-07-10

    Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin

    SciTech Connect

    Noguchi, Miho; Yu, Dong; Hirayama, Ryoichi; Ninomiya, Yasuharu; Sekine, Emiko; Kubota, Nobuo; Ando, Koichi; Okayasu, Ryuichi . E-mail: rokayasu@nirs.go.jp

    2006-12-22

    In order to investigate the mechanism of radio-sensitization by an Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), we studied repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with 17-AAG. DSBs are thought to be the critical target for radiation-induced cell death. Two human tumor cell lines DU145 and SQ-5 which showed clear radio-sensitization by 17-AAG revealed a significant inhibition of DSB repair, while normal human cells which did not show radio-sensitization by the drug indicated no change in the DSB repair kinetics with 17-AAG. We further demonstrated that BRCA2 was a novel client protein for Hsp90, and 17-AAG caused the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for homologous recombination (HR) pathway of DSB repair. Our data demonstrate for the first time that 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing.

  15. MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.

    PubMed

    Kostyrko, Kaja; Neuenschwander, Samuel; Junier, Thomas; Regamey, Alexandre; Iseli, Christian; Schmid-Siegert, Emanuel; Bosshard, Sandra; Majocchi, Stefano; Le Fourn, Valérie; Girod, Pierre-Alain; Xenarios, Ioannis; Mermod, Nicolas

    2017-02-01

    Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis-dependent microhomology-mediated end-joining (SD-MMEJ) activities. Genome-wide analysis of the integration loci and junction sequences validated the prevalent use of the SD-MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD-MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384-396. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

  16. Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase

    PubMed Central

    Lee, Kyung-Jong; Saha, Janapriya; Sun, Jingxin; Fattah, Kazi R.; Wang, Shu-Chi; Jakob, Burkhard; Chi, Linfeng; Wang, Shih-Ya; Taucher-Scholz, Gisela; Davis, Anthony J.; Chen, David J.

    2016-01-01

    Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku's affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells. PMID:26712563

  17. Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase.

    PubMed

    Lee, Kyung-Jong; Saha, Janapriya; Sun, Jingxin; Fattah, Kazi R; Wang, Shu-Chi; Jakob, Burkhard; Chi, Linfeng; Wang, Shih-Ya; Taucher-Scholz, Gisela; Davis, Anthony J; Chen, David J

    2016-02-29

    Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku's affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells.

  18. ATR-ATRIP kinase complex triggers activation of the Fanconi anemia DNA repair pathway.

    PubMed

    Shigechi, Tomoko; Tomida, Junya; Sato, Koichi; Kobayashi, Masahiko; Eykelenboom, John K; Pessina, Fabio; Zhang, Yanbin; Uchida, Emi; Ishiai, Masamichi; Lowndes, Noel F; Yamamoto, Kenichi; Kurumizaka, Hitoshi; Maehara, Yoshihiko; Takata, Minoru

    2012-03-01

    ATR kinase activates the S-phase checkpoint when replication forks stall at sites of DNA damage. This event also causes phosphorylation of the Fanconi anemia (FA) protein FANCI, triggering its monoubiquitination of the key DNA repair factor FANCD2 by the FA core E3 ligase complex, thereby promoting this central pathway of DNA repair which permits replication to be restarted. However, the interplay between ATR and the FA pathway has been unclear. In this study, we present evidence that their action is directly linked, gaining insights into this relationship in a DT40 mutant cell line that is conditionally deficient in the critical ATR-binding partner protein ATRIP. Using this system, we showed that ATRIP was crucial for DNA damage-induced FANCD2 monoubiquitination and FANCI phosphorylation. ATR kinase phosphorylated recombinant FANCI protein in vitro, which was facilitated by the presence of FANCD2. Mechanistic investigations revealed that the RPA region but not the TopBP1 region of ATRIP was required for FANCD2 monoubiquitination, whereas Chk1 phosphorylation relied upon both domains. Together, our findings identify ATR as the kinase responsible for activating the FA pathway of DNA repair.

  19. A link between double-strand break-related repair and V(D)J recombination: the scid mutation

    SciTech Connect

    Hendrickson, E.A.; Qin, X.Q.; Bump, E.A.; Schatz, D.G.; Oettinger, M.; Weaver, D.T. )

    1991-05-15

    We show here that mammalian site-specific recombination and DNA-repair pathways share a common factor. The effects of DNA-damaging agents on cell lines derived from mice homozygous for the scid (severe combined immune deficiency) mutation were studied. Surprisingly, all scid cell lines exhibited a profound hypersensitivity to DNA-damaging agents that caused double-strand breaks (x-irradiation and bleomycin) but not to other chemicals that caused single-strand breaks or cross-links. Neutral filter elution assays demonstrated that the x-irradiation hypersensitivity could be correlated with a deficiency in repairing double-strand breaks. These data suggest that the scid gene product is involved in two pathways: DNA repair of random double-strand breaks and the site-specific and lymphoid-restricted variable-(diversity)-joining (V(D)J) DNA rearrangement process. We propose that the scid gene product performs a similar function in both pathways and may be a ubiquitous protein.

  20. Rdh54, a Rad54 Homologue in Saccharomyces Cerevisiae, Is Required for Mitotic Diploid-Specific Recombination and Repair and for Meiosis

    PubMed Central

    Klein, H. L.

    1997-01-01

    Most mitotic recombination and repair genes of Saccharomyces cerevisiae show no specificity of action for the genome ploidy. We describe here a novel repair and recombination gene that is specific for recombination and repair between homologous chromosomes. The RDH54 gene is homologous to the RAD54 gene, but rdh54 mutants do not show sensitivity to methyl methanesulfonate at concentrations that sensitize a rad54 mutant. However, the rdh54 null mutation enhances the methyl methanesulfonate sensitivity of a rad54 mutant and single rdh54 mutants are sensitive to prolonged exposure at high concentrations of methyl methanesulfonate. The RDH54 gene is required for recombination, but only in a diploid. We present evidence showing that the RDH54 gene is required for interhomologue gene conversion but not intrachromosomal gene conversion. The rdh54 mutation confers diploid-specific lethalities and reduced growth in various mutant backgrounds. These phenotypes are due to attempted recombination. The RDH54 gene is also required for meiosis as homozygous mutant diploids show very poor sporulation and reduced spore viability. The role of the RDH54 gene in mitotic repair and in meiosis and the pathway in which it acts are discussed. PMID:9409819

  1. Classical and alternative end-joining pathways for repair of lymphocyte-specific and general DNA double-strand breaks.

    PubMed

    Boboila, Cristian; Alt, Frederick W; Schwer, Bjoern

    2012-01-01

    Classical nonhomologous end joining (C-NHEJ) is one of the two major known pathways for the repair of DNA double-strand breaks (DSBs) in mammalian cells. Our understanding of C-NHEJ has been derived, in significant part, through studies of programmed physiologic DNA DSBs formed during V(D)J recombination in the developing immune system. Studies of immunoglobulin heavy-chain (IgH) class-switch recombination (CSR) also have revealed that there is an "alternative" end-joining process (A-EJ) that can function, relatively robustly, in the repair of DSBs in activated mature B lymphocytes. This A-EJ process has also been implicated in the formation of oncogenic translocations found in lymphoid tumors. In this review, we discuss our current understanding of C-NHEJ and A-EJ in the context of V(D)J recombination, CSR, and the formation of chromosomal translocations. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    PubMed Central

    Grin, Inga; Ishchenko, Alexander A.

    2016-01-01

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

  3. XLF/Cernunnos: An important but puzzling participant in the nonhomologous end joining DNA repair pathway.

    PubMed

    Menon, Vijay; Povirk, Lawrence F

    2017-10-01

    DNA double strand breaks (DSBs) are one of the most deleterious DNA lesions that promote cell death, genomic instability and carcinogenesis. The two major cellular mechanisms that repair DSBs are Nonhomologous End-Joining (NHEJ) and Homologous Recombination Repair (HRR). NHEJ is the predominant pathway, in which XLF (also called Cernunnos) is a key player. Patients with XLF mutation exhibit microcephaly, lymphopenia, and growth retardation, and are immunodeficient and radiosensitive. During NHEJ, XLF interacts with XRCC4-Ligase IV, stimulates its ligase activity, and forms DNA-binding filaments of alternating XLF and XRCC4 dimers that may serve to align broken DNA and promote ligation of noncomplementary ends. Despite its central role in NHEJ, the effects of XLF deficiency are surprisingly variable in different biological contexts, and different individual cell lines. This review summarizes the role of XLF in NHEJ, and the unexpected complexity of its interplay with other repair factors in supporting radiosurvival and V(D)J recombination. Copyright © 2017. Published by Elsevier B.V.

  4. XPF-ERCC1 participates in the Fanconi anemia pathway of cross-link repair.

    PubMed

    Bhagwat, Nikhil; Olsen, Anna L; Wang, Anderson T; Hanada, Katsuhiro; Stuckert, Patricia; Kanaar, Roland; D'Andrea, Alan; Niedernhofer, Laura J; McHugh, Peter J

    2009-12-01

    Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81(-/-), Ercc1(-/-), and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.

  5. How RecBCD Enzyme and Chi Promote DNA Break Repair and Recombination: a Molecular Biologist's View

    PubMed Central

    2012-01-01

    Summary: The repair of DNA double-strand breaks (DSBs) is essential for cell viability and important for homologous genetic recombination. In enteric bacteria such as Escherichia coli, the major pathway of DSB repair requires the RecBCD enzyme, a complex helicase-nuclease regulated by a simple unique DNA sequence called Chi. How Chi regulates RecBCD has been extensively studied by both genetics and biochemistry, and two contrasting mechanisms to generate a recombinogenic single-stranded DNA tail have been proposed: the nicking of one DNA strand at Chi versus the switching of degradation from one strand to the other at Chi. Which of these reactions occurs in cells has remained unproven because of the inability to detect intracellular DNA intermediates in bacterial recombination and DNA break repair. Here, I discuss evidence from a combination of genetics and biochemistry indicating that nicking at Chi is the intracellular (in vivo) reaction. This example illustrates the need for both types of analysis (i.e., molecular biology) to uncover the mechanism and control of complex processes in living cells. PMID:22688812

  6. EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair

    PubMed Central

    Wu, Yuehan; Lee, Suk-Hee; Williamson, Elizabeth A.; Reinert, Brian L.; Cho, Ju Hwan; Xia, Fen; Jaiswal, Aruna Shanker; Srinivasan, Gayathri; Patel, Bhavita; Brantley, Alexis; Zhou, Daohong; Shao, Lijian; Pathak, Rupak; Hauer-Jensen, Martin; Singh, Sudha; Kong, Kimi; Wu, Xaiohua; Kim, Hyun-Suk; Beissbarth, Timothy; Gaedcke, Jochen; Burma, Sandeep; Nickoloff, Jac A.; Hromas, Robert A.

    2015-01-01

    Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ. PMID:26684013

  7. Alleles of the yeast Pms1 mismatch-repair gene that differentially affect recombination- and replication-related processes.

    PubMed Central

    Welz-Voegele, Caroline; Stone, Jana E; Tran, Phuoc T; Kearney, Hutton M; Liskay, R Michael; Petes, Thomas D; Jinks-Robertson, Sue

    2002-01-01

    Mismatch-repair (MMR) systems promote eukaryotic genome stability by removing errors introduced during DNA replication and by inhibiting recombination between nonidentical sequences (spellchecker and antirecombination activities, respectively). Following a common mismatch-recognition step effected by MutS-homologous Msh proteins, homologs of the bacterial MutL ATPase (predominantly the Mlh1p-Pms1p heterodimer in yeast) couple mismatch recognition to the appropriate downstream processing steps. To examine whether the processing steps in the spellchecker and antirecombination pathways might differ, we mutagenized the yeast PMS1 gene and screened for mitotic separation-of-function alleles. Two alleles affecting only the antirecombination function of Pms1p were identified, one of which changed an amino acid within the highly conserved ATPase domain. To more specifically address the role of ATP binding/hydrolysis in MMR-related processes, we examined mutations known to compromise the ATPase activity of Pms1p or Mlh1p with respect to the mitotic spellchecker and antirecombination activities and with respect to the repair of mismatches present in meiotic recombination intermediates. The results of these analyses confirm a differential requirement for the Pms1p ATPase activity in replication vs. recombination processes, while demonstrating that the Mlh1p ATPase activity is important for all examined MMR-related functions. PMID:12454061

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

    PubMed

    Thapar, Upasna; Demple, Bruce

    2017-01-01

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

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

    PubMed Central

    Thapar, Upasna; Demple, Bruce

    2017-01-01

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

  10. Increased sensitivity to ionizing radiation by targeting the homologous recombination pathway in glioma initiating cells.

    PubMed

    Lim, Yi Chieh; Roberts, Tara L; Day, Bryan W; Stringer, Brett W; Kozlov, Sergei; Fazry, Shazrul; Bruce, Zara C; Ensbey, Kathleen S; Walker, David G; Boyd, Andrew W; Lavin, Martin F

    2014-12-01

    Glioblastoma is deemed the most malignant form of brain tumour, particularly due to its resistance to conventional treatments. A small surviving group of aberrant stem cells termed glioma initiation cells (GICs) that escape surgical debulking are suggested to be the cause of this resistance. Relatively quiescent in nature, GICs are capable of driving tumour recurrence and undergo lineage differentiation. Most importantly, these GICs are resistant to radiotherapy, suggesting that radioresistance contribute to their survival. In a previous study, we demonstrated that GICs had a restricted double strand break (DSB) repair pathway involving predominantly homologous recombination (HR) associated with a lack of functional G1/S checkpoint arrest. This unusual behaviour led to less efficient non-homologous end joining (NHEJ) repair and overall slower DNA DSB repair kinetics. To determine whether specific targeting of the HR pathway with small molecule inhibitors could increase GIC radiosensitivity, we used the Ataxia-telangiectasia mutated inhibitor (ATMi) to ablate HR and the DNA-dependent protein kinase inhibitor (DNA-PKi) to inhibit NHEJ. Pre-treatment with ATMi prior to ionizing radiation (IR) exposure prevented HR-mediated DNA DSB repair as measured by Rad51 foci accumulation. Increased cell death in vitro and improved in vivo animal survival could be observed with combined ATMi and IR treatment. Conversely, DNA-PKi treatment had minimal impact on GICs ability to resolve DNA DSB after IR with only partial reduction in cell survival, confirming the major role of HR. These results provide a mechanistic insight into the predominant form of DNA DSB repair in GICs, which when targeted may be a potential translational approach to increase patient survival. Copyright © 2014. Published by Elsevier B.V.

  11. The Nucleotide Excision Repair Pathway Limits L1 Retrotransposition

    PubMed Central

    Servant, Geraldine; Streva, Vincent A.; Derbes, Rebecca S.; Wijetunge, Madushani I.; Neeland, Marc; White, Travis B.; Belancio, Victoria P.; Roy-Engel, Astrid M.; Deininger, Prescott L.

    2017-01-01

    Long interspersed elements 1 (L1) are active mobile elements that constitute almost 17% of the human genome. They amplify through a “copy-and-paste” mechanism termed retrotransposition, and de novo insertions related to these elements have been reported to cause 0.2% of genetic diseases. Our previous data demonstrated that the endonuclease complex ERCC1-XPF, which cleaves a 3′ DNA flap structure, limits L1 retrotransposition. Although the ERCC1-XPF endonuclease participates in several different DNA repair pathways, such as single-strand annealing, or in telomere maintenance, its recruitment to DNA lesions is best characterized in the nucleotide excision repair (NER) pathway. To determine if the NER pathway prevents the insertion of retroelements in the genome, we monitored the retrotransposition efficiencies of engineered L1 elements in NER-deficient cells and in their complemented versions. Core proteins of the NER pathway, XPD and XPA, and the lesion binding protein, XPC, are involved in limiting L1 retrotransposition. In addition, sequence analysis of recovered de novo L1 inserts and their genomic locations in NER-deficient cells demonstrated the presence of abnormally large duplications at the site of insertion, suggesting that NER proteins may also play a role in the normal L1 insertion process. Here, we propose new functions for the NER pathway in the maintenance of genome integrity: limitation of insertional mutations caused by retrotransposons and the prevention of potentially mutagenic large genomic duplications at the site of retrotransposon insertion events. PMID:28049704

  12. The Nucleotide Excision Repair Pathway Limits L1 Retrotransposition.

    PubMed

    Servant, Geraldine; Streva, Vincent A; Derbes, Rebecca S; Wijetunge, Madushani I; Neeland, Marc; White, Travis B; Belancio, Victoria P; Roy-Engel, Astrid M; Deininger, Prescott L

    2017-01-01

    Long interspersed elements 1 (L1) are active mobile elements that constitute almost 17% of the human genome. They amplify through a "copy-and-paste" mechanism termed retrotransposition, and de novo insertions related to these elements have been reported to cause 0.2% of genetic diseases. Our previous data demonstrated that the endonuclease complex ERCC1-XPF, which cleaves a 3' DNA flap structure, limits L1 retrotransposition. Although the ERCC1-XPF endonuclease participates in several different DNA repair pathways, such as single-strand annealing, or in telomere maintenance, its recruitment to DNA lesions is best characterized in the nucleotide excision repair (NER) pathway. To determine if the NER pathway prevents the insertion of retroelements in the genome, we monitored the retrotransposition efficiencies of engineered L1 elements in NER-deficient cells and in their complemented versions. Core proteins of the NER pathway, XPD and XPA, and the lesion binding protein, XPC, are involved in limiting L1 retrotransposition. In addition, sequence analysis of recovered de novo L1 inserts and their genomic locations in NER-deficient cells demonstrated the presence of abnormally large duplications at the site of insertion, suggesting that NER proteins may also play a role in the normal L1 insertion process. Here, we propose new functions for the NER pathway in the maintenance of genome integrity: limitation of insertional mutations caused by retrotransposons and the prevention of potentially mutagenic large genomic duplications at the site of retrotransposon insertion events. Copyright © 2017 Servant et al.

  13. Repair of Single- and Multiple-Substitution Mismatches during Recombination in Streptococcus Pneumoniae

    PubMed Central

    Gasc, A. M.; Sicard, A. M.; Claverys, J. P.

    1989-01-01

    The use as genetic markers, during transformation of Streptococcus pneumoniae, of 19 sequences differing from wild type, located throughout the amiA locus, enabled us to examine the fate of 24 single- and 11 multiple-mismatches during recombination. Tentative mismatch ranking as a function of decreasing repair efficiency by the Hex mismatch repair system is G/T = A/C = G/G (maximum repair: 90-95%) > C/T (mostly 75 to 90% repair) > A/A (from 50 to 90% repair) > T/T (50-65% repair) > A/G (from 0 to 20% repair) > C/C. No indication of correction of the latter has been obtained. Over the limited number of samples examined, we observed no influence of the base composition of the surrounding sequence on correction efficiency for both transition mismatches and for G/G and C/C. Variations in the surrounding sequence affect repair of A/G and C/T, and, even more strongly, of A/A and T/T. No simple correlation to the G:C content of the surrounding sequence is apparent from our results, in contrast to the conclusion drawn for the Mut mismatch repair system of Escherichia coli. Examination of the fate of multiple mismatches suggests that C/C may sometimes impede recognition of otherwise corrected mismatches. PMID:2645195

  14. ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.

    PubMed Central

    Brookman, K W; Lamerdin, J E; Thelen, M P; Hwang, M; Reardon, J T; Sancar, A; Zhou, Z Q; Walter, C A; Parris, C N; Thompson, L H

    1996-01-01

    ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues. PMID:8887684

  15. Mismatch repair regulates homologous recombination, but has little influence on antigenic variation, in Trypanosoma brucei.

    PubMed

    Bell, Joanna S; McCulloch, Richard

    2003-11-14

    Antigenic variation is critical in the life of the African trypanosome, as it allows the parasite to survive in the face of host immunity and enhance its transmission to other hosts. Much of trypanosome antigenic variation uses homologous recombination of variant surface glycoprotein (VSG)-encoding genes into specialized transcription sites, but little is known about the processes that regulate it. Here we describe the effects on VSG switching when two central mismatch repair genes, MSH2 and MLH1, are mutated. We show that disruption of the parasite mismatch repair system causes an increased frequency of homologous recombination, both between perfectly matched DNA molecules and between DNA molecules with divergent sequences. Mismatch repair therefore provides an important regulatory role in homologous recombination in this ancient eukaryote. Despite this, the mismatch repair system has no detectable role in regulating antigenic variation, meaning that VSG switching is either immune to mismatch selection or that mismatch repair acts in a subtle manner, undetectable by current assays.

  16. Production of extrachromosomal microDNAs is linked to mismatch repair pathways and transcriptional activity

    PubMed Central

    Dillon, Laura W.; Kumar, Pankaj; Shibata, Yoshiyuki; Wang, Yuh-Hwa; Willcox, Smaranda; Griffith, Jack D.; Pommier, Yves; Takeda, Shunichi; Dutta, Anindya

    2015-01-01

    SUMMARY MicroDNAs are <400-base extrachromosomal circles found in mammalian cells. Tens of thousands of microDNAs have been found in all tissue types, including sperm. MicroDNAs arise preferentially from areas with high gene density, GC content, and exon density, from promoters with activating chromatin modifications and in sperm from the 5'-UTR of full-length LINE-1 elements, but are depleted from lamin-associated heterochromatin. Analysis of microDNAs from a set of human cancer cell lines revealed lineage-specific patterns of microDNA origins. A survey of microDNAs from chicken cells defective in various DNA repair proteins reveal that homologous recombination and nonhomologous end joining repair pathways are not required for microDNA production. Deletion of the MSH3 DNA mismatch repair protein results in a significant decrease in microDNA abundance, specifically from non-CpG genomic regions. Thus, microDNAs arise as part of normal cellular physiology; either from DNA breaks associated with RNA metabolism or from replication slippage followed by mismatch repair. PMID:26051933

  17. Production of Extrachromosomal MicroDNAs Is Linked to Mismatch Repair Pathways and Transcriptional Activity.

    PubMed

    Dillon, Laura W; Kumar, Pankaj; Shibata, Yoshiyuki; Wang, Yuh-Hwa; Willcox, Smaranda; Griffith, Jack D; Pommier, Yves; Takeda, Shunichi; Dutta, Anindya

    2015-06-23

    MicroDNAs are <400-base extrachromosomal circles found in mammalian cells. Tens of thousands of microDNAs have been found in all tissue types, including sperm. MicroDNAs arise preferentially from areas with high gene density, GC content, and exon density from promoters with activating chromatin modifications and in sperm from the 5'-UTR of full-length LINE-1 elements, but are depleted from lamin-associated heterochromatin. Analysis of microDNAs from a set of human cancer cell lines revealed lineage-specific patterns of microDNA origins. A survey of microDNAs from chicken cells defective in various DNA repair proteins reveals that homologous recombination and non-homologous end joining repair pathways are not required for microDNA production. Deletion of the MSH3 DNA mismatch repair protein results in a significant decrease in microDNA abundance, specifically from non-CpG genomic regions. Thus, microDNAs arise as part of normal cellular physiology—either from DNA breaks associated with RNA metabolism or from replication slippage followed by mismatch repair.

  18. The effects of mismatch repair and RAD1 genes on interchromosomal crossover recombination in Saccharomyces cerevisiae.

    PubMed

    Nicholson, Ainsley; Fabbri, Rebecca M; Reeves, Jason W; Crouse, Gray F

    2006-06-01

    We have previously shown that recombination between 400-bp substrates containing only 4-bp differences, when present in an inverted repeat orientation, is suppressed by >20-fold in wild-type strains of S. cerevisiae. Among the genes involved in this suppression were three genes involved in mismatch repair--MSH2, MSH3, and MSH6--and one in nucleotide excision repair, RAD1. We now report the involvement of these genes in interchromosomal recombination occurring via crossovers using these same short substrates. In these experiments, recombination was stimulated by a double-strand break generated by the HO endonuclease and can occur between completely identical (homologous) substrates or between nonidentical (homeologous) substrates. In addition, a unique feature of this system is that recombining DNA strands can be given a choice of either type of substrate. We find that interchromosomal crossover recombination with these short substrates is severely inhibited in the absence of MSH2, MSH3, or RAD1 and is relatively insensitive to the presence of mismatches. We propose that crossover recombination with these short substrates requires the products of MSH2, MSH3, and RAD1 and that these proteins have functions in recombination in addition to the removal of terminal nonhomology. We further propose that the observed insensitivity to homeology is a result of the difference in recombinational mechanism and/or the timing of the observed recombination events. These results are in contrast with those obtained using longer substrates and may be particularly relevant to recombination events between the abundant short repeated sequences that characterize the genomes of higher eukaryotes.

  19. Homologous recombination contributes to the repair of DNA double-strand breaks induced by high-energy iron ions

    SciTech Connect

    Zafar, Faria; Seidler, Sara B.; Kronenberg, Amy; Schild, David; Wiese, Claudia

    2010-06-29

    To test the contribution of homologous recombinational repair (HRR) in repairing DNA damaged sites induced by high-energy iron ions, we used: (1) HRR-deficient rodent cells carrying a deletion in the RAD51D gene and (2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We show that in response to iron ions, HRR contributes to cell survival in rodent cells, and that HRR-deficiency abrogates RAD51 foci formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 foci formation. For human cells irradiated with iron ions, cell survival is decreased, and, in p53 mutant cells, the levels of mutagenesis are increased when HRR is impaired. Human cells synchronized in S phase exhibit more pronounced resistance to iron ions as compared with cells in G1 phase, and this increase in radioresistance is diminished by RAD51 knockdown. These results implicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged particle irradiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival in response to high-energy high LET radiation.

  20. MET inhibition in tumor cells by PHA665752 impairs homologous recombination repair of DNA double strand breaks.

    PubMed

    Medová, Michaela; Aebersold, Daniel M; Zimmer, Yitzhak

    2012-02-01

    Abnormal activation of cellular DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems has broad implications for both cancer biology and treatment. Recent studies suggest a potential link between DNA repair and aberrant activation of the hepatocyte growth factor receptor Mesenchymal-Epithelial Transition (MET), an oncogene that is overexpressed in numerous types of human tumors and considered a prime target in clinical oncology. Using the homologous recombination (HR) direct-repeat direct-repeat green fluorescent protein ((DR)-GFP) system, we show that MET inhibition in tumor cells with deregulated MET activity by the small molecule PHA665752 significantly impairs in a dose-dependent manner HR. Using cells that express MET-mutated variants that respond differentially to PHA665752, we confirm that the observed HR inhibition is indeed MET-dependent. Furthermore, our data also suggest that decline in HR-dependent DNA repair activity is not a secondary effect due to cell cycle alterations caused by PHA665752. Mechanistically, we show that MET inhibition affects the formation of the RAD51-BRCA2 complex, which is crucial for error-free HR repair of double strand DNA lesions, presumably via downregulation and impaired translocation of RAD51 into the nucleus. Taken together, these findings assist to further support the role of MET in the cellular DNA damage response and highlight the potential future benefit of MET inhibitors for the sensitization of tumor cells to DNA damaging agents.

  1. DNA-PK, ATM and ATR collaboratively regulate p53-RPA interaction to facilitate homologous recombination DNA repair.

    PubMed

    Serrano, M A; Li, Z; Dangeti, M; Musich, P R; Patrick, S; Roginskaya, M; Cartwright, B; Zou, Y

    2013-05-09

    Homologous recombination (HR) and nonhomologous end joining (NHEJ) are two distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-dependent protein kinase (DNA-PK), the core component of NHEJ, partnering with DNA-damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), regulates HR repair of DSBs. The regulation was accomplished through modulation of the p53 and replication protein A (RPA) interaction. We show that upon DNA damage, p53 and RPA were freed from a p53-RPA complex by simultaneous phosphorylations of RPA at the N-terminus of RPA32 subunit by DNA-PK and of p53 at Ser37 and Ser46 in a Chk1/Chk2-independent manner by ATR and ATM, respectively. Neither the phosphorylation of RPA nor of p53 alone could dissociate p53 and RPA. Furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR repair and NHEJ through the co-regulation of p53-RPA interaction by DNA-PK, ATM and ATR.

  2. Live imaging of induced and controlled DNA double-strand break formation reveals extremely low repair by homologous recombination in human cells.

    PubMed

    Shahar, O D; Raghu Ram, E V S; Shimshoni, E; Hareli, S; Meshorer, E; Goldberg, M

    2012-07-26

    DNA double-strand breaks (DSBs), the most hazardous DNA lesions, may result in genomic instability, a hallmark of cancer cells. The main DSB repair pathways are non-homologous end joining (NHEJ) and homologous recombination (HR). In mammalian cells, NHEJ, which can lead to inaccurate repair, predominates. HR repair (HRR) is considered accurate and is restricted to S, G2 and M phases of the cell cycle. Despite its importance, many aspects regarding HRR remain unknown. Here, we developed a novel inducible on/off switch cell system that enables, for the first time, to induce a DSB in a rapid and reversible manner in human cells. By limiting the duration of DSB induction, we found that non-persistent endonuclease-induced DSBs are rarely repaired by HR, whereas persistent DSBs result in the published HRR frequencies (non-significant HR frequency versus frequency of ∼10%, respectively). We demonstrate that these DSBs are repaired by an accurate repair mechanism, which is distinguished from HRR (most likely, error-free NHEJ). Notably, our data reveal that HRR frequencies of endonuclease-induced DSBs in human cells are >10-fold lower than what was previously estimated by prevailing methods, which resulted in recurrent DSB formation. Our findings suggest a role for HRR mainly in repairing challenging DSBs, in contrast to uncomplicated lesions that are frequently repaired by NHEJ. Preventing HR from repairing DSBs in the complex and repetitive human genome probably has an essential role in maintaining genomic stability.

  3. Replication Checkpoint Kinase Cds1 Regulates Recombinational Repair Protein Rad60

    PubMed Central

    Boddy, Michael N.; Shanahan, Paul; Hayes McDonald, W.; Lopez-Girona, Antonia; Noguchi, Eishi; Yates III, John R.; Russell, Paul

    2003-01-01

    Genome integrity is protected by Cds1 (Chk2), a checkpoint kinase that stabilizes arrested replication forks. How Cds1 accomplishes this task is unknown. We report that Cds1 interacts with Rad60, a protein required for recombinational repair in fission yeast. Cds1 activation triggers Rad60 phosphorylation and nuclear delocalization. A Rad60 mutant that inhibits regulation by Cds1 renders cells specifically sensitive to replication fork arrest. Genetic and biochemical studies indicate that Rad60 functions codependently with Smc5 and Smc6, subunits of an SMC (structural maintenance of chromosomes) complex required for recombinational repair. These studies indicate that regulation of Rad60 is an important part of the replication checkpoint response controlled by Cds1. We propose that control of Rad60 regulates recombination events at stalled forks. PMID:12897162

  4. A Synthetic Lethal Screen Identifies DNA Repair Pathways that Sensitize Cancer Cells to Combined ATR Inhibition and Cisplatin Treatments

    PubMed Central

    Mohni, Kareem N.; Thompson, Petria S.; Luzwick, Jessica W.; Glick, Gloria G.; Pendleton, Christopher S.; Lehmann, Brian D.; Pietenpol, Jennifer A.; Cortez, David

    2015-01-01

    The DNA damage response kinase ATR may be a useful cancer therapeutic target. ATR inhibition synergizes with loss of ERCC1, ATM, XRCC1 and DNA damaging chemotherapy agents. Clinical trials have begun using ATR inhibitors in combination with cisplatin. Here we report the first synthetic lethality screen with a combination treatment of an ATR inhibitor (ATRi) and cisplatin. Combination treatment with ATRi/cisplatin is synthetically lethal with loss of the TLS polymerase ζ and 53BP1. Other DNA repair pathways including homologous recombination and mismatch repair do not exhibit synthetic lethal interactions with ATRi/cisplatin, even though loss of some of these repair pathways sensitizes cells to cisplatin as a single-agent. We also report that ATRi strongly synergizes with PARP inhibition, even in homologous recombination-proficient backgrounds. Lastly, ATR inhibitors were able to resensitize cisplatin-resistant cell lines to cisplatin. These data provide a comprehensive analysis of DNA repair pathways that exhibit synthetic lethality with ATR inhibitors when combined with cisplatin chemotherapy, and will help guide patient selection strategies as ATR inhibitors progress into the cancer clinic. PMID:25965342

  5. Double-Strand Break Repair by Interchromosomal Recombination: An In Vivo Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals

    PubMed Central

    White, Ryan R.; Sung, Patricia; Vestal, C. Greer; Benedetto, Gregory; Cornelio, Noelle; Richardson, Christine

    2013-01-01

    Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G2). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes—two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit–granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation. PMID:24349572

  6. p53 downregulates the Fanconi anaemia DNA repair pathway

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-08-01

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

  8. Gradual implementation of the meiotic recombination program via checkpoint pathways controlled by global DSB levels.

    PubMed

    Joshi, Neeraj; Brown, M Scott; Bishop, Douglas K; Börner, G Valentin

    2015-03-05

    During meiosis, Spo11-induced double-strand breaks (DSBs) are processed into crossovers, ensuring segregation of homologous chromosomes (homologs). Meiotic DSB processing entails 5' end resection and preferred strand exchange with the homolog rather than the sister chromatid (homolog bias). In many organisms, DSBs appear gradually along the genome. Here we report unexpected effects of global DSB levels on local recombination events. Early-occurring, low-abundance "scout" DSBs lack homolog bias. Their resection and interhomolog processing are controlled by the conserved checkpoint proteins Tel1(ATM) kinase and Pch2(TRIP13) ATPase. Processing pathways controlled by Mec1(ATR) kinase take over these functions only above a distinct DSB threshold, resulting in progressive strengthening of the homolog bias. We conclude that Tel1(ATM)/Pch2 and Mec1(ATR) DNA damage response pathways are sequentially activated during wild-type meiosis because of their distinct sensitivities to global DSB levels. Moreover, relative DSB order controls the DSB repair pathway choice and, ultimately, recombination outcome.

  9. Functional redundancy between repair factor XLF and damage response mediator 53BP1 in V(D)J recombination and DNA repair

    PubMed Central

    Oksenych, Valentyn; Alt, Frederick W.; Kumar, Vipul; Schwer, Bjoern; Wesemann, Duane R.; Hansen, Erica; Patel, Harin; Su, Arthur; Guo, Chunguang

    2012-01-01

    The classical nonhomologous DNA end-joining (C-NHEJ) double-strand break (DSB) repair pathway in mammalian cells maintains genome stability and is required for V(D)J recombination and lymphocyte development. Mutations in the XLF C-NHEJ factor or ataxia telangiectasia-mutated (ATM) DSB response protein cause radiosensitivity and immunodeficiency in humans. Although potential roles for XLF in C-NHEJ are unknown, ATM activates a general DSB response by phosphorylating substrates, including histone H2AX and 53BP1, which are assembled into chromatin complexes around DSBs. In mice, C-NHEJ, V(D)J recombination, and lymphocyte development are, at most, modestly impaired in the absence of XLF or ATM, but are severely impaired in the absence of both. Redundant functions of XLF and ATM depend on ATM kinase activity; correspondingly, combined XLF and H2AX deficiency severely impairs V(D)J recombination, even though H2AX deficiency alone has little impact on this process. These and other findings suggest that XLF may provide functions that overlap more broadly with assembled DSB response factors on chromatin. As one test of this notion, we generated mice and cells with a combined deficiency for XLF and 53BP1. In this context, 53BP1 deficiency, although leading to genome instability, has only modest effects on V(D)J recombination or lymphocyte development. Strikingly, we find that combined XLF/53BP1 deficiency in mice severely impairs C-NHEJ, V(D)J recombination, and lymphocyte development while also leading to general genomic instability and growth defects. We conclude that XLF is functionally redundant with multiple members of the ATM-dependent DNA damage response in facilitating C-NHEJ and discuss implications of our findings for potential functions of these factors. PMID:22308489

  10. Roles of the DNA binding proteins H-NS and StpA in homologous recombination and repair of bleomycin-induced damage in Escherichia coli.

    PubMed

    Shiraishi, Kouya; Ogata, Yasuyuki; Hanada, Katsuhiro; Kano, Yasunobu; Ikeda, Hideo

    2007-10-01

    The DNA binding protein H-NS promotes homologous recombination in Escherichia coli, but the role of its paralog StpA in this process remains unclear. Here we show that an hns mutant, but not an stpA mutant, are marginally defective in conjugational recombination and is sensitive to the double-strand-break-inducing agent bleomycin. Interestingly, the hns stpA double mutant is severely defective in homologous recombination and more bleomycin-sensitive than is the hns or stpA single mutant, indicating that the stpA mutation synergistically enhances the defects of homologous recombination and the increased bleomycin-sensitivity in the hns mutant. In addition, the transduction analysis in the hns stpA double mutant indicated that the stpA mutation also enhances the defect of recombination in the hns mutant. These results suggest that H-NS plays an important role in both homologous recombination and repair of bleomycin-induced damage, while StpA can substitute the H-NS function. The recombination analysis of hns single, stpA single, and hns stpA double mutants in the recBC sbcA and recBC sbcBC backgrounds suggested that the reduction of the hns single or hns stpA double mutants may not be due to the defect in a particular recombination pathway, but may be due to the defect in a common process of the pathways. The model for the functions of H-NS and StpA in homologous recombination and double-strand break repair is discussed.

  11. Investigations of homologous recombination pathways and their regulation.

    PubMed

    Daley, James M; Kwon, YoungHo; Niu, Hengyao; Sung, Patrick

    2013-12-13

    The DNA double-strand break (DSB), arising from exposure to ionizing radiation or various chemotherapeutic agents or from replication fork collapse, is among the most dangerous of chromosomal lesions. DSBs are highly cytotoxic and can lead to translocations, deletions, duplications, or mutations if mishandled. DSBs are eliminated by either homologous recombination (HR), which uses a homologous template to guide accurate repair, or by nonhomologous end joining (NHEJ), which simply rejoins the two broken ends after damaged nucleotides have been removed. HR generates error-free repair products and is also required for generating chromosome arm crossovers between homologous chromosomes in meiotic cells. The HR reaction includes several distinct steps: resection of DNA ends, homologous DNA pairing, DNA synthesis, and processing of HR intermediates. Each occurs in a highly regulated fashion utilizing multiple protein factors. These steps are being elucidated using a combination of genetic tools, cell-based assays, and in vitro reconstitution with highly purified HR proteins. In this review, we summarize contributions from our laboratory at Yale University in understanding HR mechanisms in eukaryotic cells.

  12. Downregulation of Homologous Recombination DNA Repair Genes by HDAC Inhibition in Prostate Cancer Is Mediated through the E2F1 Transcription Factor

    PubMed Central

    Kachhap, Sushant K.; Rosmus, Nadine; Collis, Spencer J.; Kortenhorst, Madeleine S. Q.; Wissing, Michel D.; Hedayati, Mohammad; Shabbeer, Shabana; Mendonca, Janet; Deangelis, Justin; Marchionni, Luigi; Lin, Jianqing; Höti, Naseruddin; Nortier, Johan W. R.; DeWeese, Theodore L.; Hammers, Hans; Carducci, Michael A.

    2010-01-01

    Background Histone deacetylase inhibitors (HDACis) re-express silenced tumor suppressor genes and are currently undergoing clinical trials. Although HDACis have been known to induce gene expression, an equal number of genes are downregulated upon HDAC inhibition. The mechanism behind this downregulation remains unclear. Here we provide evidence that several DNA repair genes are downregulated by HDAC inhibition and provide a mechanism involving the E2F1 transcription factor in the process. Methodology/Principal Findings Applying Analysis of Functional Annotation (AFA) on microarray data of prostate cancer cells treated with HDACis, we found a number of genes of the DNA damage response and repair pathways are downregulated by HDACis. AFA revealed enrichment of homologous recombination (HR) DNA repair genes of the BRCA1 pathway, as well as genes regulated by the E2F1 transcription factor. Prostate cancer cells demonstrated a decreased DNA repair capacity and an increased sensitization to chemical- and radio-DNA damaging agents upon HDAC inhibition. Recruitment of key HR repair proteins to the site of DNA damage, as well as HR repair capacity was compromised upon HDACi treatment. Based on our AFA data, we hypothesized that the E2F transcription factors may play a role in the downregulation of key repair genes upon HDAC inhibition in prostate cancer cells. ChIP analysis and luciferase assays reveal that the downregulation of key repair genes is mediated through decreased recruitment of the E2F1 transcription factor and not through active repression by repressive E2Fs. Conclusions/Significance Our study indicates that several genes in the DNA repair pathway are affected upon HDAC inhibition. Downregulation of the repair genes is on account of a decrease in amount and promoter recruitment of the E2F1 transcription factor. Since HDAC inhibition affects several pathways that could potentially have an impact on DNA repair, compromised DNA repair upon HDAC inhibition could

  13. Homologous recombination and double-strand break repair in the transformation of Rhizopus oryzae.

    PubMed

    Skory, C D

    2002-11-01

    Genetic transformation of the Mucorales fungi has been problematic, since DNA transformed into the host rarely integrates and usually is mitotically unstable in the absence of selective pressure. In this study, transformation of Rhizopus oryzae was investigated to determine if the fate of introduced DNA could be predicted based on double-strand break repair and recombination mechanisms found in other fungi. A transformation system was developed with uracil auxotrophs of Rhizopus oryzae that could be complemented with the pyrG gene isolated in this work. DNA transformed as circular plasmids was maintained extrachromosomally in high-molecular-weight (>23 kb) concatenated arrangement. Type-I crossover integration into the pyrG locus and type-III pyrG gene replacement events occurred in approximately 1-5% of transformants. Linearization of the plasmid pPyr225 with a single restriction enzyme that cleaves within the vector sequence almost always resulted in isolates with replicating concatenated plasmids that had been repaired by end-joining recombination that restored the restriction site. The addition of a 40-bp direct repeat on either side of this cleavage site led to repair by homologous recombination between the repeated sequences on the plasmid, resulting in loss of the restriction site. When plasmid pPyr225 was digested with two different enzymes that cleave within the vector sequence to release the pyrG containing fragment, only pyrG gene replacement recombination occurred in transformants. Linearization of plasmid pPyr225 within the pyrG gene itself gave the highest percentage (20%) of type-I integration at the pyrG locus. However, end-joining repair and gene replacement events were still the predominant types of recombination found in transformations with this plasmid topology.

  14. Chemotherapeutic compounds targeting the DNA double-strand break repair pathways: the good, the bad, and the promising.

    PubMed

    Jekimovs, Christian; Bolderson, Emma; Suraweera, Amila; Adams, Mark; O'Byrne, Kenneth J; Richard, Derek J

    2014-01-01

    The repair of DNA double-strand breaks (DSBs) is a critical cellular mechanism that exists to ensure genomic stability. DNA DSBs are the most deleterious type of insult to a cell's genetic material and can lead to genomic instability, apoptosis, or senescence. Incorrectly repaired DNA DSBs have the potential to produce chromosomal translocations and genomic instability, potentially leading to cancer. The prevalence of DNA DSBs in cancer due to unregulated growth and errors in repair opens up a potential therapeutic window in the treatment of cancers. The cellular response to DNA DSBs is comprised of two pathways to ensure DNA breaks are repaired: homologous recombination and non-homologous end joining. Identifying chemotherapeutic compounds targeting proteins involved in these DNA repair pathways has shown promise as a cancer therapy for patients, either as a monotherapy or in combination with genotoxic drugs. From the beginning, there have been a number of chemotherapeutic compounds that have yielded successful responses in the clinic, a number that have failed (CGK-733 and iniparib), and a number of promising targets for future studies identified. This review looks in detail at how the cell responds to these DNA DSBs and investigates the chemotherapeutic avenues that have been and are currently being explored to target this repair process.

  15. Xrcc3 induces cisplatin resistance by stimulation of Rad51-related recombinational repair, S-phase checkpoint activation, and reduced apoptosis.

    PubMed

    Xu, Zhi-Yuan; Loignon, Martin; Han, Fei-Yu; Panasci, Lawrence; Aloyz, Raquel

    2005-08-01

    Eukaryotic cells respond to DNA damage by activation of DNA repair, cell cycle arrest, and apoptosis. Several reports suggest that such responses may be coordinated by communication between damage repair proteins and proteins signaling other cellular responses. The Rad51-guided homologous recombination repair system plays an important role in the recognition and repair of DNA interstrand crosslinks (ICLs), and cells deficient in this repair pathway become hypersensitive to ICL-inducing agents such as cisplatin and melphalan. We investigated the possible role of the Rad51-paralog protein Xrcc3 in drug resistance. Xrcc3 overexpression in MCF-7 cells resulted in 1) a 2- to 6-fold resistance to cisplatin/melphalan, 2) a 2-fold increase in drug-induced Rad51 foci, 3) an increased cisplatin-induced S-phase arrest, 4) decreased cisplatin-induced apoptosis, and 5) increased cisplatin-induced DNA synthesis arrest. Interestingly, Xrcc3 overexpression did not alter the doubling time or cell cycle progression in the absence of DNA damage. Furthermore, Xrcc3 overexpression is associated with increased Rad51C protein levels consistent with the known interaction of these two proteins. Our results demonstrate that Xrcc3 is an important factor in DNA cross-linking drug resistance in human tumor cells and suggest that the response of the homologous recombinational repair machinery and cell cycle checkpoints to DNA cross-linking agents is intertwined.

  16. Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes.

    PubMed Central

    Zieg, J; Maples, V F; Kushner, S R

    1978-01-01

    Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products. PMID:350859

  17. PARP3 affects the relative contribution of homologous recombination and nonhomologous end-joining pathways.

    PubMed

    Beck, Carole; Boehler, Christian; Guirouilh Barbat, Josée; Bonnet, Marie-Elise; Illuzzi, Giuditta; Ronde, Philippe; Gauthier, Laurent R; Magroun, Najat; Rajendran, Anbazhagan; Lopez, Bernard S; Scully, Ralph; Boussin, François D; Schreiber, Valérie; Dantzer, Françoise

    2014-05-01

    The repair of toxic double-strand breaks (DSB) is critical for the maintenance of genome integrity. The major mechanisms that cope with DSB are: homologous recombination (HR) and classical or alternative nonhomologous end joining (C-NHEJ versus A-EJ). Because these pathways compete for the repair of DSB, the choice of the appropriate repair pathway is pivotal. Among the mechanisms that influence this choice, deoxyribonucleic acid (DNA) end resection plays a critical role by driving cells to HR, while accurate C-NHEJ is suppressed. Furthermore, end resection promotes error-prone A-EJ. Increasing evidence define Poly(ADP-ribose) polymerase 3 (PARP3, also known as ARTD3) as an important player in cellular response to DSB. In this work, we reveal a specific feature of PARP3 that together with Ku80 limits DNA end resection and thereby helps in making the choice between HR and NHEJ pathways. PARP3 interacts with and PARylates Ku70/Ku80. The depletion of PARP3 impairs the recruitment of YFP-Ku80 to laser-induced DNA damage sites and induces an imbalance between BRCA1 and 53BP1. Both events result in compromised accurate C-NHEJ and a concomitant increase in DNA end resection. Nevertheless, HR is significantly reduced upon PARP3 silencing while the enhanced end resection causes mutagenic deletions during A-EJ. As a result, the absence of PARP3 confers hypersensitivity to anti-tumoral drugs generating DSB. © The Author(s) 2014. Published by Oxford University Press.

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

    PubMed Central

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

    2015-01-01

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

  19. The Arabidopsis DNA mismatch repair gene PMS1 restricts somatic recombination between homeologous sequences.

    PubMed

    Li, Liangliang; Dion, Eric; Richard, Gabriel; Domingue, Olivier; Jean, Martine; Belzile, François J

    2009-04-01

    The eukaryotic DNA mismatch repair (MMR) system contributes to maintaining the fidelity of genetic information by correcting replication errors and preventing illegitimate recombination events. This study aimed to examine the function(s) of the Arabidopsis thaliana PMS1 gene (AtPMS1), one of three homologs of the bacterial MutL gene in plants. Two independent mutant alleles (Atpms1-1 and Atpms1-2) were obtained and one of these (Atpms1-1) was studied in detail. The mutant exhibited a reduction in seed set and a bias against the transmission of the mutant allele. Somatic recombination, both homologous and homeologous, was examined using a set of reporter constructs. Homologous recombination remained unchanged in the mutant while homeologous recombination was between 1.7- and 4.8-fold higher than in the wild type. This increase in homeologous recombination frequency was not correlated with the degree of sequence divergence. In RNAi lines, a range of increases in homeologous recombination were observed with two lines showing a 3.3-fold and a 3.6-fold increase. These results indicate that the AtPMS1 gene contributes to an antirecombination activity aimed at restricting recombination between diverged sequences.

  20. The impact of sequence divergence and DNA mismatch repair on homeologous recombination in Arabidopsis.

    PubMed

    Li, Liangliang; Jean, Martine; Belzile, François

    2006-03-01

    We examined the effects of substrate divergence and DNA mismatch repair (MMR) on recombination in Arabidopsis thaliana. Relative to the frequency observed in plants with a homologous construct (0% divergence), recombination was decreased 4.1-, 9.6-, 11.7- or 20.3-fold, respectively, in lines with constructs containing 0.5%, 2%, 4% or 9% divergence between the recombination substrates. To evaluate the contribution of the MMR system in this decrease, 12 independent reporter lines (two or three lines per reporter construct) were crossed to an AtMSH2 T-DNA insertional mutant. We examined the recombination frequency in progeny homozygous for a reporter T-DNA and homozygous either for the wild type or the mutant allele of AtMSH2. The loss of MMR activity led to a two- to ninefold increase in homeologous recombination and the size of the increase did not seem to correlate with the amount of divergence. Inversely, complementation of the insertional mutant with a wild-type cDNA of AtMSH2 reduced recombination. Our results demonstrate clearly that sequence divergence can dramatically reduce the recombination frequency in plants and that the MMR system plays a part in this decrease.

  1. A pathway for repair of NAD(P)H in plants.

    PubMed

    Colinas, Maite; Shaw, Holly V; Loubéry, Sylvain; Kaufmann, Markus; Moulin, Michael; Fitzpatrick, Teresa B

    2014-05-23

    Unwanted enzyme side reactions and spontaneous decomposition of metabolites can lead to a build-up of compounds that compete with natural enzyme substrates and must be dealt with for efficient metabolism. It has recently been realized that there are enzymes that process such compounds, formulating the concept of metabolite repair. NADH and NADPH are vital cellular redox cofactors but can form non-functional hydrates (named NAD(P)HX) spontaneously or enzymatically that compete with enzymes dependent on NAD(P)H, impairing normal enzyme function. Here we report on the functional characterization of components of a potential NAD(P)H repair pathway in plants comprising a stereospecific dehydratase (NNRD) and an epimerase (NNRE), the latter being fused to a vitamin B6 salvage enzyme. Through the use of the recombinant proteins, we show that the ATP-dependent NNRD and NNRE act concomitantly to restore NAD(P)HX to NAD(P)H. NNRD behaves as a tetramer and NNRE as a dimer, but the proteins do not physically interact. In vivo fluorescence analysis demonstrates that the proteins are localized to mitochondria and/or plastids, implicating these as the key organelles where this repair is required. Expression analysis indicates that whereas NNRE is present ubiquitously, NNRD is restricted to seeds but appears to be dispensable during the normal Arabidopsis life cycle.

  2. Genetic Recombination in Bacillus subtilis 168: Effect of ΔhelD on DNA Repair and Homologous Recombination

    PubMed Central

    Carrasco, Begoña; Fernández, Silvia; Petit, Marie-Agnes; Alonso, Juan C.

    2001-01-01

    The B. subtilis ΔhelD allele rendered cells proficient in transformational recombination and moderately sensitive to methyl methanesulfonate when present in an otherwise Rec+ strain. The ΔhelD allele was introduced into rec-deficient strains representative of the α (recF strain), β (addA addB), γ (recH), ɛ (ΔrecU), and ζ (ΔrecS) epistatic groups. The ΔhelD mutation increased the sensitivity to DNA-damaging agents of addAB, ΔrecU, and ΔrecS cells, did not affect the survival of recH cells, and decreased the sensitivity of recF cells. ΔhelD also partially suppressed the DNA repair phenotype of other mutations classified within the α epistatic group, namely the recL, ΔrecO, and recR mutations. The ΔhelD allele marginally reduced plasmid transformation (three- to sevenfold) of mutations classified within the α, β, and γ epistatic groups. Altogether, these data indicate that the loss of helicase IV might stabilize recombination repair intermediates formed in the absence of recFLOR and render recFLOR, addAB, and recH cells impaired in plasmid transformation. PMID:11544244

  3. Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair

    PubMed Central

    Carrasco, Begoña; Yadav, Tribhuwan; Serrano, Ester; Alonso, Juan C.

    2015-01-01

    Genetic data have revealed that the absence of Bacillus subtilis RecO and one of the end-processing avenues (AddAB or RecJ) renders cells as sensitive to DNA damaging agents as the null recA, suggesting that both end-resection pathways require RecO for recombination. RecA, in the rATP·Mg2+ bound form (RecA·ATP), is inactive to catalyze DNA recombination between linear double-stranded (ds) DNA and naked complementary circular single-stranded (ss) DNA. We showed that RecA·ATP could not nucleate and/or polymerize on SsbA·ssDNA or SsbB·ssDNA complexes. RecA·ATP nucleates and polymerizes on RecO·ssDNA·SsbA complexes more efficiently than on RecO·ssDNA·SsbB complexes. Limiting SsbA concentrations were sufficient to stimulate RecA·ATP assembly on the RecO·ssDNA·SsbB complexes. RecO and SsbA are necessary and sufficient to ‘activate’ RecA·ATP to catalyze DNA strand exchange, whereas the AddAB complex, RecO alone or in concert with SsbB was not sufficient. In presence of AddAB, RecO and SsbA are still necessary for efficient RecA·ATP-mediated three-strand exchange recombination. Based on genetic and biochemical data, we proposed that SsbA and RecO (or SsbA, RecO and RecR in vivo) are crucial for RecA activation for both, AddAB and RecJ–RecQ (RecS) recombinational repair pathways. PMID:26001966

  4. Polynucleotide phosphorylase is implicated in homologous recombination and DNA repair in Escherichia coli.

    PubMed

    Carzaniga, Thomas; Sbarufatti, Giulia; Briani, Federica; Dehò, Gianni

    2017-04-04

    Polynucleotide phosphorylase (PNPase, encoded by pnp) is generally thought of as an enzyme dedicated to RNA metabolism. The pleiotropic effects of PNPase deficiency is imputed to altered processing and turnover of mRNAs and small RNAs, which in turn leads to aberrant gene expression. However, it has long since been known that this enzyme may also catalyze template-independent polymerization of dNDPs into ssDNA and the reverse phosphorolytic reaction. Recently, PNPase has been implicated in DNA recombination, repair, mutagenesis and resistance to genotoxic agents in diverse bacterial species, raising the possibility that PNPase may directly, rather than through control of gene expression, participate in these processes. In this work we present evidence that in Escherichia coli PNPase enhances both homologous recombination upon P1 transduction and error prone DNA repair of double strand breaks induced by zeocin, a radiomimetic agent. Homologous recombination does not require PNPase phosphorolytic activity and is modulated by its RNA binding domains whereas error prone DNA repair of zeocin-induced DNA damage is dependent on PNPase catalytic activity and cannot be suppressed by overexpression of RNase II, the other major enzyme (encoded by rnb) implicated in exonucleolytic RNA degradation. Moreover, E. coli pnp mutants are more sensitive than the wild type to zeocin. This phenotype depends on PNPase phosphorolytic activity and is suppressed by rnb, thus suggesting that zeocin detoxification may largely depend on RNA turnover. Our data suggest that PNPase may participate both directly and indirectly through regulation of gene expression to several aspects of DNA metabolism such as recombination, DNA repair and resistance to genotoxic agents.

  5. Extracellular signal-related kinase positively regulates ataxia telangiectasia mutated, homologous recombination repair, and the DNA damage response.

    PubMed

    Golding, Sarah E; Rosenberg, Elizabeth; Neill, Steven; Dent, Paul; Povirk, Lawrence F; Valerie, Kristoffer

    2007-02-01

    The accurate joining of DNA double-strand breaks by homologous recombination repair (HRR) is critical to the long-term survival of the cell. The three major mitogen-activated protein (MAP) kinase (MAPK) signaling pathways, extracellular signal-regulated kinase (ERK), p38, and c-Jun-NH(2)-kinase (JNK), regulate cell growth, survival, and apoptosis. To determine the role of MAPK signaling in HRR, we used a human in vivo I-SceI-based repair system. First, we verified that this repair platform is amenable to pharmacologic manipulation and show that the ataxia telangiectasia mutated (ATM) kinase is critical for HRR. The ATM-specific inhibitor KU-55933 compromised HRR up to 90% in growth-arrested cells, whereas this effect was less pronounced in cycling cells. Then, using well-characterized MAPK small-molecule inhibitors, we show that ERK1/2 and JNK signaling are important positive regulators of HRR in growth-arrested cells. On the other hand, inhibition of the p38 MAPK pathway generated an almost 2-fold stimulation of HRR. When ERK1/2 signaling was stimulated by oncogenic RAF-1, an approximately 2-fold increase in HRR was observed. KU-55933 partly blocked radiation-induced ERK1/2 phosphorylation, suggesting that ATM regulates ERK1/2 signaling. Furthermore, inhibition of MAP/ERK kinase (MEK)/ERK signaling resulted in severely reduced levels of phosphorylated (S1981) ATM foci but not gamma-H2AX foci, and suppressed ATM phosphorylation levels >85% throughout the cell cycle. Collectively, these results show that MAPK signaling positively and negatively regulates HRR in human cells. More specifically, ATM-dependent signaling through the RAF/MEK/ERK pathway is critical for efficient HRR and for radiation-induced ATM activation, suggestive of a regulatory feedback loop between ERK and ATM.

  6. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination

    PubMed Central

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-01-01

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

  7. Autophagy confers DNA damage repair pathways to protect the hematopoietic system from nuclear radiation injury

    PubMed Central

    Lin, Weiwei; Yuan, Na; Wang, Zhen; Cao, Yan; Fang, Yixuan; Li, Xin; Xu, Fei; Song, Lin; Wang, Jian; Zhang, Han; Yan, Lili; Xu, Li; Zhang, Xiaoying; Zhang, Suping; Wang, Jianrong

    2015-01-01

    Autophagy is essentially a metabolic process, but its in vivo role in nuclear radioprotection remains unexplored. We observed that ex vivo autophagy activation reversed the proliferation inhibition, apoptosis, and DNA damage in irradiated hematopoietic cells. In vivo autophagy activation improved bone marrow cellularity following nuclear radiation exposure. In contrast, defective autophagy in the hematopoietic conditional mouse model worsened the hematopoietic injury, reactive oxygen species (ROS) accumulation and DNA damage caused by nuclear radiation exposure. Strikingly, in vivo defective autophagy caused an absence or reduction in regulatory proteins critical to both homologous recombination (HR) and non-homologous end joining (NHEJ) DNA damage repair pathways, as well as a failure to induce these proteins in response to nuclear radiation. In contrast, in vivo autophagy activation increased most of these proteins in hematopoietic cells. DNA damage assays confirmed the role of in vivo autophagy in the resolution of double-stranded DNA breaks in total bone marrow cells as well as bone marrow stem and progenitor cells upon whole body irradiation. Hence, autophagy protects the hematopoietic system against nuclear radiation injury by conferring and intensifying the HR and NHEJ DNA damage repair pathways and by removing ROS and inhibiting apoptosis. PMID:26197097

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

    PubMed

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

    2014-01-01

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

  9. Regulation of Immunoglobulin Class-Switch Recombination: Choreography of Noncoding Transcription, Targeted DNA Deamination, and Long-Range DNA Repair

    PubMed Central

    Matthews, Allysia J.; Zheng, Simin; DiMenna, Lauren J.; Chaudhuri, Jayanta

    2014-01-01

    Upon encountering antigens, mature IgM-positive B lymphocytes undergo class-switch recombination (CSR) wherein exons encoding the default Cμ constant coding gene segment of the immunoglobulin (Ig) heavy-chain (Igh) locus are excised and replaced with a new constant gene segment (referred to as “Ch genes”, e.g., Cγ, Cε, or Cα). The B cell thereby changes from expressing IgM to one producing IgG, IgE, or IgA, with each antibody isotype having a different effector function during an immune reaction. CSR is a DNA deletional-recombination reaction that proceeds through the generation of DNA double-strand breaks (DSBs) in repetitive switch (S) sequences preceding each Ch gene and is completed by end-joining between donor Sμ and acceptor S regions. CSR is a multistep reaction requiring transcription through S regions, the DNA cytidine deaminase AID, and the participation of several general DNA repair pathways including base excision repair, mismatch repair, and classical nonhomologous end-joining. In this review, we discuss our current understanding of how transcription through S regions generates substrates for AID-mediated deamination and how AID participates not only in the initiation of CSR but also in the conversion of deaminated residues into DSBs. Additionally, we review the multiple processes that regulate AID expression and facilitate its recruitment specifically to the Ig loci, and how deregulation of AID specificity leads to oncogenic translocations. Finally, we summarize recent data on the potential role of AID in the maintenance of the pluripotent stem cell state during epigenetic reprogramming. PMID:24507154

  10. Regulation of immunoglobulin class-switch recombination: choreography of noncoding transcription, targeted DNA deamination, and long-range DNA repair.

    PubMed

    Matthews, Allysia J; Zheng, Simin; DiMenna, Lauren J; Chaudhuri, Jayanta

    2014-01-01

    Upon encountering antigens, mature IgM-positive B lymphocytes undergo class-switch recombination (CSR) wherein exons encoding the default Cμ constant coding gene segment of the immunoglobulin (Ig) heavy-chain (Igh) locus are excised and replaced with a new constant gene segment (referred to as "Ch genes", e.g., Cγ, Cɛ, or Cα). The B cell thereby changes from expressing IgM to one producing IgG, IgE, or IgA, with each antibody isotype having a different effector function during an immune reaction. CSR is a DNA deletional-recombination reaction that proceeds through the generation of DNA double-strand breaks (DSBs) in repetitive switch (S) sequences preceding each Ch gene and is completed by end-joining between donor Sμ and acceptor S regions. CSR is a multistep reaction requiring transcription through S regions, the DNA cytidine deaminase AID, and the participation of several general DNA repair pathways including base excision repair, mismatch repair, and classical nonhomologous end-joining. In this review, we discuss our current understanding of how transcription through S regions generates substrates for AID-mediated deamination and how AID participates not only in the initiation of CSR but also in the conversion of deaminated residues into DSBs. Additionally, we review the multiple processes that regulate AID expression and facilitate its recruitment specifically to the Ig loci, and how deregulation of AID specificity leads to oncogenic translocations. Finally, we summarize recent data on the potential role of AID in the maintenance of the pluripotent stem cell state during epigenetic reprogramming.

  11. Histone dosage regulates DNA damage sensitivity in a checkpoint-independent manner by the homologous recombination pathway

    PubMed Central

    Liang, Dun; Burkhart, Sarah Lyn; Singh, Rakesh Kumar; Kabbaj, Marie-Helene Miquel; Gunjan, Akash

    2012-01-01

    In eukaryotes, multiple genes encode histone proteins that package genomic deoxyribonucleic acid (DNA) and regulate its accessibility. Because of their positive charge, ‘free’ (non-chromatin associated) histones can bind non-specifically to the negatively charged DNA and affect its metabolism, including DNA repair. We have investigated the effect of altering histone dosage on DNA repair in budding yeast. An increase in histone gene dosage resulted in enhanced DNA damage sensitivity, whereas deletion of a H3–H4 gene pair resulted in reduced levels of free H3 and H4 concomitant with resistance to DNA damaging agents, even in mutants defective in the DNA damage checkpoint. Studies involving the repair of a HO endonuclease-mediated DNA double-strand break (DSB) at the MAT locus show enhanced repair efficiency by the homologous recombination (HR) pathway on a reduction in histone dosage. Cells with reduced histone dosage experience greater histone loss around a DSB, whereas the recruitment of HR factors is concomitantly enhanced. Further, free histones compete with the HR machinery for binding to DNA and associate with certain HR factors, potentially interfering with HR-mediated repair. Our findings may have important implications for DNA repair, genomic stability, carcinogenesis and aging in human cells that have dozens of histone genes. PMID:22850743

  12. Pseudosynapsis and Decreased Stringency of Meiotic Repair Pathway Choice on the Hemizygous Sex Chromosome of Caenorhabditis elegans Males

    PubMed Central

    Checchi, Paula M.; Lawrence, Katherine S.; Van, Mike V.; Larson, Braden J.; Engebrecht, JoAnne

    2014-01-01

    During meiosis, accurate chromosome segregation relies on homology to mediate chromosome pairing, synapsis, and crossover recombination. Crossovers are dependent upon formation and repair of double-strand breaks (DSBs) by homologous recombination (HR). In males of many species, sex chromosomes are largely hemizygous, yet DSBs are induced along nonhomologous regions. Here we analyzed the genetic requirements for meiotic DSB repair on the completely hemizygous X chromosome of Caenorhabditis elegans males. Our data reveal that the kinetics of DSB formation, chromosome pairing, and synapsis are tightly linked in the male germ line. Moreover, DSB induction on the X is concomitant with a brief period of pseudosynapsis that may allow X sister chromatids to masquerade as homologs. Consistent with this, neither meiotic kleisins nor the SMC-5/6 complex are essential for DSB repair on the X. Furthermore, early processing of X DSBs is dependent on the CtIP/Sae2 homolog COM-1, suggesting that as with paired chromosomes, HR is the preferred pathway. In contrast, the X chromosome is refractory to feedback mechanisms that ensure crossover formation on autosomes. Surprisingly, neither RAD-54 nor BRC-2 are essential for DSB repair on the X, suggesting that unlike autosomes, the X is competent for repair in the absence of HR. When both RAD-54 and the structure-specific nuclease XPF-1 are abrogated, X DSBs persist, suggesting that single-strand annealing is engaged in the absence of HR. Our findings indicate that alteration in sister chromatid interactions and flexibility in DSB repair pathway choice accommodate hemizygosity on sex chromosomes. PMID:24939994

  13. Preferential repair of UV damage in highly transcribed DNA diminishes UV-induced intrachromosomal recombination in mammalian cells.

    PubMed Central

    Deng, W P; Nickoloff, J A

    1994-01-01

    The relationships among transcription, recombination, DNA damage, and repair in mammalian cells were investigated. We monitored the effects of transcription on UV-induced intrachromosomal recombination between neomycin repeats including a promoterless allele and an inducible heteroallele regulated by the mouse mammary tumor virus promoter. Although transcription and UV light separately stimulated recombination, increasing transcription levels reduced UV-induced recombination. Preferential repair of UV damage in transcribed strands was shown in highly transcribed DNA, suggesting that recombination is stimulated by unrepaired UV damage and that increased DNA repair in highly transcribed alleles removes recombinogenic lesions. This study indicates that the genetic consequences of DNA damage depend on transcriptional states and provides a basis for understanding tissue- and gene-specific responses to DNA-damaging agents. Images PMID:8264606

  14. Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells.

    PubMed

    Truong, Lan N; Li, Yongjiang; Shi, Linda Z; Hwang, Patty Yi-Hwa; He, Jing; Wang, Hailong; Razavian, Niema; Berns, Michael W; Wu, Xiaohua

    2013-05-07

    Microhomology-mediated end joining (MMEJ) is a major pathway for Ku-independent alternative nonhomologous end joining, which contributes to chromosomal translocations and telomere fusions, but the underlying mechanism of MMEJ in mammalian cells is not well understood. In this study, we demonstrated that, distinct from Ku-dependent classical nonhomologous end joining, MMEJ--even with very limited end resection--requires cyclin-dependent kinase activities and increases significantly when cells enter S phase. We also showed that MMEJ shares the initial end resection step with homologous recombination (HR) by requiring meiotic recombination 11 homolog A (Mre11) nuclease activity, which is needed for subsequent recruitment of Bloom syndrome protein (BLM) and exonuclease 1 (Exo1) to DNA double-strand breaks (DSBs) to promote extended end resection and HR. MMEJ does not require S139-phosphorylated histone H2AX (γ-H2AX), suggesting that initial end resection likely occurs at DSB ends. Using a MMEJ and HR competition repair substrate, we demonstrated that MMEJ with short end resection is used in mammalian cells at the level of 10-20% of HR when both HR and nonhomologous end joining are available. Furthermore, MMEJ is used to repair DSBs generated at collapsed replication forks. These studies suggest that MMEJ not only is a backup repair pathway in mammalian cells, but also has important physiological roles in repairing DSBs to maintain cell viability, especially under genomic stress.

  15. Studies of protein-protein interactions in Fanconi anemia pathway to unravel the DNA interstrand crosslink repair mechanism.

    PubMed

    Siddiqui, Mohd Quadir; Rajpurohit, Yogendra S; Thapa, Pankaj S; Maurya, Ganesh Kumar; Banerjee, Kuheli; Khan, Mudassar Ali; Panda, Pragnya; Hasan, Syed K; Gadewal, Nikhil; Misra, Hari S; Varma, Ashok K

    2017-11-01

    Fanconi anemia (FA), a cancer predisposition syndrome exhibits hallmark feature of radial chromosome formation, and hypersensitivity to DNA crosslinking agents. A set of FA pathway proteins mainly FANCI, FANCD2 and BRCA2 are expressed to repair the covalent crosslink between the dsDNA. However, FA, BRCA pathways play an important role in DNA ICL repair as well as in homologous recombination repair, but the presumptive role of FA-BRCA proteins has not clearly explored particularly in context to function associated protein-protein interactions (PPIs). Here, in-vivo, in-vitro and in-silico studies have been performed for functionally relevant domains of FANCI, FANCD2 and BRCA2. To our conclusion, FANCI ARM repeat interacts with FANCD2 CUE domain and BRCA2 C-terminal region. Interestingly, FANCD2 CUE domain also interacts strongly with BRCA2 C-terminal region. Interactions between BRCA2 CTR and functionally relevant mutations Ser222Ala (cell cycle checkpoint mutant) and Leu231Arg (DNA ICL repair mutant) present in FANCD2 CUE domain have been analysed. To our finding, these mutations abrogate the binding between FANCD2 CUE domain and BRCA2 CTR. Furthermore, (1) different domain of FANCI, FANCD2 and BRCA2 are playing important role in PPIs, (2) mutations cause the impairment in the PPIs which in turn may disrupt the DNA ICL repair mechanism. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Shared epithelial pathways to lung repair and disease.

    PubMed

    Spella, Magda; Lilis, Ioannis; Stathopoulos, Georgios T

    2017-06-30

    Chronic lung diseases present tremendous health burdens and share a common pathobiology of dysfunctional epithelial repair. Lung adenocarcinoma, the leading cancer killer worldwide, is caused mainly by chemical carcinogens of tobacco smoke that induce mutations in pulmonary epithelial cells leading to uncontrolled epithelial proliferation. Lung epithelial cells that possess the capacity for self-renewal and regeneration of other lung cell types are believed to underlie the pathobiology of chronic obstructive, fibrotic and neoplastic lung disorders. However, the understanding of lung epithelial progenitor cell hierarchy and turnover is incomplete and a comprehensive model of the cellular and transcriptional events that underlie lung regeneration and carcinogenesis is missing. The mapping of these processes is extremely important, since their modulation would potentially allow effective cure and/or prevention of chronic lung diseases. In this review we describe current knowledge on cellular and molecular pathways at play during lung repair and carcinogenesis and summarise the critical lung cell populations with regenerative and cancerous potential. Copyright ©ERS 2017.

  17. Human MutL-complexes monitor homologous recombination independently of mismatch repair.

    PubMed

    Siehler, Simone Yasmin; Schrauder, Michael; Gerischer, Ulrike; Cantor, Sharon; Marra, Giancarlo; Wiesmüller, Lisa

    2009-02-01

    The role of mismatch repair proteins has been well studied in the context of DNA repair following DNA polymerase errors. Particularly in yeast, MSH2 and MSH6 have also been implicated in the regulation of genetic recombination, whereas MutL homologs appeared to be less important. So far, little is known about the role of the human MutL homolog hMLH1 in recombination, but recently described molecular interactions suggest an involvement. To identify activities of hMLH1 in this process, we applied an EGFP-based assay for the analysis of different mechanisms of DNA repair, initiated by a targeted double-stranded DNA break. We analysed 12 human cellular systems, differing in the hMLH1 and concomitantly in the hPMS1 and hPMS2 status via inducible protein expression, genetic reconstitution, or RNA interference. We demonstrate that hMLH1 and its complex partners hPMS1 and hPMS2 downregulate conservative homologous recombination (HR), particularly when involving DNA sequences with only short stretches of uninterrupted homology. Unexpectedly, hMSH2 is dispensable for this effect. Moreover, the damage-signaling kinase ATM and its substrates BLM and BACH1 are not strictly required, but the combined effect of ATM/ATR-signaling components may mediate the anti-recombinogenic effect. Our data indicate a protective role of hMutL-complexes in a process which may lead to detrimental genome rearrangements, in a manner which does not depend on mismatch repair.

  18. Isolation of the functional human excision repair gene ERCC5 by intercosmid recombination

    SciTech Connect

    Mudgett, J.S.; MacInnes, M.A. )

    1990-12-01

    The complete human nucleotide exicision repair gene ERCC5 was isolated as a functional gene on overlapping cosmids. ERCC5 corrects the excision repair deficiency of Chinese hamster ovary cell line UV135, of complementation group 5. Cosmids that contained human sequences were obtained from a UV-resistant cell line derived from UV135 cells transformed with human genomic DNA. Individually, none of the cosmids complemented the UV135 repair defect; cosmid groups were formed to represent putative human genomic regions, and specific pairs of cosmids that effectively transformed UV135 cells to UV resistance were identified. Analysis of transformants derived from the active cosmid pairs showed that the functional 32-kbp ERCC5 gene was reconstructed by homologous intercosmid recombination. The cloned human sequences exhibited 100% concordance with the locus designated genetically as ERCC5 located on human chromosome 13q. Cosmid-transformed UV135 host cells repaired cytotoxic damage to levels about 70% of normal and repaired UV-irradiated shuttle vector DNA to levels about 82% of normal.

  19. Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells

    PubMed Central

    Fattah, Farjana; Lee, Eu Han; Weisensel, Natalie; Wang, Yongbao; Lichter, Natalie; Hendrickson, Eric A.

    2010-01-01

    The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways—the main Ku heterodimer-dependent or “classic” NHEJ (C-NHEJ) pathway and an “alternative” NHEJ (A-NHEJ) pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PKcs, XLF, and LIGIV), and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PKcs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PKcs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice. PMID:20195511

  20. Functions of PARylation in DNA Damage Repair Pathways.

    PubMed

    Wei, Huiting; Yu, Xiaochun

    2016-06-01

    Protein poly ADP-ribosylation (PARylation) is a widespread post-translational modification at DNA lesions, which is catalyzed by poly(ADP-ribose) polymerases (PARPs). This modification regulates a number of biological processes including chromatin reorganization, DNA damage response (DDR), transcriptional regulation, apoptosis, and mitosis. PARP1, functioning as a DNA damage sensor, can be activated by DNA lesions, forming PAR chains that serve as a docking platform for DNA repair factors with high biochemical complexity. Here, we highlight molecular insights into PARylation recognition, the expanding role of PARylation in DDR pathways, and the functional interaction between PARylation and ubiquitination, which will offer us a better understanding of the biological roles of this unique post-translational modification. Copyright © 2016 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

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

    SciTech Connect

    Noda, Taichi; Takahashi, Akihisa; Kondo, Natsuko; Mori, Eiichiro; Okamoto, Noritomo; Nakagawa, Yosuke; Ohnishi, Ken; Zdzienicka, Malgorzata Z.; Thompson, Larry H.; Helleday, Thomas; Asada, Hideo; and others

    2011-01-07

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

  2. Comparative Genomics of DNA Recombination and Repair in Cyanobacteria: Biotechnological Implications

    PubMed Central

    Cassier-Chauvat, Corinne; Veaudor, Théo; Chauvat, Franck

    2016-01-01

    Cyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product. Hence, it is important to study DNA recombination and repair in cyanobacteria for both basic and applied research. This review reports what is known in a few widely studied model cyanobacteria and what can be inferred by mining the sequenced genomes of morphologically and physiologically diverse strains. We show that cyanobacteria possess many E. coli-like DNA recombination and repair genes, and possibly other genes not yet identified. E. coli-homolog genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity. Many genes are extremely well conserved in cyanobacteria (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD), even in small genomes, suggesting that they encode the core DNA repair process. In addition to these core genes, the marine Prochlorococcus and Synechococcus strains harbor recBCD (DNA recombination), umuCD (mutational DNA replication), as well as the key SOS genes lexA (regulation of the SOS system) and sulA (postponing of cell division until completion of DNA reparation). Hence, these strains could possess an E. coli-type SOS system. In contrast, several cyanobacteria endowed with larger genomes lack typical SOS genes. For examples, the two studied Gloeobacter strains lack alkB, lexA, and sulA; and Synechococcus PCC7942 has neither lexA nor recCD. Furthermore, the Synechocystis PCC6803 lexA product does not regulate DNA repair genes. Collectively, these findings

  3. Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells

    NASA Technical Reports Server (NTRS)

    Dar, M. E.; Winters, T. A.; Jorgensen, T. J.

    1997-01-01

    Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human disease. Homozygotes suffer from a number of neurological disorders, as well as very high cancer incidence. Heterozygotes may also have a higher than normal risk of cancer, particularly for the breast. The gene responsible for the disease (ATM) has been cloned, but its role in mechanisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defect; however, overall levels of DSB rejoining appear normal. We used the shuttle vector, pZ189, containing an oxidatively-induced DSB, to compare the integrity of DSB rejoining in one normal and two A-T fibroblast cells lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutations found in all three cell lines were deletions (44-63%). The DNA sequence analysis indicated that 17 of the 17 plasmids with deletion mutations in normal cells occurred between short direct-repeat sequences (removing one of the repeats plus the intervening sequences), implicating illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve direct-repeats sequences, implicating a defect in the illegitimate recombination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a mechanistic understanding of this lethal disease.

  4. Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells

    NASA Technical Reports Server (NTRS)

    Dar, M. E.; Winters, T. A.; Jorgensen, T. J.

    1997-01-01

    Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human disease. Homozygotes suffer from a number of neurological disorders, as well as very high cancer incidence. Heterozygotes may also have a higher than normal risk of cancer, particularly for the breast. The gene responsible for the disease (ATM) has been cloned, but its role in mechanisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defect; however, overall levels of DSB rejoining appear normal. We used the shuttle vector, pZ189, containing an oxidatively-induced DSB, to compare the integrity of DSB rejoining in one normal and two A-T fibroblast cells lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutations found in all three cell lines were deletions (44-63%). The DNA sequence analysis indicated that 17 of the 17 plasmids with deletion mutations in normal cells occurred between short direct-repeat sequences (removing one of the repeats plus the intervening sequences), implicating illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve direct-repeats sequences, implicating a defect in the illegitimate recombination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a mechanistic understanding of this lethal disease.

  5. Self-Organization of Meiotic Recombination Initiation: General Principles and Molecular Pathways

    PubMed Central

    Keeney, Scott; Lange, Julian; Mohibullah, Neeman

    2015-01-01

    Recombination in meiosis is a fascinating case study for the coordination of chromosomal duplication, repair, and segregation with each other and with progression through a cell-division cycle. Meiotic recombination initiates with formation of developmentally programmed DNA double-strand breaks (DSBs) at many places across the genome. DSBs are important for successful meiosis but are also dangerous lesions that can mutate or kill, so cells ensure that DSBs are made only at the right times, places, and amounts. This review examines the complex web of pathways that accomplish this control. We explore how chromosome breakage is integrated with meiotic progression and how feedback mechanisms spatially pattern DSB formation and make it homeostatic, robust, and error-correcting. Common regulatory themes recur in different organisms or in different contexts in the same organism. We review this evolutionary and mechanistic conservation but also highlight where control modules have diverged. The framework that emerges helps explain how meiotic chromosomes behave as a self-organizing system. PMID:25421598

  6. A quantitative model of the major pathways for radiation-induced DNA double-strand break repair.

    PubMed

    Belov, Oleg V; Krasavin, Eugene A; Lyashko, Marina S; Batmunkh, Munkhbaatar; Sweilam, Nasser H

    2015-02-07

    We have developed a model approach to simulate the major pathways of DNA double-strand break (DSB) repair in mammalian and human cells. The proposed model shows a possible mechanistic explanation of the basic regularities of DSB processing through the non-homologous end-joining (NHEJ), homologous recombination (HR), single-strand annealing (SSA) and two alternative end-joining pathways. It reconstructs the time-courses of radiation-induced foci specific to particular repair processes including the major intermediate stages. The model is validated for ionizing radiations of a wide range of linear energy transfer (0.2-236 keV/µm) including a relatively broad spectrum of heavy ions. The appropriate set of reaction rate constants was suggested to satisfy the kinetics of DSB rejoining for the considered types of exposure. The simultaneous assessment of several repair pathways allows to describe their possible biological relations in response to irradiation. With the help of the proposed approach, we reproduce several experimental data sets on γ-H2AX foci remaining in different types of cells including those defective in NHEJ, HR, or SSA functions. The results produced confirm the hypothesis suggesting existence of at least two alternative Ku-independent end-joining pathways.

  7. Importance of the cell cycle phase for the choice of the appropriate DSB repair pathway, for genome stability maintenance: the trans-S double-strand break repair model.

    PubMed

    Delacôte, Fabien; Lopez, Bernard S

    2008-01-01

    A DNA double-strand break (DSB) is a highly harmful lesion that can lead to genome rearrangements. Two main pathways compete for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). Depending on the cell cycle phase, the choice of one DSB repair pathway over the other will secure genome stability maintenance or in contrast will increase the risk of genetic instability. HR with the sister chromatid is an efficient way to maintain genome stability, for damage occurring at a post-replication stage. However, in G(1) checkpoint-defective cells, DSBs produced in the G(1) phase and not repaired by NHEJ, can progress through S phase and be processed by HR in late S/G(2) phase. We propose the "trans-S DSB repair" model to account for these data. In this situation HR cannot use the sister chromatid (which is also broken at the same locus) and is thus forced to use ectopic homologous sequences dispersed through the genome, increasing the risk of genetic instability. This shows that the two DSB repair pathways can compete through the cell cycle and underlines the importance of the association between the cell cycle checkpoint and the appropriate DNA repair pathway for genome stability maintenance.

  8. Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli*

    PubMed Central

    Robertson, Adam B.; Matson, Steven W.

    2012-01-01

    The Escherichia coli very short patch (VSP) repair pathway corrects thymidine-guanine mismatches that result from spontaneous hydrolytic deamination damage of 5-methyl cytosine. The VSP repair pathway requires the Vsr endonuclease, DNA polymerase I, a DNA ligase, MutS, and MutL to function at peak efficiency. The biochemical roles of most of these proteins in the VSP repair pathway have been studied extensively. However, these proteins have not been studied together in the context of VSP repair in an in vitro system. Using purified components of the VSP repair system in a reconstitution reaction, we have begun to develop an understanding of the role played by each of these proteins in the VSP repair pathway and have gained insights into their interactions. In this report we demonstrate an in vitro reconstitution of the VSP repair pathway using a plasmid DNA substrate. Surprisingly, the repair track length can be modulated by the concentration of DNA ligase. We propose roles for MutL and MutS in coordination of this repair pathway. PMID:22846989

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

    PubMed

    Stirling, Peter C; Hieter, Philip

    2016-07-22

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

  10. Structure of REC2, a recombinational repair gene of Ustilago maydis, and its function in homologous recombination between plasmid and chromosomal sequences.

    PubMed Central

    Rubin, B P; Ferguson, D O; Holloman, W K

    1994-01-01

    Mutation in the REC2 gene of Ustilago maydis leads to defects in DNA repair, recombination, and meiosis. Analysis of the primary sequence of the Rec2 protein reveals a region with significant homology to bacterial RecA protein and to the yeast recombination proteins Dmc1, Rad51, and Rad57. This homologous region in the U. maydis Rec2 protein was found to be functionally sensitive to mutation, lending support to the hypothesis that Rec2 has a functional RecA-like domain essential for activity in recombination and repair. Homologous recombination between plasmid and chromosomal DNA sequences is reduced substantially in the rec2 mutant following transformation. The frequency can be restored to a level approaching, but not exceeding, that observed in the wild-type strain if transformation is performed with cells containing multiple copies of REC2. Images PMID:8065360

  11. Recombinational repair of radiation-induced double-strand breaks occurs in the absence of extensive resection

    PubMed Central

    Westmoreland, James W.; Resnick, Michael A.

    2016-01-01

    Recombinational repair provides accurate chromosomal restitution after double-strand break (DSB) induction. While all DSB recombination repair models include 5′-3′ resection, there are no studies that directly assess the resection needed for repair between sister chromatids in G-2 arrested cells of random, radiation-induced ‘dirty’ DSBs. Using our Pulse Field Gel Electrophoresis-shift approach, we determined resection at IR-DSBs in WT and mutants lacking exonuclease1 or Sgs1 helicase. Lack of either reduced resection length by half, without decreased DSB repair or survival. In the exo1Δ sgs1Δ double mutant, resection was barely detectable, yet it only took an additional hour to achieve a level of repair comparable to WT and there was only a 2-fold dose-modifying effect on survival. Results with a Dnl4 deletion strain showed that remaining repair was not due to endjoining. Thus, similar to what has been shown for a single, clean HO-induced DSB, a severe reduction in resection tract length has only a modest effect on repair of multiple, dirty DSBs in G2-arrested cells. Significantly, this study provides the first opportunity to directly relate resection length at DSBs to the capability for global recombination repair between sister chromatids. PMID:26503252

  12. Recombinational repair of radiation-induced double-strand breaks occurs in the absence of extensive resection.

    PubMed

    Westmoreland, James W; Resnick, Michael A

    2016-01-29

    Recombinational repair provides accurate chromosomal restitution after double-strand break (DSB) induction. While all DSB recombination repair models include 5'-3' resection, there are no studies that directly assess the resection needed for repair between sister chromatids in G-2 arrested cells of random, radiation-induced 'dirty' DSBs. Using our Pulse Field Gel Electrophoresis-shift approach, we determined resection at IR-DSBs in WT and mutants lacking exonuclease1 or Sgs1 helicase. Lack of either reduced resection length by half, without decreased DSB repair or survival. In the exo1Δ sgs1Δ double mutant, resection was barely detectable, yet it only took an additional hour to achieve a level of repair comparable to WT and there was only a 2-fold dose-modifying effect on survival. Results with a Dnl4 deletion strain showed that remaining repair was not due to endjoining. Thus, similar to what has been shown for a single, clean HO-induced DSB, a severe reduction in resection tract length has only a modest effect on repair of multiple, dirty DSBs in G2-arrested cells. Significantly, this study provides the first opportunity to directly relate resection length at DSBs to the capability for global recombination repair between sister chromatids. Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  13. Positive Cofactor 4 (PC4) is critical for DNA repair pathway re-routing in DT40 cells

    PubMed Central

    Caldwell, Randolph B.; Braselmann, Herbert; Schoetz, Ulrike; Heuer, Steffen; Scherthan, Harry; Zitzelsberger, Horst

    2016-01-01

    PC4 is an abundant single-strand DNA binding protein that has been implicated in transcription and DNA repair. Here, we show that PC4 is involved in the cellular DNA damage response. To elucidate the role, we used the DT40 chicken B cell model, which produces clustered DNA lesions at Ig loci via the action of activation-induced deaminase. Our results help resolve key aspects of immunoglobulin diversification and suggest an essential role of PC4 in repair pathway choice. We show that PC4 ablation in gene conversion (GC)-active cells significantly disrupts GC but has little to no effect on targeted homologous recombination. In agreement, the global double-strand break repair response, as measured by γH2AX foci analysis, is unperturbed 16 hours post irradiation. In cells with the pseudo-genes removed (GC inactive), PC4 ablation reduced the overall mutation rate while simultaneously increasing the transversion mutation ratio. By tagging the N-terminus of PC4, gene conversion and somatic hypermutation are all but abolished even when native non-tagged PC4 is present, indicating a dominant negative effect. Our data point to a very early and deterministic role for PC4 in DNA repair pathway re-routing. PMID:27374870

  14. Isolation of novel human and mouse genes of the recA/RAD51 recombination-repair gene family.

    PubMed Central

    Cartwright, R; Dunn, A M; Simpson, P J; Tambini, C E; Thacker, J

    1998-01-01

    Genes from the recA/RAD51 family play essential roles in homologous recombination in all organisms. Using sequence homologies from eukaryotic members of this family we have identified fragments of two additional mammalian genes with homology to RAD51. Cloning the full-length cDNAs for both human and mouse genes showed that the sequences are highly conserved, and that the predicted proteins have characteristic features of this gene family. One of the novel genes (R51H2) occurs in two forms in human cDNA, differing extensively at the 3' end, probably due to an unusual form of alternative splicing. The new genes (R51H2 and R51H3) were mapped to human chromosomes 14q23-24 and 17q1.2, respectively. Expression studies showed that R51H2 is expressed at lower levels than R51H3 , but that expression of both genes occurs at elevated levels in the testis compared with other tissues. The combination of gene structure conservation and the transcript expression patterns suggests that these new members of the recA/RAD51 family may also function in homologous recombination-repair pathways. PMID:9512535

  15. Effects of suppressing the DNA mismatch repair system on homeologous recombination in tomato.

    PubMed

    Tam, Sheh May; Hays, John B; Chetelat, Roger T

    2011-12-01

    In plant breeding, the ability to manipulate genetic (meiotic) recombination would be beneficial for facilitating gene transfer from wild relatives of crop plants. The DNA mismatch repair (MMR) system helps maintain genetic integrity by correcting base mismatches that arise via DNA synthesis or damage, and antagonizes recombination between homeologous (divergent) DNA sequences. Previous studies have established that the genomes of cultivated tomato (Solanum lycopersicum) and the wild relative S. lycopersicoides are substantially diverged (homeologous) such that recombination between their chromosomes is strongly reduced. Here, we report the effects on homeologous recombination of suppressing endogenous MMR genes in S. lycopersicum via RNAi-induced silencing of SlMSH2 and SlMSH7 or overexpressing dominant negatives of Arabidopsis MSH2 (AtMSH2-DN) in an alien substitution line (SL-8) of S. lycopersicoides in tomato. We show that certain inhibitions of MMR (RNAi of SlMSH7, AtMSH2-DN) are associated with modest increases in homeologous recombination, ranging from 3.8 to 29.2% (average rate of 17.8%) compared to controls. Unexpectedly, only the AtMSH2-DN proteins but not RNAi-induced silencing of MSH2 was found to increase homeologous recombination. The ratio of single to double crossovers (SCO:DCO ratio) decreased by approximately 50% in progeny of the AtMSH2-DN parents. An increase in the frequency of heterozygous SL-8 plants was also observed in the progeny of the SlMSH7-RNAi parents. Our findings may contribute to acceleration of introgression in cultivated tomato.

  16. The effect of thermal dose on hyperthermia-mediated inhibition of DNA repair through homologous recombination.

    PubMed

    van den Tempel, Nathalie; Laffeber, Charlie; Odijk, Hanny; van Cappellen, Wiggert A; van Rhoon, Gerard C; Franckena, Martine; Kanaar, Roland

    2017-07-04

    Hyperthermia has a number of biological effects that sensitize tumors to radiotherapy in the range between 40-44 °C. One of these effects is heat-induced degradation of BRCA2 that in turn causes reduced RAD51 focus formation, which results in an attenuation of DNA repair through homologous recombination. Prompted by this molecular insight into how hyperthermia attenuates homologous recombination, we now quantitatively explore time and temperature dynamics of hyperthermia on BRCA2 levels and RAD51 focus formation in cell culture models, and link this to their clonogenic survival capacity after irradiation (0-6 Gy). For treatment temperatures above 41 °C, we found a decrease in cell survival, an increase in sensitization towards irradiation, a decrease of BRCA2 protein levels, and altered RAD51 focus formation. When the temperatures exceeded 43 °C, we found that hyperthermia alone killed more cells directly, and that processes other than homologous recombination were affected by the heat. This study demonstrates that optimal inhibition of HR is achieved by subjecting cells to hyperthermia at 41-43 °C for 30 to 60 minutes. Our data provides a guideline for the clinical application of novel combination treatments that could exploit hyperthermia's attenuation of homologous recombination, such as the combination of hyperthermia with PARP-inhibitors for non-BRCA mutations carriers.

  17. Distinct histone modifications define initiation and repair of meiotic recombination in the mouse.

    PubMed

    Buard, Jérôme; Barthès, Pauline; Grey, Corinne; de Massy, Bernard

    2009-09-02

    Little is known about the factors determining the location and activity of the rapidly evolving meiotic crossover hotspots that shape genome diversity. Here, we show that several histone modifications are enriched at the active mouse Psmb9 hotspot, and we distinguish those marks that precede from those that follow hotspot recombinational activity. H3K4Me3, H3K4Me2 and H3K9Ac are specifically enriched in the chromatids that carry an active initiation site, and in the absence of DNA double-strand breaks (DSBs) in Spo11(-/-) mice. We thus propose that these marks are part of the substrate for recombination initiation at the Psmb9 hotspot. In contrast, hyperacetylation of H4 is increased as a consequence of DSB formation, as shown by its dependency on Spo11 and by the enrichment detected on both recombining chromatids. In addition, the comparison with another hotspot, Hlx1, strongly suggests that H3K4Me3 and H4 hyperacetylation are common features of DSB formation and repair, respectively. Altogether, the chromatin signatures of the Psmb9 and Hlx1 hotspots provide a basis for understanding the distribution of meiotic recombination.

  18. Distinct histone modifications define initiation and repair of meiotic recombination in the mouse

    PubMed Central

    Buard, Jérôme; Barthès, Pauline; Grey, Corinne; de Massy, Bernard

    2009-01-01

    Little is known about the factors determining the location and activity of the rapidly evolving meiotic crossover hotspots that shape genome diversity. Here, we show that several histone modifications are enriched at the active mouse Psmb9 hotspot, and we distinguish those marks that precede from those that follow hotspot recombinational activity. H3K4Me3, H3K4Me2 and H3K9Ac are specifically enriched in the chromatids that carry an active initiation site, and in the absence of DNA double-strand breaks (DSBs) in Spo11−/− mice. We thus propose that these marks are part of the substrate for recombination initiation at the Psmb9 hotspot. In contrast, hyperacetylation of H4 is increased as a consequence of DSB formation, as shown by its dependency on Spo11 and by the enrichment detected on both recombining chromatids. In addition, the comparison with another hotspot, Hlx1, strongly suggests that H3K4Me3 and H4 hyperacetylation are common features of DSB formation and repair, respectively. Altogether, the chromatin signatures of the Psmb9 and Hlx1 hotspots provide a basis for understanding the distribution of meiotic recombination. PMID:19644444

  19. Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair.

    PubMed

    Davis, Luther; Maizels, Nancy

    2014-03-11

    DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9(D10A) nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors.

  20. Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair

    PubMed Central

    Davis, Luther; Maizels, Nancy

    2014-01-01

    DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9D10A nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors. PMID:24556991

  1. A novel protein, Rsf1/Pxd1, is critical for the single-strand annealing pathway of double-strand break repair in Schizosaccharomyces pombe.

    PubMed

    Wang, Hanqian; Zhang, Zhanlu; Zhang, Lan; Zhang, Qiuxue; Zhang, Liang; Zhao, Yangmin; Wang, Weibu; Fan, Yunliu; Wang, Lei

    2015-06-01

    The process of single-strand annealing (SSA) repairs DNA double-strand breaks that are flanked by direct repeat sequences through the coordinated actions of a series of proteins implicated in recombination, mismatch repair and nucleotide excision repair (NER). Many of the molecular and mechanistic insights gained in SSA repair have principally come from studies in the budding yeast Saccharomyces cerevisiae. However, there is little molecular understanding of the SSA pathway in the fission yeast Schizosaccharomyces pombe. To further our understanding of this important process, we established a new chromosome-based SSA assay in fission yeast. Our genetic analyses showed that, although many homologous components participate in SSA repair in these species indicating that some evolutionary conservation, Saw1 and Slx4 are not principal agents in the SSA repair pathway in fission yeast. This is in marked contrast to the function of Saw1 and Slx4 in budding yeast. Additionally, a novel genus-specific protein, Rsf1/Pxd1, physically interacts with Rad16, Swi10 and Saw1 in vitro and in vivo. We find that Rsf1/Pxd1 is not required for NER and demonstrate that, in fission yeast, Rsf1/Pxd1, but not Saw1, plays a critical role in SSA recombination.

  2. N-Nitrosocarbaryl-induced mutagenesis in Haemophilus influenzae strains deficient in repair and recombination.

    PubMed

    Beattie, K L

    1975-02-01

    Mutagenesis was studied in repair- and recombination-deficient strains of Haemophilus influenzae after treatment with N-nitrosocarbaryl (NC). Three different strains of H. influenzae carrying mutations affecting excision-repair of UV-induced pyrimidine dimers exhibited normal repair of premutational lesions (as detected by decreased mutation yield resulting from post-treatment DNA synthesis delay) and normal nonreplicative mutation fixation. This indicated that neither of these phenomena are caused by the smae repair mechanism that removes UV-induced pyrimidine dimers from the DNA. The recombination-deficient mutant recI is apparently deficient in the replication-dependent mode of NC-induced mutation fixation. This conclusion is based on the following results: (I) NC-induced mutagenesis is lower in the recI strain than in rec+ cells. (2) Repair of premutational lesions (which depends on the existence of replication-dependent mutation fixation for its detection) was not detected in the recI strain. (3) When nonreplicative mutation fixation and final mutation frequency were measured in the same experiment, about I/4 to I/3 of the final mutation yield could be accounted for by nonreplicative mutation fixation in the rec+ strain, whereas all of the mutation could be accounted for in the recI strain by the nonreplicative mutation fixation. (4) When mutation fixation in strain dna9 recI was followed at the permissive (36 degrees) and nonpermissive (41 degrees) temperatures, it became apparent that in the recI strain replication-dependent mutation fixation occurs at early times, but these newly fixed mutations are unstable and disappear at later times, leaving only the mutations fixed by the nonreplicative process. The recI strain exhibits normal repair of NC-induced single-strand breaks or alkali-labile bonds in the DNA labeled before treatment, but is slow in joining discontinuties present in DNA synthesized after treatment. The results are consistent with the idea that

  3. Collaborative roles of gammaH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair.

    PubMed

    Sonoda, Eiichiro; Zhao, Guang Yu; Kohzaki, Masaoki; Dhar, Pawan Kumar; Kikuchi, Koji; Redon, Christophe; Pilch, Duane R; Bonner, William M; Nakano, Atsushi; Watanabe, Masami; Nakayama, Tatsuo; Takeda, Shunichi; Takami, Yasunari

    2007-03-01

    One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (gammaH2AX) at DNA double-strand breaks (DSBs). However, the role of gammaH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX (H2AX(-/S139A)) and examined their DNA repair capacity. H2AX(-/S139A) cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3, also delays Rad51 focus formation. To test the interaction of Xrcc3 and gammaH2AX, we disrupted XRCC3 in H2AX(-/S139A) cells. XRCC3(-/-)/H2AX(-/S139A) mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3(-/-)/H2AX(-/S139A) cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly.

  4. TopBP1 associates with NBS1 and is involved in homologous recombination repair

    SciTech Connect

    Morishima, Ken-ichi; Sakamoto, Shuichi; Kobayashi, Junya; Izumi, Hideki; Suda, Tetsuji; Matsumoto, Yoshiyuki; Tauchi, Hiroshi; Ide, Hiroshi; Komatsu, Kenshi; Matsuura, Shinya

    2007-11-03

    TopBP1 is involved in DNA replication and DNA damage checkpoint. Recent studies have demonstrated that TopBP1 is a direct positive effecter of ATR. However, it is not known how TopBP1 recognizes damaged DNA. Here, we show that TopBP1 formed nuclear foci after exposure to ionizing radiation, but such TopBP1 foci were abolished in Nijmegen breakage syndrome cells. We also show that TopBP1 physically associated with NBS1 in vivo. These results suggested that NBS1 might regulate TopBP1 recruitment to the sites of DNA damage. TopBP1-depleted cells showed hypersensitivity to Mitomycin C and ionizing radiation, an increased frequency of sister-chromatid exchange level, and a reduced frequency of DNA double-strand break induced homologous recombination repair. Together, these results suggested that TopBP1 might be a mediator of DNA damage signaling from NBS1 to ATR and promote homologous recombination repair.

  5. Resolvase OsGEN1 Mediates DNA Repair by Homologous Recombination1[OPEN

    PubMed Central

    Lu, Pingli

    2017-01-01

    Yen1/GEN1 are canonical Holliday junction resolvases that belong to the RAD2/XPG family. In eukaryotes, such as budding yeast, mice, worms, and humans, Yen1/GEN1 work together with Mus81-Mms4/MUS81-EME1 and Slx1-Slx4/SLX1-SLX4 in DNA repair by homologous recombination to maintain genome stability. In plants, the biological function of Yen1/GEN1 remains largely unclear. In this study, we characterized the loss of function mutants of OsGEN1 and OsSEND1, a pair of paralogs of Yen1/GEN1 in rice (Oryza sativa). We first investigated the role of OsGEN1 during meiosis and found a reduction in chiasma frequency by ∼6% in osgen1 mutants, compared to the wild type, suggesting a possible involvement of OsGEN1 in the formation of crossovers. Postmeiosis, OsGEN1 foci were detected in wild-type microspore nuclei, but not in the osgen1 mutant concomitant with an increase in double-strand breaks. Persistent double-strand breaks led to programmed cell death of the male gametes and complete male sterility. In contrast, depletion of OsSEND1 had no effects on plant development and did not enhance osgen1 defects. Our results indicate that OsGEN1 is essential for homologous recombinational DNA repair at two stages of microsporogenesis in rice. PMID:28049740

  6. Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability.

    PubMed

    Su, Xiaofeng A; Dion, Vincent; Gasser, Susan M; Freudenreich, Catherine H

    2015-05-15

    Secondary structure-forming DNA sequences such as CAG repeats interfere with replication and repair, provoking fork stalling, chromosome fragility, and recombination. In budding yeast, we found that expanded CAG repeats are more likely than unexpanded repeats to localize to the nuclear periphery. This positioning is transient, occurs in late S phase, requires replication, and is associated with decreased subnuclear mobility of the locus. In contrast to persistent double-stranded breaks, expanded CAG repeats at the nuclear envelope associate with pores but not with the inner nuclear membrane protein Mps3. Relocation requires Nup84 and the Slx5/8 SUMO-dependent ubiquitin ligase but not Rad51, Mec1, or Tel1. Importantly, the presence of the Nup84 pore subcomplex and Slx5/8 suppresses CAG repeat fragility and instability. Repeat instability in nup84, slx5, or slx8 mutant cells arises through aberrant homologous recombination and is distinct from instability arising from the loss of ligase 4-dependent end-joining. Genetic and physical analysis of Rad52 sumoylation and binding at the CAG tract suggests that Slx5/8 targets sumoylated Rad52 for degradation at the pore to facilitate recovery from acute replication stress by promoting replication fork restart. We thereby confirmed that the relocation of damage to nuclear pores plays an important role in a naturally occurring repair process.

  7. ATM-dependent chromatin remodeler Rsf-1 facilitates DNA damage checkpoints and homologous recombination repair.

    PubMed

    Min, Sunwoo; Jo, Sujin; Lee, Ho-Soo; Chae, Sunyoung; Lee, Jong-Soo; Ji, Jae-Hoon; Cho, Hyeseong

    2014-01-01

    As a member of imitation switch (ISWI) family in ATP-dependent chromatin remodeling factors, RSF complex consists of SNF2h ATPase and Rsf-1. Although it has been reported that SNF2h ATPase is recruited to DNA damage sites (DSBs) in a poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent manner in DNA damage response (DDR), the function of Rsf-1 is still elusive. Here we show that Rsf-1 is recruited to DSBs confirmed by various cellular analyses. Moreover, the initial recruitment of Rsf-1 and SNF2h to DSBs shows faster kinetics than that of γH2AX after micro-irradiation. Signals of Rsf-1 and SNF2h are retained over 30 min after micro-irradiation, whereas γH2AX signals are gradually reduced at 10 min. In addition, Rsf-1 is accumulated at DSBs in ATM-dependent manner, and the putative pSQ motifs of Rsf-1 by ATM are required for its accumulation at DSBs. Furtheremore, depletion of Rsf-1 attenuates the activation of DNA damage checkpoint signals and cell survival upon DNA damage. Finally, we demonstrate that Rsf-1 promotes homologous recombination repair (HRR) by recruiting resection factors RPA32 and Rad51. Thus, these findings reveal a new function of chromatin remodeler Rsf-1 as a guard in DNA damage checkpoints and homologous recombination repair.

  8. Expansion of a chromosomal repeat in Escherichia coli: roles of replication, repair, and recombination functions

    PubMed Central

    Poteete, Anthony R

    2009-01-01

    Background Previous studies of gene amplification in Escherichia coli have suggested that it occurs in two steps: duplication and expansion. Expansion is thought to result from homologous recombination between the repeated segments created by duplication. To explore the mechanism of expansion, a 7 kbp duplication in the chromosome containing a leaky mutant version of the lac operon was constructed, and its expansion into an amplified array was studied. Results Under selection for lac function, colonies bearing multiple copies of the mutant lac operon appeared at a constant rate of approximately 4 to 5 per million cells plated per day, on days two through seven after plating. Expansion was not seen in a recA strain; null mutations in recBCD and ruvC reduced the rate 100- and 10-fold, respectively; a ruvC recG double mutant reduced the rate 1000-fold. Expansion occurred at an increased rate in cells lacking dam, polA, rnhA, or uvrD functions. Null mutations of various other cellular recombination, repair, and stress response genes had little effect upon expansion. The red recombination genes of phage lambda could substitute for recBCD in mediating expansion. In the red-substituted cells, expansion was only partially dependent upon recA function. Conclusion These observations are consistent with the idea that the expansion step of gene amplification is closely related, mechanistically, to interchromosomal homologous recombination events. They additionally provide support for recently described models of RecA-independent Red-mediated recombination at replication forks. PMID:19236706

  9. USP7 inhibition alters homologous recombination repair and targets CLL cells independently of ATM/p53 functional status.

    PubMed

    Agathanggelou, Angelo; Smith, Edward; Davies, Nicholas J; Kwok, Marwan; Zlatanou, Anastasia; Oldreive, Ceri E; Mao, Jingwen; Da Costa, David; Yadollahi, Sina; Perry, Tracey; Kearns, Pamela; Skowronska, Anna; Yates, Elliot; Parry, Helen; Hillmen, Peter; Reverdy, Celine; Delansorne, Remi; Paneesha, Shankara; Pratt, Guy; Moss, Paul; Taylor, A Malcolm R; Stewart, Grant S; Stankovic, Tatjana

    2017-07-13

    The role of deubiquitylase ubiquitin-specific protease 7 (USP7) in the regulation of the p53-dependent DNA damage response (DDR) pathway is well established. Whereas previous studies have mostly focused on the mechanisms underlying how USP7 directly controls p53 stability, we recently showed that USP7 modulates the stability of the DNA damage responsive E3 ubiquitin ligase RAD18. This suggests that targeting USP7 may have therapeutic potential even in tumors with defective p53 or ibrutinib resistance. To test this hypothesis, we studied the effect of USP7 inhibition in chronic lymphocytic leukemia (CLL) where the ataxia telangiectasia mutated (ATM)-p53 pathway is inactivated with relatively high frequency, leading to treatment resistance and poor clinical outcome. We demonstrate that USP7 is upregulated in CLL cells, and its loss or inhibition disrupts homologous recombination repair (HRR). Consequently, USP7 inhibition induces significant tumor-cell killing independently of ATM and p53 through the accumulation of genotoxic levels of DNA damage. Moreover, USP7 inhibition sensitized p53-defective, chemotherapy-resistant CLL cells to clinically achievable doses of HRR-inducing chemotherapeutic agents in vitro and in vivo in a murine xenograft model. Together, these results identify USP7 as a promising therapeutic target for the treatment of hematological malignancies with DDR defects, where ATM/p53-dependent apoptosis is compromised. © 2017 by The American Society of Hematology.

  10. Smc5–Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination

    PubMed Central

    De Piccoli, Giacomo; Cortes-Ledesma, Felipe; Ira, Gregory; Torres-Rosell, Jordi; Uhle, Stefan; Farmer, Sarah; Hwang, Ji-Young; Machin, Felix; Ceschia, Audrey; McAleenan, Alexandra; Cordon-Preciado, Violeta; Clemente-Blanco, Andrés; Vilella-Mitjana, Felip; Ullal, Pranav; Jarmuz, Adam; Leitao, Beatriz; Bressan, Debra; Dotiwala, Farokh; Papusha, Alma; Zhao, Xiaolan; Myung, Kyungjae; Haber, James E.; Aguilera, Andrés; Aragón, Luis

    2015-01-01

    DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5–Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5–Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5–Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events. PMID:16892052

  11. Homologous recombination repairs secondary replication induced DNA double-strand breaks after ionizing radiation

    PubMed Central

    Groth, Petra; Orta, Manuel Luís; Elvers, Ingegerd; Majumder, Muntasir Mamun; Lagerqvist, Anne; Helleday, Thomas

    2012-01-01

    Ionizing radiation (IR) produces direct two-ended DNA double-strand breaks (DSBs) primarily repaired by non-homologous end joining (NHEJ). It is, however, well established that homologous recombination (HR) is induced and required for repair of a subset of DSBs formed following IR. Here, we find that HR induced by IR is drastically reduced when post-DNA damage replication is inhibited in mammalian cells. Both IR-induced RAD51 foci and HR events in the hprt gene are reduced in the presence of replication polymerase inhibitor aphidicolin (APH). Interestingly, we also detect reduced IR-induced toxicity in HR deficient cells when inhibiting post-DNA damage replication. When studying DSB formation following IR exposure, we find that apart from the direct DSBs the treatment also triggers formation of secondary DSBs peaking at 7–9 h after exposure. These secondary DSBs are restricted to newly replicated DNA and abolished by inhibiting post-DNA damage replication. Further, we find that IR-induced RAD51 foci are decreased by APH only in cells replicating at the time of IR exposure, suggesting distinct differences between IR-induced HR in S- and G2-phases of the cell cycle. Altogether, our data indicate that secondary replication-associated DSBs formed following exposure to IR are major substrates for IR-induced HR repair. PMID:22505579

  12. Homologous recombination repairs secondary replication induced DNA double-strand breaks after ionizing radiation.

    PubMed

    Groth, Petra; Orta, Manuel Luís; Elvers, Ingegerd; Majumder, Muntasir Mamun; Lagerqvist, Anne; Helleday, Thomas

    2012-08-01

    Ionizing radiation (IR) produces direct two-ended DNA double-strand breaks (DSBs) primarily repaired by non-homologous end joining (NHEJ). It is, however, well established that homologous recombination (HR) is induced and required for repair of a subset of DSBs formed following IR. Here, we find that HR induced by IR is drastically reduced when post-DNA damage replication is inhibited in mammalian cells. Both IR-induced RAD51 foci and HR events in the hprt gene are reduced in the presence of replication polymerase inhibitor aphidicolin (APH). Interestingly, we also detect reduced IR-induced toxicity in HR deficient cells when inhibiting post-DNA damage replication. When studying DSB formation following IR exposure, we find that apart from the direct DSBs the treatment also triggers formation of secondary DSBs peaking at 7-9 h after exposure. These secondary DSBs are restricted to newly replicated DNA and abolished by inhibiting post-DNA damage replication. Further, we find that IR-induced RAD51 foci are decreased by APH only in cells replicating at the time of IR exposure, suggesting distinct differences between IR-induced HR in S- and G2-phases of the cell cycle. Altogether, our data indicate that secondary replication-associated DSBs formed following exposure to IR are major substrates for IR-induced HR repair.

  13. Real-time analysis of double-strand DNA break repair by homologous recombination.

    PubMed

    Hicks, Wade M; Yamaguchi, Miyuki; Haber, James E

    2011-02-22

    The ability to induce synchronously a single site-specific double-strand break (DSB) in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during DSB repair. Special attention has been paid to the switching of mating-type genes in Saccharomyces cerevisiae, a process initiated by the HO endonuclease by cleaving the MAT locus. A DSB in MATa is repaired by homologous recombination--specifically, by gene conversion--using a heterochromatic donor, HMLα. Repair results in the replacement of the a-specific sequences (Ya) by Yα and switching from MATa to MATα. We report that MAT switching requires the DNA replication factor Dpb11, although it does not require the Cdc7-Dbf4 kinase or the Mcm and Cdc45 helicase components. Using Southern blot, PCR, and ChIP analysis of samples collected every 10 min, we extend previous studies of this process to identify the times for the loading of Rad51 recombinase protein onto the DSB ends at MAT, the subsequent strand invasion by the Rad51 nucleoprotein filament into the donor sequences, the initiation of new DNA synthesis, and the removal of the nonhomologous Y sequences. In addition we report evidence for the transient displacement of well-positioned nucleosomes in the HML donor locus during strand invasion.

  14. A Cross-Cancer Genetic Association Analysis of the DNA repair and DNA Damage Signaling Pathways for Lung, Ovary, Prostate, Breast and Colorectal Cancer

    PubMed Central

    Scarbrough, Peter M.; Weber, Rachel Palmieri; Iversen, Edwin S.; Brhane, Yonathan; Amos, Christopher I.; Kraft, Peter; Hung, Rayjean J.; Sellers, Thomas A.; Witte, John S.; Pharoah, Paul; Henderson, Brian E.; Gruber, Stephen B.; Hunter, David J.; Garber, Judy E.; Joshi, Amit D.; McDonnell, Kevin; Easton, Doug F.; Eeles, Ros; Kote-Jarai, Zsofia; Muir, Kenneth; Doherty, Jennifer A.; Schildkraut, Joellen M.

    2015-01-01

    Background DNA damage is an established mediator of carcinogenesis, though GWAS have identified few significant loci. This cross-cancer site, pooled analysis was performed to increase the power to detect common variants of DNA repair genes associated with cancer susceptibility. Methods We conducted a cross-cancer analysis of 60,297 SNPs, at 229 DNA repair gene regions, using data from the NCI Genetic Associations and Mechanisms in Oncology (GAME-ON) Network. Our analysis included data from 32 GWAS and 48,734 controls and 51,537 cases across five cancer sites (breast, colon, lung, ovary, and prostate). Because of the unavailability of individual data, data were analyzed at the aggregate level. Meta-analysis was performed using the Association analysis for SubSETs (ASSET) software. To test for genetic associations that might escape individual variant testing due to small effect sizes, pathway analysis of eight DNA repair pathways was performed using hierarchical modeling. Results We identified three susceptibility DNA repair genes, RAD51B (p < 5.09 × 10−6), MSH5 (p < 5.09 × 10−6) and BRCA2 (p = 5.70 × 10−6). Hierarchical modeling identified several pleiotropic associations with cancer risk in the base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination pathways. Conclusions Only three susceptibility loci were identified which had all been previously reported. In contrast, hierarchical modeling identified several pleiotropic cancer risk associations in key DNA repair pathways. Impact Results suggest that many common variants in DNA repair genes are likely associated with cancer susceptibility through small effect sizes that do not meet stringent significance testing criteria. PMID:26637267

  15. IGF-1R inhibition enhances radiosensitivity and delays double-strand break repair by both non-homologous end-joining and homologous recombination.

    PubMed

    Chitnis, M M; Lodhia, K A; Aleksic, T; Gao, S; Protheroe, A S; Macaulay, V M

    2014-11-06

    Inhibition of type 1 insulin-like growth factor receptor (IGF-1R) enhances tumor cell sensitivity to ionizing radiation. It is not clear how this effect is mediated, nor whether this approach can be applied effectively in the clinic. We previously showed that IGF-1R depletion delays repair of radiation-induced DNA double-strand breaks (DSBs), unlikely to be explained entirely by reduction in homologous recombination (HR) repair. The current study tested the hypothesis that IGF-1R inhibition induces a repair defect that involves non-homologous end joining (NHEJ). IGF-1R inhibitor AZ12253801 blocked cell survival and radiosensitized IGF-1R-overexpressing murine fibroblasts but not isogenic IGF-1R-null cells, supporting specificity for IGF-1R. IGF-1R inhibition enhanced radiosensitivity in DU145, PC3 and 22Rv1 prostate cancer cells, comparable to effects of Ataxia Telangiectasia Mutated inhibition. AZ12253801-treated DU145 cells showed delayed resolution of γH2AX foci, apparent within 1 h of irradiation and persisting for 24 h. In contrast, IGF-1R inhibition did not influence radiosensitivity or γH2AX focus resolution in LNCaP-LN3 cells, suggesting that radiosensitization tracks with the ability of IGF-1R to influence DSB repair. To differentiate effects on repair from growth and cell-survival responses, we tested AZ12253801 in DU145 cells at sub-SF50 concentrations that had no early (⩽48 h) effects on cell cycle distribution or apoptosis induction. Irradiated cultures contained abnormal mitoses, and after 5 days IGF-1R-inhibited cells showed enhanced radiation-induced polyploidy and nuclear fragmentation, consistent with the consequences of entry into mitosis with incompletely repaired DNA. AZ12253801 radiosensitized DNA-dependent protein kinase (DNA-PK)-proficient but not DNA-PK-deficient glioblastoma cells, and did not radiosensitize DNA-PK-inhibited DU145 cells, suggesting that in the context of DSB repair, IGF-1R functions in the same pathway as DNA

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

    PubMed Central

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

    2001-01-01

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

  17. Molecular Mechanisms of the Whole DNA Repair System: A Comparison of Bacterial and Eukaryotic Systems

    PubMed Central

    Morita, Rihito; Nakane, Shuhei; Shimada, Atsuhiro; Inoue, Masao; Iino, Hitoshi; Wakamatsu, Taisuke; Fukui, Kenji; Nakagawa, Noriko; Masui, Ryoji; Kuramitsu, Seiki

    2010-01-01

    DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly in Thermus thermophilus HB8. PMID:20981145

  18. Emerging roles for centromere-associated proteins in DNA repair and genetic recombination.

    PubMed

    Osman, Fekret; Whitby, Matthew C

    2013-12-01

    Centromere proteins CENP-S and CENP-X are members of the constitutive centromere-associated network, which is a conserved group of proteins that are needed for the assembly and function of kinetochores at centromeres. Intriguingly CENP-S and CENP-X have alter egos going by the names of MHF1 (FANCM-associated histone-fold protein 1) and MHF2 respectively. In this guise they function with a DNA translocase called FANCM (Fanconi's anemia complementation group M) to promote DNA repair and homologous recombination. In the present review we discuss current knowledge of the biological roles of CENP-S and CENP-X and how their dual existence may be a common feature of CCAN (constitutive centromere-associated network) proteins.

  19. Replication factor A is required in vivo for DNA replication, repair, and recombination.

    PubMed Central

    Longhese, M P; Plevani, P; Lucchini, G

    1994-01-01

    Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes. Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFA1 gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae. This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination. Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase alpha-primase complex and with DNA polymerase delta. Images PMID:7969128

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

    PubMed Central

    Sorrell, Melanie; Berman, Zachary

    2014-01-01

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

  1. Promyelocytic leukemia nuclear bodies support a late step in DNA double-strand break repair by homologous recombination.

    PubMed

    Yeung, Percy Luk; Denissova, Natalia G; Nasello, Cara; Hakhverdyan, Zhanna; Chen, J Don; Brenneman, Mark A

    2012-05-01

    The PML protein and PML nuclear bodies (PML-NB) are implicated in multiple cellular functions relevant to tumor suppression, including DNA damage response. In most cases of acute promyelocytic leukemia, the PML and retinoic acid receptor alpha (RARA) genes are translocated, resulting in expression of oncogenic PML-RARα fusion proteins. PML-NB fail to form normally, and promyelocytes remain in an undifferentiated, abnormally proliferative state. We examined the involvement of PML protein and PML-NB in homologous recombinational repair (HRR) of chromosomal DNA double-strand breaks. Transient overexpression of wild-type PML protein isoforms produced hugely enlarged or aggregated PML-NB and reduced HRR by ~2-fold, suggesting that HRR depends to some extent upon normal PML-NB structure. Knockdown of PML by RNA interference sharply attenuated formation of PML-NB and reduced HRR by up to 20-fold. However, PML-knockdown cells showed apparently normal induction of H2AX phosphorylation and RAD51 foci after DNA damage by ionizing radiation. These findings indicate that early steps in HRR, including recognition of DNA double-strand breaks, initial processing of ends, and assembly of single-stranded DNA/RAD51 nucleoprotein filaments, do not depend upon PML-NB. The HRR deficit in PML-depleted cells thus reflects inhibition of later steps in the repair pathway. Expression of PML-RARα fusion proteins disrupted PML-NB structure and reduced HRR by up to 10-fold, raising the possibility that defective HRR and resulting genomic instability may figure in the pathogenesis, progression and relapse of acute promyelocytic leukemia.

  2. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

    PubMed

    Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

    2016-02-17

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems.

  3. Methotrexate induces DNA damage and inhibits homologous recombination repair in choriocarcinoma cells

    PubMed Central

    Xie, Lisha; Zhao, Tiancen; Cai, Jing; Su, You; Wang, Zehua; Dong, Weihong

    2016-01-01

    Objective The objective of this study was to investigate the mechanism of sensitivity to methotrexate (MTX) in human choriocarcinoma cells regarding DNA damage response. Methods Two choriocarcinoma cancer cell lines, JAR and JEG-3, were utilized in this study. An MTX-sensitive osteosarcoma cell line MG63, an MTX-resistant epithelial ovarian cancer cell line A2780 and an MTX-resistant cervical adenocarcinoma cell line Hela served as controls. Cell viability assay was carried out to assess MTX sensitivity of cell lines. MTX-induced DNA damage was evaluated by comet assay. Quantitative reverse transcription polymerase chain reaction was used to detect the mRNA levels of BRCA1, BRCA2, RAD51 and RAD52. The protein levels of γH2AX, RAD 51 and p53 were analyzed by Western blot. Results Remarkable DNA strand breaks were observed in MTX-sensitive cell lines (JAR, JEG-3 and MG63) but not in MTX-resistant cancer cells (A2780 and Hela) after 48 h of MTX treatment. Only in the choriocarcinoma cells, the expression of homologous recombination (HR) repair gene RAD51 was dramatically suppressed by MTX in a dose- and time-dependent manner, accompanied with the increase in p53. Conclusion The MTX-induced DNA strand breaks accompanied by deficiencies in HR repair may contribute to the hypersensitivity to chemotherapy in choriocarcinoma. PMID:27895503

  4. A Test of the Double-Strand Break Repair Model for Meiotic Recombination in Saccharomyces Cerevisiae

    PubMed Central

    Gilbertson, L. A.; Stahl, F. W.

    1996-01-01

    We tested predictions of the double-strand break repair (DSBR) model for meiotic recombination by examining the segregation patterns of small palindromic insertions, which frequently escape mismatch repair when in heteroduplex DNA. The palindromes flanked a well characterized DSB site at the ARG4 locus. The ``canonical'' DSBR model, in which only 5' ends are degraded and resolution of the four-stranded intermediate is by Holliday junction resolvase, predicts that hDNA will frequently occur on both participating chromatids in a single event. Tetrads reflecting this configuration of hDNA were rare. In addition, a class of tetrads not predicted by the canonical DSBR model was identified. This class represented events that produced hDNA in a ``trans'' configuration, on opposite strands of the same duplex on the two sides of the DSB site. Whereas most classes of convertant tetrads had typical frequencies of associated crossovers, tetrads with trans hDNA were parental for flanking markers. Modified versions of the DSBR model, including one that uses a topoisomerase to resolve the canonical DSBR intermediate, are supported by these data. PMID:8878671

  5. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity

    PubMed Central

    Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal S. M.; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K.

    2016-01-01

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear–plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

  6. RecF recombination pathway in Escherichia coli cells lacking RecQ, UvrD and HelD helicases.

    PubMed

    Buljubašić, Maja; Repar, Jelena; Zahradka, Ksenija; Dermić, Damir; Zahradka, Davor

    2012-04-01

    In recBCD sbcB sbcC(D) mutants of Escherichia coli homologous recombination proceeds via RecF pathway, which is thought to require RecQ, UvrD and HelD helicases at its initial stage. It was previously suggested that depletion of all three helicases totally abolishes the RecF pathway. The present study (re)examines the roles of these helicases in transductional recombination, and in recombinational repair of UV-induced DNA damage in the RecF pathway. The study has employed the ΔrecBCD ΔsbcB sbcC201 and ΔrecBCD sbcB15 sbcC201 strains, carrying combinations of mutations in recQ, uvrD, and helD genes. We show that in ΔrecBCD ΔsbcB sbcC201 strains, recombination requires exclusively the RecQ helicase. In ΔrecBCD sbcB15 sbcC201 strains, RecQ may be partially substituted by UvrD helicase. The HelD helicase is dispensable for recombination in both backgrounds. Our results also suggest that significant portion of recombination events in the RecF pathway is independent of RecQ, UvrD and HelD. These events are initiated either by RecJ nuclease alone or by RecJ nuclease associated with an unknown helicase. Inactivation of exonuclease VII by a xseA mutation further decreases the requirement for helicase activity in the RecF pathway. We suggest that elimination of nucleases acting on 3' single-strand DNA ends reduces the necessity for helicases in initiation of recombination.

  7. Repairing DNA double-strand breaks by the prokaryotic non-homologous end-joining pathway.

    PubMed

    Brissett, Nigel C; Doherty, Aidan J

    2009-06-01

    The NHEJ (non-homologous end-joining) pathway is one of the major mechanisms for repairing DSBs (double-strand breaks) that occur in genomic DNA. In common with eukaryotic organisms, many prokaryotes possess a conserved NHEJ apparatus that is essential for the repair of DSBs arising in the stationary phase of the cell cycle. Although the bacterial NHEJ complex is much more minimal than its eukaryotic counterpart, both pathways share a number of common mechanistic features. The relative simplicity of the prokaryotic NHEJ complex makes it a tractable model system for investigating the cellular and molecular mechanisms of DSB repair. The present review describes recent advances in our understanding of prokaryotic end-joining, focusing primarily on biochemical, structural and cellular aspects of the mycobacterial NHEJ repair pathway.

  8. Arsenic exposure disrupts the normal function of the FA/BRCA repair pathway.

    PubMed

    Peremartí, Jana; Ramos, Facundo; Marcos, Ricard; Hernández, Alba

    2014-11-01

    Chronic arsenic exposure is known to enhance the genotoxicity/carcinogenicity of other DNA-damaging agents by inhibiting DNA repair activities. Interference with nucleotide excision repair and base excision repair are well documented, but interactions with other DNA repair pathways are poorly explored so far. The Fanconi anemia FA/BRCA pathway is a DNA repair mechanism required for maintaining genomic stability and preventing cancer. Here, interactions between arsenic compounds and the FA/BRCA pathway were explored by using isogenic FANCD2(-/-) (FA/BRCA-deficient) and FANCD2(+/+) (FA/BRCA-corrected) human fibroblasts. To study whether arsenic disrupts the normal FA/BRCA function, FANCD2(+/+) cells were preexposed to subtoxic concentrations of the trivalent arsenic compounds methylarsonous acid (MMA(III)) and arsenic trioxide (ATO) for 2 weeks. The cellular response to mitomicin-C, hydroxyurea, or diepoxybutane, typical inducers of the studied pathway, was then evaluated and compared to that of FANCD2(-/-) cells. Our results show that preexposure to the trivalent arsenicals MMA(III) and ATO induces in corrected cells, a cellular FA/BRCA-deficient phenotype characterized by hypersensitivity, enhanced accumulation in the G2/M compartment and increased genomic instability--measured as micronuclei. Overall, our data demonstrate that environmentally relevant arsenic exposures disrupt the normal function of the FA/BRCA activity, supporting a novel source of arsenic co- and carcinogenic effects. This is the first study linking arsenic exposure with the FA/BRCA DNA repair pathway.

  9. BRCA2-dependent homologous recombination is required for repair of Arsenite-induced replication lesions in mammalian cells

    PubMed Central

    Ying, Songmin; Myers, Katie; Bottomley, Sarah; Helleday, Thomas; Bryant, Helen E.

    2009-01-01

    Arsenic exposure constitutes one of the most widespread environmental carcinogens, and is associated with increased risk of many different types of cancers. Here we report that arsenite (As[III]) can induce both replication-dependent DNA double-strand breaks (DSB) and homologous recombination (HR) at doses as low as 5 µM (0.65 mg/l), which are within the typical doses often found in drinking water in contaminated areas. We show that the production of DSBs is dependent on active replication and is likely to be the result of conversion of a DNA single-strand break (SSB) into a toxic DSB when encountered by a replication fork. We demonstrate that HR is required for the repair of these breaks and show that a functional HR pathway protects against As[III]-induced cytotoxicity. In addition, BRCA2-deficient cells are sensitive to As[III] and we suggest that As[III] could be exploited as a therapy for HR-deficient tumours such as BRCA1 and BRCA2 mutated breast and ovarian cancers. PMID:19553191

  10. Role for the mammalian Swi5-Sfr1 complex in DNA strand break repair through homologous recombination.

    PubMed

    Akamatsu, Yufuko; Jasin, Maria

    2010-10-14

    In fission yeast, the Swi5-Sfr1 complex plays an important role in homologous recombination (HR), a pathway crucial for the maintenance of genomic integrity. Here we identify and characterize mammalian Swi5 and Sfr1 homologues. Mouse Swi5 and Sfr1 are nuclear proteins that form a complex in vivo and in vitro. Swi5 interacts in vitro with Rad51, the DNA strand-exchange protein which functions during HR. By generating Swi5(-/-) and Sfr1(-/-) embryonic stem cell lines, we found that both proteins are mutually interdependent for their stability. Importantly, the Swi5-Sfr1 complex plays a role in HR when Rad51 function is perturbed in vivo by expression of a BRC peptide from BRCA2. Swi5(-/-) and Sfr1(-/-) cells are selectively sensitive to agents that cause DNA strand breaks, in particular ionizing radiation, camptothecin, and the Parp inhibitor olaparib. Consistent with a role in HR, sister chromatid exchange induced by Parp inhibition is attenuated in Swi5(-/-) and Sfr1(-/-) cells, and chromosome aberrations are increased. Thus, Swi5-Sfr1 is a newly identified complex required for genomic integrity in mammalian cells with a specific role in the repair of DNA strand breaks.

  11. Ring-shaped architecture of RecR: implications for its role in homologous recombinational DNA repair

    PubMed Central

    Lee, Byung Il; Kim, Kyoung Hoon; Park, Soo Jeong; Eom, Soo Hyun; Song, Hyun Kyu; Suh, Se Won

    2004-01-01

    RecR, together with RecF and RecO, facilitates RecA loading in the RecF pathway of homologous recombinational DNA repair in procaryotes . The human Rad52 protein is a functional counterpart of RecFOR. We present here the crystal structure of RecR from Deinococcus radiodurans (DR RecR). A monomer of DR RecR has a two-domain structure: the N-terminal domain with a helix–hairpin–helix (HhH) motif and the C-terminal domain with a Cys4 zinc-finger motif, a Toprim domain and a Walker B motif. Four such monomers form a ring-shaped tetramer of 222 symmetry with a central hole of 30−35 Å diameter. In the crystal, two tetramers are concatenated, implying that the RecR tetramer is capable of opening and closing. We also show that DR RecR binds to both dsDNA and ssDNA, and that its HhH motif is essential for DNA binding. PMID:15116069

  12. Camptothecin enhances the frequency of oligonucleotide-directed gene repair in mammalian cells by inducing DNA damage and activating homologous recombination.

    PubMed

    Ferrara, Luciana; Kmiec, Eric B

    2004-01-01

    Camptothecin (CPT) is an anticancer drug that promotes DNA breakage at replication forks and the formation of lesions that activate the processes of homologous recombination (HR) and nonhomologous end joining. We have taken advantage of the CPT-induced damage response by coupling it to gene repair directed by synthetic oligonucleotides, a process in which a mutant base pair is converted into a wild-type one. Here, we show that pretreating DLD-1 cells with CPT leads to a significant stimulation in the frequency of correction of an integrated mutant enhanced green fluorescent protein gene. The stimulation is dose-dependent and coincident with the formation of double-strand DNA breaks. Caffeine, but not vanillin, blocks the enhancement of gene repair suggesting that, in this system, HR is the pathway most responsible for elevating the frequency of correction. The involvement of HR is further proven by studies in which wortmannin was seen to inhibit gene repair at high concentrations but not at lower levels that are known to inhibit DNA-PK activity. Taken together, our results suggest that DNA damage induced by CPT activates a cellular response that stimulates gene repair in mammalian cells.

  13. The major DNA repair pathway after both proton and carbon-ion radiation is NHEJ, but the HR pathway is more relevant in carbon ions.

    PubMed

    Gerelchuluun, Ariungerel; Manabe, Eri; Ishikawa, Takaaki; Sun, Lue; Itoh, Kazuya; Sakae, Takeji; Suzuki, Kenshi; Hirayama, Ryoichi; Asaithamby, Aroumougame; Chen, David J; Tsuboi, Koji

    2015-03-01

    The purpose of this study was to identify the roles of non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways in repairing DNA double-strand breaks (DSBs) induced by exposure to high-energy protons and carbon ions (C ions) versus gamma rays in Chinese hamster cells. Two Chinese hamster cell lines, ovary AA8 and lung fibroblast V79, as well as various mutant sublines lacking DNA-PKcs (V3), X-ray repair cross-complementing protein-4 [XRCC4 (XR1), XRCC3 (irs1SF) and XRCC2 (irs1)] were exposed to gamma rays ((137)Cs), protons (200 MeV; 2.2 keV/μm) and C ions (290 MeV; 50 keV/μm). V3 and XR1 cells lack the NHEJ pathway, whereas irs1 and irs1SF cells lack the HR pathway. After each exposure, survival was measured using a clonogenic survival assay, in situ DSB induction was evaluated by immunocytochemical analysis of histone H2AX phosphorylation at serine 139 (γ-H2AX foci) and chromosome aberrations were examined using solid staining. The findings from this study showed that clonogenic survival clearly depended on the NHEJ and HR pathway statuses, and that the DNA-PKcs(-/-) cells (V3) were the most sensitive to all radiation types. While protons and γ rays yielded almost the same biological effects, C-ion exposure greatly enhanced the sensitivity of wild-type and HR-deficient cells. However, no significant enhancement of sensitivity in cell killing was seen after C-ion irradiation of NHEJ deficient cells. Decreases in the number of γ-H2AX foci after irradiation occurred more slowly in the NHEJ deficient cells. In particular, V3 cells had the highest number of residual γ-H2AX foci at 24 h after C-ion irradiation. Chromosomal aberrations were significantly higher in both the NHEJ- and HR-deficient cell lines than in wild-type cell lines in response to all radiation types. Protons and gamma rays induced the same aberration levels in each cell line, whereas C ions introduced higher but not significantly different aberration levels. Our results

  14. An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head.

    PubMed

    Mace, Kimberly A; Pearson, Joseph C; McGinnis, William

    2005-04-15

    We used wounded Drosophila embryos to define an evolutionarily conserved pathway for repairing the epidermal surface barrier. This pathway includes a wound response enhancer from the Ddc gene that requires grainy head (grh) function and binding sites for the Grh transcription factor. At the signaling level, tyrosine kinase and extracellular signal-regulated kinase (ERK) activities are induced in epidermal cells near wounds, and activated ERK is required for a robust wound response. The conservation of this Grh-dependent pathway suggests that the repair of insect cuticle and mammal skin is controlled by an ancient, shared control system for constructing and healing the animal body surface barrier.

  15. PI3K/Akt/mTOR pathway inhibitors enhance radiosensitivity in radioresistant prostate cancer cells through inducing apoptosis, reducing autophagy, suppressing NHEJ and HR repair pathways.

    PubMed

    Chang, L; Graham, P H; Hao, J; Ni, J; Bucci, J; Cozzi, P J; Kearsley, J H; Li, Y

    2014-10-02

    The PI3K/Akt/mTOR pathway has a central role in cancer metastasis and radiotherapy. To develop effective therapeutics to improve radiosensitivity, understanding the possible pathways of radioresistance involved and the effects of a combination of the PI3K/Akt/mTOR inhibitors with radiotherapy on prostate cancer (CaP) radioresistant cells is needed. We found that compared with parent CaP cells, CaP-radioresistant cells demonstrated G0/G1 and S phase arrest, activation of cell cycle check point, autophagy and DNA repair pathway proteins, and inactivation of apoptotic proteins. We also demonstrated that compared with combination of single PI3K or mTOR inhibitors (BKM120 or Rapamycin) and radiation, low-dose of dual PI3K/mTOR inhibitors (BEZ235 or PI103) combined with radiation greatly improved treatment efficacy by repressing colony formation, inducing more apoptosis, leading to the arrest of the G2/M phase, increased double-strand break levels and less inactivation of cell cycle check point, autophagy and non-homologous end joining (NHEJ)/homologous recombination (HR) repair pathway proteins in CaP-radioresistant cells. This study describes the possible pathways associated with CaP radioresistance and demonstrates the putative mechanisms of the radiosensitization effect in CaP-resistant cells in the combination treatment. The findings from this study suggest that the combination of dual PI3K/Akt/mTOR inhibitors (BEZ235 or PI103) with radiotherapy is a promising modality for the treatment of CaP to overcome radioresistance.

  16. Functional redundancy between the XLF and DNA-PKcs DNA repair factors in V(D)J recombination and nonhomologous DNA end joining.

    PubMed

    Oksenych, Valentyn; Kumar, Vipul; Liu, Xiangyu; Guo, Chunguang; Schwer, Bjoern; Zha, Shan; Alt, Frederick W

    2013-02-05

    Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway that is required for assembly of antigen receptor variable region gene segments by V(D)J recombination. Recombination activating gene endonuclease initiates V(D)J recombination by generating DSBs between two V(D)J coding gene segments and flanking recombination signal sequences (RS), with the two coding ends and two RS ends joined by C-NHEJ to form coding joins and signal joins, respectively. During C-NHEJ, recombination activating gene factor generates two coding ends as covalently sealed hairpins and RS ends as blunt 5'-phosphorylated DSBs. Opening and processing of coding end hairpins before joining by C-NHEJ requires the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). However, C-NHEJ of RS ends, which do not require processing, occurs relatively normally in the absence of DNA-PKcs. The XRCC4-like factor (XLF) is a C-NHEJ component that is not required for C-NHEJ of chromosomal signal joins or coding joins because of functional redundancy with ataxia telangiectasia mutated kinase, a protein that also has some functional overlap with DNA-PKcs in this process. Here, we show that XLF has dramatic functional redundancy with DNA-PKcs in the V(D)J SJ joining process, which is nearly abrogated in their combined absence. Moreover, we show that XLF functionally overlaps with DNA-PKcs in normal mouse development, promotion of genomic stability in mouse fibroblasts, and in IgH class switch recombination in mature B cells. Our findings suggest that DNA-PKcs has fundamental roles in C-NHEJ processes beyond end processing that have been masked by functional overlaps with XLF.

  17. The DNA excision repair system of the highly radioresistant bacterium Deinococcus radiodurans is facilitated by the pentose phosphate pathway.

    PubMed

    Zhang, Y-M; Liu, J-K; Wong, T-Y

    2003-06-01

    Deinococcus radiodurans is highly resistant to radiation and mutagenic chemicals. Mutants defective in the putative glucose-6-phosphate dehydrogenase gene (zwf-) and the aldolase gene (fda-) were generated by homologous recombination. These mutants were used to test the cells' resistance to agents that cause dimer formation and DNA strand breaks. The zwf - mutants were more sensitive to agents that induce DNA excision repair, such as UV irradiation and H2O2, but were as resistant to DNA strand break-causing agents such as methylmethanesulphonic acid (MMS) and mitomycin C (MMC) as the wild-type cells. Analysis of the cytoplasmic fraction of zwf- cells showed that the concentrations of inosine monophosphate (IMP) and uridine monophosphate (UMP) were only 30% of those found in the wild-type cells. The fda- mutants were slightly more resistant to UV light and H2O2. Results suggested that the deinococcal pentose phosphate pathway augmented the DNA excision repair system by providing cells with adequate metabolites for the DNA mismatch repair.

  18. Involvement of a periplasmic protein kinase in DNA strand break repair and homologous recombination in Escherichia coli.

    PubMed

    Khairnar, Nivedita P; Kamble, Vidya A; Mangoli, Suhas H; Apte, Shree K; Misra, Hari S

    2007-07-01

    The involvement of signal transduction in the repair of radiation-induced damage to DNA has been known in eukaryotes but remains understudied in bacteria. This article for the first time demonstrates a role for the periplasmic lipoprotein (YfgL) with protein kinase activity transducing a signal for DNA strand break repair in Escherichia coli. Purified YfgL protein showed physical as well as functional interaction with pyrroloquinoline-quinone in solution and the protein kinase activity of YfgL was strongly stimulated in the presence of pyrroloquinoline-quinone. Transgenic E. coli cells producing Deinococcus radiodurans pyrroloquinoline-quinone synthase showed nearly four log cycle improvement in UVC dark survival and 10-fold increases in gamma radiation resistance as compared with untransformed cells. Pyrroloquinoline-quinone enhanced the UV resistance of E. coli through the YfgL protein and required the active recombination repair proteins. The yfgL mutant showed higher sensitivity to UVC, mitomycin C and gamma radiation as compared with wild-type cells and showed a strong impairment in homologous DNA recombination. The mutant expressing an active YfgL in trans recovered the lost phenotypes to nearly wild-type levels. The results strongly suggest that the periplasmic phosphoquinolipoprotein kinase YfgL plays an important role in radiation-induced DNA strand break repair and homologous recombination in E. coli.

  19. Meiotic recombination involving heterozygous large insertions in Saccharomyces cerevisiae: formation and repair of large, unpaired DNA loops.

    PubMed Central

    Kearney, H M; Kirkpatrick, D T; Gerton, J L; Petes, T D

    2001-01-01

    Meiotic recombination in Saccharomyces cerevisiae involves the formation of heteroduplexes, duplexes containing DNA strands derived from two different homologues. If the two strands of DNA differ by an insertion or deletion, the heteroduplex will contain an unpaired DNA loop. We found that unpaired loops as large as 5.6 kb can be accommodated within a heteroduplex. Repair of these loops involved the nucleotide excision repair (NER) enzymes Rad1p and Rad10p and the mismatch repair (MMR) proteins Msh2p and Msh3p, but not several other NER (Rad2p and Rad14p) and MMR (Msh4p, Msh6p, Mlh1p, Pms1p, Mlh2p, Mlh3p) proteins. Heteroduplexes were also formed with DNA strands derived from alleles containing two different large insertions, creating a large "bubble"; repair of this substrate was dependent on Rad1p. Although meiotic recombination events in yeast are initiated by double-strand DNA breaks (DSBs), we showed that DSBs occurring within heterozygous insertions do not stimulate interhomologue recombination. PMID:11514439

  20. DNA repair and damage pathways in breast cancer development and therapy.

    PubMed

    Majidinia, Maryam; Yousefi, Bahman

    2017-06-01

    DNA damage/repair constitutes several key pathways working in concert to eliminate DNA lesions and maintain genome stability and integrity. Defective components in DNA damage and repair machinery are an underlying cause for the development and progression of different types of cancers, and breast cancer is no exception. In this paper, we will briefly explain the importance of DNA damage and repair, introduce the current classification schemes for breast cancer, and review the known defects in the repair machinery that have been associated with the risk of breast cancer. Finally, we discuss how the understanding of these pathways can help to design therapeutics for specific targeting of breast cancer tumors. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Nanotechnology in cancer therapy: targeting the inhibition of key DNA repair pathways.

    PubMed

    Aziz, K; Nowsheen, S; Georgakilas, A G

    2010-10-01

    Cancer therapy has been changing over the decades as we move away from the administration of broad spectrum cytotoxic drugs and towards the use of therapy targeted for each tumor type. After the induction of DNA damage through chemotherapeutic agents, tumor cells can survive due to their proficient DNA repair pathways, some of which are dysregulated in cancer. Latest improvements in nanotechnology and drug discovery has led to the discovery of some very unique, highly specific and innovative drugs as inhibitors of various DNA repair pathways like base excision repair and double strand break repair. In this review we look at the efficacy and potency of these small chemical molecules to target the processing of DNA damage induced by standard therapeutic agents. Emphasis is given to those drugs currently under clinical trials. We also discuss the future directions of using this nanotechnology to increase the therapeutic ratio in cancer treatment.

  2. Specific targeted gene repair using single-stranded DNA oligonucleotides at an endogenous locus in mammalian cells uses homologous recombination.

    PubMed

    McLachlan, Jennifer; Fernandez, Serena; Helleday, Thomas; Bryant, Helen E

    2009-12-03

    The feasibility of introducing point mutations in vivo using single-stranded DNA oligonucleotides (ssON) has been demonstrated but the efficiency and mechanism remain elusive and potential side effects have not been fully evaluated. Understanding the mechanism behind this potential therapy may help its development. Here, we demonstrate the specific repair of an endogenous non-functional hprt gene by a ssON in mammalian cells, and show that the frequency of such an event is enhanced when cells are in S-phase of the cell cycle. A potential barrier in using ssONs as gene therapy could be non-targeted mutations or gene rearrangements triggered by the ssON. Both the non-specific mutation frequencies and the frequency of gene rearrangements were largely unaffected by ssONs. Furthermore, we find that the introduction of a mutation causing the loss of a functional endogenous hprt gene by a ssON occurred at a similarly low but statistically significant frequency in wild type cells and in cells deficient in single strand break repair, nucleotide excision repair and mismatch repair. However, this mutation was not induced in XRCC3 mutant cells deficient in homologous recombination. Thus, our data suggest ssON-mediated targeted gene repair is more efficient in S-phase and involves homologous recombination.

  3. Initiation of DNA double strand break repair: signaling and single-stranded resection dictate the choice between homologous recombination, non-homologous end-joining and alternative end-joining.

    PubMed

    Grabarz, Anastazja; Barascu, Aurélia; Guirouilh-Barbat, Josée; Lopez, Bernard S

    2012-01-01

    A DNA double strand break (DSB) is a highly toxic lesion, which can generate genetic instability and profound genome rearrangements. However, DSBs are required to generate diversity during physiological processes such as meiosis or the establishment of the immune repertoire. Thus, the precise regulation of a complex network of processes is necessary for the maintenance of genomic stability, allowing genetic diversity but protecting against genetic instability and its consequences on oncogenesis. Two main strategies are employed for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by single-stranded DNA (ssDNA) resection and requires sequence homology with an intact partner, while NHEJ requires neither resection at initiation nor a homologous partner. Thus, resection is an pivotal step at DSB repair initiation, driving the choice of the DSB repair pathway employed. However, an alternative end-joining (A-EJ) pathway, which is highly mutagenic, has recently been described; A-EJ is initiated by ssDNA resection but does not require a homologous partner. The choice of the appropriate DSB repair system, for instance according the cell cycle stage, is essential for genome stability maintenance. In this context, controlling the initial events of DSB repair is thus an essential step that may be irreversible, and the wrong decision should lead to dramatic consequences. Here, we first present the main DSB repair mechanisms and then discuss the importance of the choice of the appropriate DSB repair pathway according to the cell cycle phase. In a third section, we present the early steps of DSB repair i.e., DSB signaling, chromatin remodeling, and the regulation of ssDNA resection. In the last part, we discuss the competition between the different DSB repair mechanisms. Finally, we conclude with the importance of the fine tuning of this network for genome stability maintenance and for tumor protection in fine.

  4. Genistein sensitizes sarcoma cells in vitro and in vivo by enhancing apoptosis and by inhibiting DSB repair pathways.

    PubMed

    Liu, X X; Sun, C; Jin, X D; Li, P; Zheng, X G; Zhao, T; Li, Q

    2016-06-01

    The aim of this work was to investigate the radiosensitization effects of genistein on mice sarcoma cells and the corresponding biological mechanisms in vitro and in vivo Using the non-toxic dosage of 10 μM genistein, the sensitizer enhancement ratios after exposure to X-rays at 50% cell survival (IC50) was 1.45 for S180 cells. For mice cotreated with genistein and X-rays, the excised tumor tissues had reduced blood vessels and decreased size and volume compared with the control and irradiation-only groups. Moreover, a significant increase in apoptosis was accompanied by upregulation of Bax and downregulation of Bcl-2 in the mitochondria, and lots of cytochrome c being transferred to the cytoplasm. Furthermore, X-rays combined with genistein inhibited the activity of DNA-PKcs, so DNA-injured sites were dominated by Ku70/80, leading to incompleteness of homologous recombination (HR) and non-homologous end-joining (NHEJ) repairs and the eventual occurrence of cell apoptosis. Our study, for the first time, demonstrated that genistein sensitized sarcoma cells to X-rays and that this radiosensitizing effect depended on induction of the mitochondrial apoptosis pathway and inhibition of the double-strand break (DSB) repair pathways.

  5. Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis

    SciTech Connect

    Cole, G.M.; Mortimer, R.K. ); Schild, D. )

    1989-07-01

    The DNA repair and recombination genes of Saccharomyces cerevisiae, RAD52 and RAD54, were transcriptionally induced approximately 10- to 15-fold in sporulating MATa/{alpha} cells. Congenic MATa/a cells, which did not sporulate, did not show similar increases. Assays of {beta}-galactosidase activity in strains harboring either a RAD52- or RAD54-lacZ gene fusion indicated that this induction occurred at a time concomitant with a commitment to meiotic recombination, as measured by prototroph formation from his1 heteroalleles.

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

    SciTech Connect

    Jones, IM

    2002-01-09

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

  7. Human RecQ Helicases in DNA Repair, Recombination, and Replication

    PubMed Central

    Croteau, Deborah L.; Popuri, Venkateswarlu; Opresko, Patricia L.; Bohr, Vilhelm A.

    2015-01-01

    RecQ helicases are an important family of genome surveillance proteins conserved from bacteria to humans. Each of the five human RecQ helicases plays critical roles in genome maintenance and stability, and the RecQ protein family members are often referred to as guardians of the genome. The importance of these proteins in cellular homeostasis is underscored by the fact that defects in BLM, WRN, and RECQL4 are linked to distinct heritable human disease syndromes. Each human RecQ helicase has a unique set of protein-interacting partners, and these interactions dictate its specialized functions in genome maintenance, including DNA repair, recombination, replication, and transcription. Human RecQ helicases also interact with each other, and these interactions have significant impact on enzyme function. Future research goals in this field include a better understanding of the division of labor among the human RecQ helicases and learning how human RecQ helicases collaborate and cooperate to enhance genome stability. PMID:24606147

  8. Structure and regulation of the Escherichia coli ruv operon involved in DNA repair and recombination.

    PubMed Central

    Shinagawa, H; Makino, K; Amemura, M; Kimura, S; Iwasaki, H; Nakata, A

    1988-01-01

    The ruv gene of Escherichia coli, which is involved in DNA repair and recombination, was cloned on a plasmid vector. The DNA of the ruv region was sequenced; it had two open reading frames in tandem that could code for 22- and 37-kilodalton proteins. The proteins encoded by these open reading frames were identified by the maxicell method. The two genes were aligned in the same orientation and regulated by the SOS system, so the two genes probably constitute an operon. The distal one complemented the ruv mutations. Transcription of the operon was studied both in vivo and in vitro. Two transcription initiation sites were identified upstream of the coding frames, and the transcription from both sites was repressed by the LexA repressor. A DNA sequence that is homologous to the SOS box and bound by LexA protein was found in the regulatory region of the operon. The amino acid sequence of Ruv protein deduced from the DNA sequence shows a high degree of homology to the consensus sequence shared by ATP-binding proteins. Images PMID:2842314

  9. ATR suppresses endogenous DNA damage and allows completion of homologous recombination repair.

    PubMed

    Brown, Adam D; Sager, Brian W; Gorthi, Aparna; Tonapi, Sonal S; Brown, Eric J; Bishop, Alexander J R

    2014-01-01

    DNA replication fork stalling or collapse that arises from endogenous damage poses a serious threat to genome stability, but cells invoke an intricate signaling cascade referred to as the DNA damage response (DDR) to prevent such damage. The gene product ataxia telangiectasia and Rad3-related (ATR) responds primarily to replication stress by regulating cell cycle checkpoint control, yet it's role in DNA repair, particularly homologous recombination (HR), remains unclear. This is of particular interest since HR is one way in which replication restart can occur in the presence of a stalled or collapsed fork. Hypomorphic mutations in human ATR cause the rare autosomal-recessive disease Seckel syndrome, and complete loss of Atr in mice leads to embryonic lethality. We recently adapted the in vivo murine pink-eyed unstable (pun) assay for measuring HR frequency to be able to investigate the role of essential genes on HR using a conditional Cre/loxP system. Our system allows for the unique opportunity to test the effect of ATR loss on HR in somatic cells under physiological conditions. Using this system, we provide evidence that retinal pigment epithelium (RPE) cells lacking ATR have decreased density with abnormal morphology, a decreased frequency of HR and an increased level of chromosomal damage.

  10. Incorporation of Human Recombinant Tropoelastin into Silk Fibroin Membranes with the View to Repairing Bruch's Membrane.

    PubMed

    Shadforth, Audra M A; Suzuki, Shuko; Alzonne, Raphaelle; Edwards, Grant A; Richardson, Neil A; Chirila, Traian V; Harkin, Damien G

    2015-09-16

    Bombyx mori silk fibroin membranes provide a potential delivery vehicle for both cells and extracellular matrix (ECM) components into diseased or injured tissues. We have previously demonstrated the feasibility of growing retinal pigment epithelial cells (RPE) on fibroin membranes with the view to repairing the retina of patients afflicted with age-related macular degeneration (AMD). The goal of the present study was to investigate the feasibility of incorporating the ECM component elastin, in the form of human recombinant tropoelastin, into these same membranes. Two basic strategies were explored: (1) membranes prepared from blended solutions of fibroin and tropoelastin; and (2) layered constructs prepared from sequentially cast solutions of fibroin, tropoelastin, and fibroin. Optimal conditions for RPE attachment were achieved using a tropoelastin-fibroin blend ratio of 10 to 90 parts by weight. Retention of tropoelastin within the blend and layered constructs was confirmed by immunolabelling and Fourier-transform infrared spectroscopy (FTIR). In the layered constructs, the bulk of tropoelastin was apparently absorbed into the initially cast fibroin layer. Blend membranes displayed higher elastic modulus, percentage elongation, and tensile strength (p < 0.01) when compared to the layered constructs. RPE cell response to fibroin membranes was not affected by the presence of tropoelastin. These findings support the potential use of fibroin membranes for the co-delivery of RPE cells and tropoelastin.

  11. Berberine Radiosensitizes Human Esophageal Cancer Cells by Downregulating Homologous Recombination Repair Protein RAD51

    PubMed Central

    Liu, Zhaojian; Wang, Yu; Zhao, Minnan; Hao, Chunyan; Feng, Shuai; Guo, Haiyang; Xu, Bing; Yang, Qifeng; Gong, Yaoqin; Shao, Changshun

    2011-01-01

    Background Esophageal squamous cell carcinomas (ESCC) have poor prognosis. While combined modality of chemotherapy and radiotherapy increases survival, most patients die within five years. Development of agents that confer cancer cell-specific chemo- and radiosensitivity may improve the therapy of ESCC. We here reported the discovery of berberine as a potent radiosensitizing agent on ESCC cells. Principal Findings Berberine at low concentrations (<15 µM) substantially radiosensitized ESCC cells. X-ray induced DNA double-strand breaks (DSBs) persist longer in ESCC cells pretreated with berberine. Berberine pretreatment led to a significant downregulation of RAD51, a key player in homologous recombination repair, in ESCC cells, but not in non-malignant human cells. Downregulation of RAD51 by RNA interference similarly radiosensitized the cancer cells, and, conversely, introduction of exogenous RAD51 was able to significantly counteract the radiosensitizing effect of berberine, thus establishing RAD51 as a key determinant in radiation sensitivity. We also observed that RAD51 was commonly overexpressed in human ESCC tissues, suggesting that it is necessary to downregulate RAD51 to achieve high radio- or chemotherapeutic efficacy of ESCC in clinic, because overexpression of RAD51 is known to confer radio- and chemoresistance. Conclusions/Significance Berberine can effectively downregulate RAD51 in conferring radiosensitivity on esophageal cancer cells. Its clinical application as an adjuvant in chemotherapy and radiotherapy of esophageal cancers should be explored. PMID:21858113

  12. Triapine potentiates platinum-based combination therapy by disruption of homologous recombination repair

    PubMed Central

    Ratner, Elena S; Zhu, Yong-Lian; Penketh, Philip G; Berenblum, Julie; Whicker, Margaret E; Huang, Pamela H; Lee, Yashang; Ishiguro, Kimiko; Zhu, Rui; Sartorelli, Alan C; Lin, Z Ping

    2016-01-01

    Background: Platinum resistance may be attributable to inherent or acquired proficiency in homologous recombination repair (HRR) in epithelial ovarian cancer (EOC). The objective of this study was to evaluate the efficacy of the small molecule inhibitor triapine to disrupt HRR and sensitise BRCA wild-type EOC cells to platinum-based combination therapy in vitro and in vivo. Methods: The sensitivity of BRCA wild-type cancer cells to olaparib, cisplatin, carboplatin, doxorubicin, or etoposide in combination with triapine was evaluated by clonogenic survival assays. The effects of triapine on HRR activity in cells were measured with a DR-GFP reporter assay. The ability of triapine to enhance the effects of the carboplatin-doxil combination on EOC tumour growth delay was determined using a xenograft tumour mouse model. Results: Platinum resistance is associated with wild-type BRCA status. Triapine inhibits HRR activity and enhances the sensitivity of BRCA wild-type cancer cells to cisplatin, olaparib, and doxorubicin. However, sequential combination of triapine and cisplatin is necessary to achieve synergism. Moreover, triapine potentiates platinum-based combination therapy against BRCA wild-type EOC cells and produces significant delay of EOC tumour growth. Conclusions: Triapine promises to augment the clinical efficacy of platinum-based combination regimens for treatment of platinum-resistant EOC with wild-type BRCA and proficient HRR activity. PMID:26964031

  13. Human RecQ helicases in DNA repair, recombination, and replication.

    PubMed

    Croteau, Deborah L; Popuri, Venkateswarlu; Opresko, Patricia L; Bohr, Vilhelm A

    2014-01-01

    RecQ helicases are an important family of genome surveillance proteins conserved from bacteria to humans. Each of the five human RecQ helicases plays critical roles in genome maintenance and stability, and the RecQ protein family members are often referred to as guardians of the genome. The importance of these proteins in cellular homeostasis is underscored by the fact that defects in BLM, WRN, and RECQL4 are linked to distinct heritable human disease syndromes. Each human RecQ helicase has a unique set of protein-interacting partners, and these interactions dictate its specialized functions in genome maintenance, including DNA repair, recombination, replication, and transcription. Human RecQ helicases also interact with each other, and these interactions have significant impact on enzyme function. Future research goals in this field include a better understanding of the division of labor among the human RecQ helicases and learning how human RecQ helicases collaborate and cooperate to enhance genome stability.

  14. RecQ helicases; at the crossroad of genome replication, repair, and recombination.

    PubMed

    Rezazadeh, Sarallah

    2012-04-01

    DNA helicases are ubiquitous enzymes that unwind double-stranded DNA in an ATP-dependent and directionally specific manner. Such an action is essential for the processes of DNA repair, recombination, transcription, and DNA replication. Here, I focus on a subgroup of DNA helicases, the RecQ family, which is highly conserved in evolution. Members of this conserved family of proteins have a key role in protecting and stabilizing the genome against deleterious changes. Deficiencies in RecQ helicases can lead to high levels of genomic instability and, in humans, to premature aging and increased susceptibility to cancer. Their diverse roles in DNA metabolism, which include a role in telomere maintenance, reflect interactions with multiple cellular proteins, some of which are multifunctional and also have very diverse functions. In this review, protein structural motifs and the roles of different domains will be discussed first. The Review moves on to speculate about the different models to explain why RecQ helicases are required to protect against genome instability.

  15. Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi.

    PubMed

    Chiurillo, Miguel A; Moraes Barros, Roberto R; Souza, Renata T; Marini, Marjorie M; Antonio, Cristiane R; Cortez, Danielle R; Curto, María Á; Lorenzi, Hernán A; Schijman, Alejandro G; Ramirez, José L; da Silveira, José F

    2016-01-01

    Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a T. cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-SceI meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families.

  16. Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi

    PubMed Central

    Chiurillo, Miguel A.; Moraes Barros, Roberto R.; Souza, Renata T.; Marini, Marjorie M.; Antonio, Cristiane R.; Cortez, Danielle R.; Curto, María Á.; Lorenzi, Hernán A.; Schijman, Alejandro G.; Ramirez, José L.; da Silveira, José F.

    2016-01-01

    Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a T. cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-SceI meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families. PMID:28066363

  17. Evidence for repair of ultraviolet light-damaged herpes virus in human fibroblasts by a recombination mechanism

    SciTech Connect

    Hall, J.D.; Featherston, J.D.; Almy, R.E.

    1980-09-01

    Human cells were either singly or multiply infected with herpes simplex virus (HSV-1) damaged by ultraviolet (uv) light, and the fraction of cells able to produce infectious virus was measured. The fraction of virus-producing cells was considerably greater for multiply infected cells than for singly infected cells at each uv dose examined. These high survival levels of uv-irradiated virus in multiply infected cells demonstrated that multiplicity-dependent repair, possibly due to genetic exchanges between damaged HSV-1 genomes, was occurring in these cells. To test whether uv light is recombinogenic for HSV-1, the effect of uv irradiation on the yield of temperature-resistant viral recombinants in cells infected with pairs of temperature-sensitive mutants was also investigated. The results of these experiments showed that the defective functions in these mutant host cells are not required for multiplicity-dependent repair or uv-stimulated viral recombination in herpes-infected cells.

  18. DNA-protein crosslinks processed by nucleotide excision repair and homologous recombination with base and strand preference in E. coli model system.

    PubMed

    Fang, Qingming

    2013-01-01

    Bis-electrophiles including dibromoethane and epibromohydrin can react with O(6)-alkylguanine-DNA alkyltransferase (AGT) and form AGT-DNA crosslinks in vitro and in vivo. The presence of human AGT (hAGT) paradoxically increases the mutagenicity and cytotoxicity of bis-electrophiles in cells. Here we establish a bacterial system to study the repair mechanism and cellular responses to DNA-protein crosslinks (DPCs) in vivo. Results show that both nucleotide excision repair (NER) and homologous recombination (HR) pathways can process hAGT-DNA crosslinks with HR playing a dominant role. Mutation spectra show that HR has no strand preference but NER favors processing of the DPCs in the transcribed strand; UvrA, UvrB and Mfd can interfere with small size DPCs but only UvrA can interfere with large size DPCs in the transcribed strand processed by HR. Further, we found that DPCs at TA deoxynucleotide sites are very inefficiently processed by NER and the presence of NER can interfere with these DNA lesions processed by HR. These data indicate that NER and HR can process DPCs cooperatively and competitively and NER processes DPCs with base and strand preference. Therefore, the formation of hAGT-DNA crosslinks can be a plausible and specific system to study the repair mechanism and effects of DPCs precisely in vivo.

  19. Homologous recombinational repair factors are recruited and loaded onto the viral DNA genome in Epstein-Barr virus replication compartments.

    PubMed

    Kudoh, Ayumi; Iwahori, Satoko; Sato, Yoshitaka; Nakayama, Sanae; Isomura, Hiroki; Murata, Takayuki; Tsurumi, Tatsuya

    2009-07-01

    Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.

  20. New single nucleotide polymorphisms (SNPs) in homologous recombination repair genes detected by microarray analysis in Polish breast cancer patients.

    PubMed

    Romanowicz, Hanna; Strapagiel, Dominik; Słomka, Marcin; Sobalska-Kwapis, Marta; Kępka, Ewa; Siewierska-Górska, Anna; Zadrożny, Marek; Bieńkiewicz, Jan; Smolarz, Beata

    2016-11-30

    Breast cancer is the most common cause of malignancy and mortality in women worldwide. This study aimed at localising homologous recombination repair (HR) genes and their chromosomal loci and correlating their nucleotide variants with susceptibility to breast cancer. In this study, authors analysed the association between single nucleotide polymorphisms (SNPs) in homologous recombination repair genes and the incidence of breast cancer in the population of Polish women. Blood samples from 94 breast cancer patients were analysed as test group. Individuals were recruited into the study at the Department of Oncological Surgery and Breast Diseases of the Institute of the Polish Mother's Memorial Hospital in Lodz, Poland. Healthy controls (n = 500) were obtained from the Biobank Laboratory, Department of Molecular Biophysics, University of Lodz. Then, DNA of breast cancer patients was compared with one of the disease-free women. The test was supported by microarray analysis. Statistically significant correlations were identified between breast cancer and 3 not described previously SNPs of homologous recombination repair genes BRCA1 and BRCA2: rs59004709, rs4986852 and rs1799950. Further studies on larger groups are warranted to support the hypothesis of correlation between the abovementioned genetic variants and breast cancer risk.

  1. The ATPase activity of Fml1 is essential for its roles in homologous recombination and DNA repair

    PubMed Central

    Nandi, Saikat; Whitby, Matthew C.

    2012-01-01

    In fission yeast, the DNA helicase Fml1, which is an orthologue of human FANCM, is a key component of the machinery that drives and governs homologous recombination (HR). During the repair of DNA double-strand breaks by HR, it limits the occurrence of potentially deleterious crossover recombinants, whereas at stalled replication forks, it promotes HR to aid their recovery. Here, we have mutated conserved residues in Fml1’s Walker A (K99R) and Walker B (D196N) motifs to determine whether its activities are dependent on its ability to hydrolyse ATP. Both Fml1K99R and Fml1D196N are proficient for DNA binding but totally deficient in DNA unwinding and ATP hydrolysis. In vivo both mutants exhibit a similar reduction in recombination at blocked replication forks as a fml1Δ mutant indicating that Fml1’s motor activity, fuelled by ATP hydrolysis, is essential for its pro-recombinogenic role. Intriguingly, both fml1K99R and fml1D196N mutants exhibit greater sensitivity to genotoxins and higher levels of crossing over during DSB repair than a fml1Δ strain. These data suggest that without its motor activity, the binding of Fml1 to its DNA substrate can impede alternative mechanisms of repair and crossover avoidance. PMID:22844101

  2. A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52.

    PubMed Central

    Firmenich, A A; Elias-Arnanz, M; Berg, P

    1995-01-01

    To understand the mechanisms involved in homologous recombination, we have performed a search for Saccharomyces cerevisiae mutants unable to carry out plasmid-to-chromosome gene conversion. For this purpose, we have developed a colony color assay in which recombination is induced by the controlled delivery of double-strand breaks (DSBs). Recombination occurs between a chromosomal mutant ade2 allele and a second plasmid-borne ade2 allele where DSBs are introduced via the site-specific HO endonuclease. Besides isolating a number of new alleles in known rad genes, we identified a novel allele of the RFA1 gene, rfa1-44, which encodes the large subunit of the heterotrimeric yeast single-stranded DNA-binding protein RPA. Characterization of rfa1-44 revealed that it is, like members of the RAD52 epistasis group, sensitive to X rays, high doses of UV, and HO-induced DSBs. In addition, rfa1-44 shows a reduced ability to undergo sporulation and HO-induced gene conversion. The mutation was mapped to a single-base substitution resulting in an aspartate at amino acid residue 77 instead of glycine. Moreover, all radiation sensitivities and repair defects of rfa1-44 are suppressed by RAD52 in a dose-dependent manner, and one RAD52 mutant allele, rad52-34, displays nonallelic noncomplementation when crossed with rfa1-44. Presented is a model accounting for this genetic interaction in which Rfa1, in a complex with Rad52, serves to assemble other proteins of the recombination-repair machinery at the site of DSBs and other kinds of DNA damage. We believe that our findings and those of J. Smith and R. Rothstein (Mol. Cell. Biol. 15:1632-1641, 1995) are the first in vivo demonstrations of the involvement of a eukaryotic single-stranded binding protein in recombination and repair processes. PMID:7862153

  3. Genetic instability is prevented by Mrc1-dependent spatio-temporal separation of replicative and repair activities of homologous recombination

    PubMed Central

    Prado, Félix

    2014-01-01

    Homologous recombination (HR) is required to protect and restart stressed replication forks. Paradoxically, the Mrc1 branch of the S phase checkpoints, which is activated by replicative stress, prevents HR repair at breaks and arrested forks. Indeed, the mechanisms underlying HR can threaten genome integrity if not properly regulated. Thus, understanding how cells avoid genetic instability associated with replicative stress, a hallmark of cancer, is still a challenge. Here I discuss recent results that support a model by which HR responds to replication stress through replicative and repair activities that operate at different stages of the cell cycle (S and G2, respectively) and in distinct subnuclear structures. Remarkably, the replication checkpoint appears to control this scenario by inhibiting the assembly of HR repair centers at stressed forks during S phase, thereby avoiding genetic instability. PMID:24615940

  4. Bone Injury and Repair Trigger Central and Peripheral NPY Neuronal Pathways

    PubMed Central

    Alencastre, Inês S.; Neto, Estrela; Ribas, João; Ferreira, Sofia; Vasconcelos, Daniel M.; Sousa, Daniela M.; Summavielle, Teresa; Lamghari, Meriem

    2016-01-01

    Bone repair is a specialized type of wound repair controlled by complex multi-factorial events. The nervous system is recognized as one of the key regulators of bone mass, thereby suggesting a role for neuronal pathways in bone homeostasis. However, in the context of bone injury and repair, little is known on the interplay between the nervous system and bone. Here, we addressed the neuropeptide Y (NPY) neuronal arm during the initial stages of bone repair encompassing the inflammatory response and ossification phases in femoral-defect mouse model. Spatial and temporal analysis of transcriptional and protein levels of NPY and its receptors, Y1R and Y2R, reported to be involved in bone homeostasis, was performed in bone, dorsal root ganglia (DRG) and hypothalamus after femoral injury. The results showed that NPY system activity is increased in a time- and space-dependent manner during bone repair. Y1R expression was trigged in both bone and DRG throughout the inflammatory phase, while a Y2R response was restricted to the hypothalamus and at a later stage, during the ossification step. Our results provide new insights into the involvement of NPY neuronal pathways in bone repair. PMID:27802308

  5. Ubiquitin-specific peptidase 20 regulates Rad17 stability, checkpoint kinase 1 phosphorylation and DNA repair by homologous recombination.

    PubMed

    Shanmugam, Ilanchezhian; Abbas, Mohammad; Ayoub, Farhan; Mirabal, Susan; Bsaili, Manal; Caulder, Erin K; Weinstock, David M; Tomkinson, Alan E; Hromas, Robert; Shaheen, Monte

    2014-08-15

    Rad17 is a subunit of the Rad9-Hus1-Rad1 clamp loader complex, which is required for Chk1 activation after DNA damage. Rad17 has been shown to be regulated by the ubiquitin-proteasome system. We have identified a deubiquitylase, USP20 that is required for Rad17 protein stability in the steady-state and post DNA damage. We demonstrate that USP20 and Rad17 interact, and that this interaction is enhanced by UV exposure. We show that USP20 regulation of Rad17 is at the protein level in a proteasome-dependent manner. USP20 depletion results in poor activation of Chk1 protein by phosphorylation, consistent with Rad17 role in ATR-mediated phosphorylation of Chk1. Similar to other DNA repair proteins, USP20 is phosphorylated post DNA damage, and its depletion sensitizes cancer cells to damaging agents that form blocks ahead of the replication forks. Similar to Chk1 and Rad17, which enhance recombinational repair of collapsed replication forks, we demonstrate that USP20 depletion impairs DNA double strand break repair by homologous recombination. Together, our data establish a new function of USP20 in genome maintenance and DNA repair.

  6. Unloading of homologous recombination factors is required for restoring double-stranded DNA at damage repair loci.

    PubMed

    Vasianovich, Yulia; Altmannova, Veronika; Kotenko, Oleksii; Newton, Matthew D; Krejci, Lumir; Makovets, Svetlana

    2017-01-17

    Cells use homology-dependent DNA repair to mend chromosome breaks and restore broken replication forks, thereby ensuring genome stability and cell survival. DNA break repair via homology-based mechanisms involves nuclease-dependent DNA end resection, which generates long tracts of single-stranded DNA required for checkpoint activation and loading of homologous recombination proteins Rad52/51/55/57. While recruitment of the homologous recombination machinery is well characterized, it is not known how its presence at repair loci is coordinated with downstream re-synthesis of resected DNA We show that Rad51 inhibits recruitment of proliferating cell nuclear antigen (PCNA), the platform for assembly of the DNA replication machinery, and that unloading of Rad51 by Srs2 helicase is required for efficient PCNA loading and restoration of resected DNA As a result, srs2Δ mutants are deficient in DNA repair correlating with extensive DNA processing, but this defect in srs2Δ mutants can be suppressed by inactivation of the resection nuclease Exo1. We propose a model in which during re-synthesis of resected DNA, the replication machinery must catch up with the preceding processing nucleases, in order to close the single-stranded gap and terminate further resection. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  7. RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria.

    PubMed

    Gupta, Richa; Shuman, Stewart; Glickman, Michael S

    2015-10-01

    Mycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation of adnAB or recO individually causes partial impairment of HR, but loss of adnAB and recO in combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNA in vitro, also participates in the SSA pathway. The functions of RecF and RecR, which, in other model bacteria, function in concert with RecO as mediators of RecA loading, have not been examined in mycobacteria. Here, we present a genetic analysis of recF and recR in mycobacterial recombination. We find that RecF, like RecO, participates in the AdnAB-independent arm of the HR pathway and in SSA. In contrast, RecR is required for all HR in mycobacteria and for SSA. The essentiality of RecR as an agent of HR is yet another distinctive feature of mycobacterial DNA repair.IMPORTANCE This study clarifies the molecular requirements for homologous recombination in mycobacteria. Specifically, we demonstrate that RecF and RecR play important roles in both the RecA-dependent homologous recombination and RecA-independent single-strand annealing pathways. Coupled with our previous findings (R. Gupta, M. Ryzhikov, O. Koroleva, M. Unciuleac, S. Shuman, S. Korolev, and M. S. Glickman, Nucleic Acids Res 41:2284-2295, 2013, http://dx.doi.org/10.1093/nar/gks1298), these results revise our view of mycobacterial recombination and place the RecFOR system in a central position in homology-dependent DNA repair.

  8. DNA ligase IV and artemis act cooperatively to suppress homologous recombination in human cells: implications for DNA double-strand break repair.

    PubMed

    Kurosawa, Aya; Saito, Shinta; So, Sairei; Hashimoto, Mitsumasa; Iwabuchi, Kuniyoshi; Watabe, Haruka; Adachi, Noritaka

    2013-01-01

    Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.

  9. DNA Ligase IV and Artemis Act Cooperatively to Suppress Homologous Recombination in Human Cells: Implications for DNA Double-Strand Break Repair

    PubMed Central

    Kurosawa, Aya; Saito, Shinta; So, Sairei; Hashimoto, Mitsumasa; Iwabuchi, Kuniyoshi; Watabe, Haruka; Adachi, Noritaka

    2013-01-01

    Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR. PMID:23967291

  10. Evaluation of DNA Repair Function as a Predictor of Response in a Clinical Trial of PARP Inhibitor Monotherapy for Recurrent Ovarian Carcinoma

    DTIC Science & Technology

    2014-10-01

    recombination (HR) pathway of DNA repair. Previous work had shown that cancer cells with deleterious FA/HR pathway mutations are hypersensitive to poly...homologous recombination , nonhomologous end-joining (NHEJ), immunohistochemistry, poly(ADP-ribose) polymerase, Ku70, Ku80, PARP1, 53BP1, DNA -PK, Artemis...regulates five different DNA repair pathways (1, 2). Building on preclinical observations that defects in homologous recombination (HR) repair, which

  11. Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination.

    PubMed

    Colloms, Sean D; Merrick, Christine A; Olorunniji, Femi J; Stark, W Marshall; Smith, Margaret C M; Osbourn, Anne; Keasling, Jay D; Rosser, Susan J

    2014-02-01

    Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.

  12. Articular cartilage repair with recombinant human type II collagen/polylactide scaffold in a preliminary porcine study.

    PubMed

    Muhonen, Virpi; Salonius, Eve; Haaparanta, Anne-Marie; Järvinen, Elina; Paatela, Teemu; Meller, Anna; Hannula, Markus; Björkman, Mimmi; Pyhältö, Tuomo; Ellä, Ville; Vasara, Anna; Töyräs, Juha; Kellomäki, Minna; Kiviranta, Ilkka

    2016-05-01

    The purpose of this study was to investigate the potential of a novel recombinant human type II collagen/polylactide scaffold (rhCo-PLA) in the repair of full-thickness cartilage lesions with autologous chondrocyte implantation technique (ACI). The forming repair tissue was compared to spontaneous healing (spontaneous) and repair with a commercial porcine type I/III collagen membrane (pCo). Domestic pigs (4-month-old, n = 20) were randomized into three study groups and a circular full-thickness chondral lesion with a diameter of 8 mm was created in the right medial femoral condyle. After 3 weeks, the chondral lesions were repaired with either rhCo-PLA or pCo together with autologous chondrocytes, or the lesion was only debrided and left untreated for spontaneous repair. The repair tissue was evaluated 4 months after the second operation. Hyaline cartilage formed most frequently in the rhCo-PLA treatment group. Biomechanically, there was a trend that both treatment groups resulted in better repair tissue than spontaneous healing. Adverse subchondral bone reactions developed less frequently in the spontaneous group (40%) and the rhCo-PLA treated group (50%) than in the pCo control group (100%). However, no statistically significant differences were found between the groups. The novel rhCo-PLA biomaterial showed promising results in this proof-of-concept study, but further studies will be needed in order to determine its effectiveness in articular cartilage repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:745-753, 2016.

  13. How Trypanosoma cruzi deals with oxidative stress: Antioxidant defence and DNA repair pathways.

    PubMed

    Machado-Silva, Alice; Cerqueira, Paula Gonçalves; Grazielle-Silva, Viviane; Gadelha, Fernanda Ramos; Peloso, Eduardo de Figueiredo; Teixeira, Santuza Maria Ribeiro; Machado, Carlos Renato

    2016-01-01

    Trypanosoma cruzi, the causative agent of Chagas disease, is an obligatory intracellular parasite with a digenetic life cycle. Due to the variety of host environments, it faces several sources of oxidative stress. In addition to reactive oxygen species (ROS) produced by its own metabolism, T. cruzi must deal with high ROS levels generated as part of the host's immune responses. Hence, the conclusion that T. cruzi has limited ability to deal with ROS (based on the lack of a few enzymes involved with oxidative stress responses) seems somewhat paradoxical. Actually, to withstand such variable sources of oxidative stress, T. cruzi has developed complex defence mechanisms. This includes ROS detoxification pathways that are distinct from the ones in the mammalian host, DNA repair pathways and specialized polymerases, which not only protect its genome from the resulting oxidative damage but also contribute to the generation of genetic diversity within the parasite population. Recent studies on T. cruzi's DNA repair pathways as mismatch repair (MMR) and GO system suggested that, besides a role associated with DNA repair, some proteins of these pathways may also be involved in signalling oxidative damage. Recent data also suggested that an oxidative environment might be beneficial for parasite survival within the host cell as it contributes to iron mobilization from the host's intracellular storages. Besides contributing to the understanding of basic aspects of T. cruzi biology, these studies are highly relevant since oxidative stress pathways are part of the poorly understood mechanisms behind the mode of action of drugs currently used against this parasite. By unveiling new peculiar aspects of T. cruzi biology, emerging data on DNA repair pathways and other antioxidant defences from this parasite have revealed potential new targets for a much needed boost in drug development efforts towards a better treatment for Chagas disease. Copyright © 2015. Published by Elsevier B.V.

  14. Genetic Analysis of δheld and δuvrd Mutations in Combination with Other Genes in the Recf Recombination Pathway in Escherichia Coli: Suppression of a Ruvb Mutation by a Uvrd Deletion

    PubMed Central

    Mendonca, V. M.; Matson, S. W.

    1995-01-01

    Helicase II (uvrD gene product) and helicase IV (helD gene product) have been shown previously to be involved in the RecF pathway of recombination. To better understand the role of these two proteins in homologous recombination in the RecF pathway [recBCsbcB(C) background], we investigated the interactions between helD, uvrD and the following RecF pathway genes: recF, recO, recN and ruvAB. We observed synergistic interactions between uvrD and the recF, recN, recO and recG genes in both conjugational recombination and the repair of methylmethane sulfonate (MMS)-induced DNA damage. No synergistic interactions were detected between helD and the recF, recO and recN genes when conjugational recombination was analyzed. We did, however, detect synergistic interactions between helD and recF/recO in recombinational repair. Suprisingly, the uvrD deletion completely suppressed the phenotype of a ruvB mutation in a recBCsbcB(C) background. Both conjugational recombination efficiency and MMS-damaged DNA repair proficiency returned to wild-type levels in the δuvrDruvB9 double mutant. Suppression of the effects of the ruvB mutation by a uvrD deletion was dependent on the recG and recN genes and not dependent on the recF/O/R genes. These data are discussed in the context of two ``RecF'' homologous recombination pathways operating in a recBCsbcB(C) strain background. PMID:8647383

  15. Mismatch repair proteins MSH2, MLH1, and EXO1 are important for class-switch recombination events occurring in B cells that lack nonhomologous end joining.

    PubMed

    Eccleston, Jennifer; Yan, Catherine; Yuan, Karen; Alt, Frederick W; Selsing, Erik

    2011-02-15

    In the absence of core nonhomologous end-joining (NHEJ) factors, Ab gene class-switch recombination (CSR) uses an alternative end-joining (A-EJ) pathway to recombine switch (S) region DNA breaks. Previous reports showing decreased S-junction microhomologies in MSH2-deficient mice and an exonuclease 1 (EXO1) role in yeast microhomology-mediated end joining suggest that mismatch repair (MMR) proteins might influence A-EJ-mediated CSR. We have directly investigated whether MMR proteins collectively or differentially influence the A-EJ mechanism of CSR by analyzing CSR in mice deficient in both XRCC4 and individual MMR proteins. We find CSR is reduced and that Igh locus chromosome breaks are reduced in the MMR/XRCC4 double-deficient B cells compared with B cells deficient in XRCC4 alone, suggesting MMR proteins function upstream of double-strand break formation to influence CSR efficiency in these cells. Our results show that MLH1, EXO1, and MSH2 are all important for efficient A-EJ-mediated CSR, and we propose that MMR proteins convert DNA nicks and point mutations into dsDNA breaks for both C-NHEJ and A-EJ pathways of CSR. We also find Mlh1-XRCC4(-) B cells have an increased frequency of direct S junctions, suggesting that MLH1 proteins may have additional functions that influence A-EJ-mediated CSR.

  16. Targeting the DNA repair pathway in Ewing sarcoma.

    PubMed

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

    2014-11-06

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

  17. Transplantation of genetically engineered cardiac fibroblasts producing recombinant human erythropoietin to repair the infarcted myocardium

    PubMed Central

    Ruvinov, Emil; Sharabani-Yosef, Orna; Nagler, Arnon; Einbinder, Tom; Feinberg, Micha S; Holbova, Radka; Douvdevani, Amos; Leor, Jonathan

    2008-01-01

    Background Erythropoietin possesses cellular protection properties. The aim of the present study was to test the hypothesis that in situ expression of recombinant human erythropoietin (rhEPO) would improve tissue repair in rat after myocardial infarction (MI). Methods and results RhEPO-producing cardiac fibroblasts were generated ex vivo by transduction with retroviral vector. The anti-apoptotic effect of rhEPO-producing fibroblasts was evaluated by co-culture with rat neonatal cardiomyocytes exposed to H2O2-induced oxidative stress. Annexin V/PI assay and DAPI staining showed that compared with control, rhEPO forced expression markedly attenuated apoptosis and improved survival of cultured cardiomyocytes. To test the effect of rhEPO on the infarcted myocardium, Sprague-Dawley rats were subjected to permanent coronary artery occlusion, and rhEPO-producing fibroblasts, non-transduced fibroblasts, or saline, were injected into the scar tissue seven days after infarction. One month later, immunostaining identified rhEPO expression in the implanted engineered cells but not in controls. Compared with non-transduced fibroblasts or saline injection, implanted rhEPO-producing fibroblasts promoted vascularization in the scar, and prevented cell apoptosis. By two-dimensional echocardiography and postmortem morphometry, transplanted EPO-engineered fibroblasts did not prevent left ventricular (LV) dysfunction and adverse LV remodeling 5 and 9 weeks after MI. Conclusion In situ expression of rhEPO enhances vascularization and reduces cell apoptosis in the infarcted myocardium. However, local EPO therapy is insufficient for functional improvement after MI in rat. PMID:19014419

  18. Targeting the DNA Repair Pathway in Ewing Sarcoma

    PubMed Central

    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; Thiagarajan, Suresh; Shirinifard, Abbas; Sablauer, Andras; Shelat, Anang A.; Dyer, Michael A.

    2015-01-01

    Ewing sarcoma (EWS) is a tumor of the bone and soft-tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using 3 different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice. PMID:25437539

  19. Involvement of DNA-dependent RNA polymerase in a recA-independent pathway of genetic recombination in Escheria coli.

    PubMed

    Ikeda, H; Kobayashi, I

    1977-09-01

    Recombinant DNA molecule of phage lambda formed in Escherichia coli in the presence of chloramphenicol and/or rifampin can be assayed by their biological activity. recA- cells were found to be capable of forming recombinant lambda phage DNA in the presence of chloramphenicol. The relatively high recA-independent recombination observed in this system contrasts with the relatively low recA-independent recombination when recombinant phage particles rather than recombinant DNA are titrated. Formation of the recombinant DNA was suppressed by the the addition of rifampin. The introduction of the rif-r mutation into host bacteria made their recombination activity rifampin-resistant. These results show that DNA-dependent RNA polymerase (EC 2.7.7.6) is involved in this recA-independent pathway of recombination, which is named the "Rpo pathway." This is distinct from Red, Int, RecBC, RecE, or Der pathways of recombination. Crossover was much more frequent in the N-PL-cI and cI-PR-O regions than in the A-D and O-S regions. The crossover seems to occur in the regions that are transcribed actively. Some local change of DNA structure caused by transcription might be required for the Rpo pathway of recombination.

  20. Involvement of DNA-dependent RNA polymerase in a recA-independent pathway of genetic recombination in Escheria coli.

    PubMed Central

    Ikeda, H; Kobayashi, I

    1977-01-01

    Recombinant DNA molecule of phage lambda formed in Escherichia coli in the presence of chloramphenicol and/or rifampin can be assayed by their biological activity. recA- cells were found to be capable of forming recombinant lambda phage DNA in the presence of chloramphenicol. The relatively high recA-independent recombination observed in this system contrasts with the relatively low recA-independent recombination when recombinant phage particles rather than recombinant DNA are titrated. Formation of the recombinant DNA was suppressed by the the addition of rifampin. The introduction of the rif-r mutation into host bacteria made their recombination activity rifampin-resistant. These results show that DNA-dependent RNA polymerase (EC 2.7.7.6) is involved in this recA-independent pathway of recombination, which is named the "Rpo pathway." This is distinct from Red, Int, RecBC, RecE, or Der pathways of recombination. Crossover was much more frequent in the N-PL-cI and cI-PR-O regions than in the A-D and O-S regions. The crossover seems to occur in the regions that are transcribed actively. Some local change of DNA structure caused by transcription might be required for the Rpo pathway of recombination. PMID:333450

  1. Arabidopsis ARP endonuclease functions in a branched base excision DNA repair pathway completed by LIG1.

    PubMed

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

    2011-11-01

    Base excision repair (BER) is an essential cellular defence mechanism against DNA damage, but it is poorly understood in plants. We used an assay that monitors repair of damaged bases and abasic (apurinic/apyrimidinic, AP) sites in Arabidopsis to characterize post-excision events during plant BER. We found that Apurinic endonuclease-redox protein (ARP) is the major AP endonuclease activity in Arabidopsis cell extracts, and is required for AP incision during uracil BER in vitro. Mutant plants that are deficient in ARP grow normally but are hypersensitive to 5-fluorouracil, a compound that favours mis-incorporation of uracil into DNA. We also found that, after AP incision, the choice between single-nucleotide or long-patch DNA synthesis (SN- or LP-BER) is influenced by the 5' end of the repair gap. When the 5' end is blocked and not amenable to β-elimination, the SN sub-pathway is abrogated, and repair is accomplished through LP-BER only. Finally, we provide evidence that Arabidopsis DNA ligase I (LIG1) is required for both SN- and LP-BER. lig1 RNAi-silenced lines show very reduced uracil BER, and anti-LIG1 antibody abolishes repair in wild-type cell extracts. In contrast, knockout lig4(-/-) mutants exhibit normal BER and nick ligation levels. Our results suggest that a branched BER pathway completed by a member of the DNA ligase I family may be an ancient feature in eukaryotic species.

  2. Loss of CtIP disturbs homologous recombination repair and sensitizes breast cancer cells to PARP inhibitors.

    PubMed

    Wang, Junhui; Ding, Qianshan; Fujimori, Hiroaki; Motegi, Akira; Miki, Yoshio; Masutani, Mitsuko

    2016-02-16

    Breast cancer is one of the leading causes of death worldwide, and therefore, new and improved approaches for the treatment of breast cancer are desperately needed. CtIP (RBBP8) is a multifunctional protein that is involved in various cellular functions, including transcription, DNA replication, DNA repair and the G1 and G2 cell cycle checkpoints. CtIP plays an important role in homologous recombination repair by interacting with tumor suppressor protein BRCA1. Here, we analyzed the expression profile of CtIP by data mining using published microarray data sets. We found that CtIP expression is frequently decreased in breast cancer patients, and the patient group with low-expressing CtIP mRNA is associated with a significantly lower survival rate. The knockdown of CtIP in breast cancer MCF7 cells reduced Rad51 foci numbers and enhanced f H2AX foci formation after f-irradiation, suggesting that deficiency of CtIP decreases homologous recombination repair and delays DNA double strand break repair. To explore the effect of CtIP on PARP inhibitor therapy for breast cancer, CtIP-depleted MCF7 cells were treated with PARP inhibitor olaparib (AZD2281) or veliparib (ABT-888). As in BRCA mutated cells, PARP inhibitors showed cytotoxicity to CtIP-depleted cells by preventing cells from repairing DNA damage, leading to decreased cell viability. Further, a xenograft tumor model in mice with MCF7 cells demonstrated significantly increased sensitivity towards PARP inhibition under CtIP deficiency. In summary, this study shows that low level of CtIP expression is associated with poor prognosis in breast cancer, and provides a rationale for establishing CtIP expression as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients.

  3. A Delicate Balance Between Repair and Replication Factors Regulates Recombination Between Divergent DNA Sequences in Saccharomyces cerevisiae

    PubMed Central

    Chakraborty, Ujani; George, Carolyn M.; Lyndaker, Amy M.; Alani, Eric

    2016-01-01

    Single-strand annealing (SSA) is an important homologous recombination mechanism that repairs DNA double strand breaks (DSBs) occurring between closely spaced repeat sequences. During SSA, the DSB is acted upon by exonucleases to reveal complementary sequences that anneal and are then repaired through tail clipping, DNA synthesis, and ligation steps. In baker’s yeast, the Msh DNA mismatch recognition complex and the Sgs1 helicase act to suppress SSA between divergent sequences by binding to mismatches present in heteroduplex DNA intermediates and triggering a DNA unwinding mechanism known as heteroduplex rejection. Using baker’s yeast as a model, we have identified new factors and regulatory steps in heteroduplex rejection during SSA. First we showed that Top3-Rmi1, a topoisomerase complex that interacts with Sgs1, is required for heteroduplex rejection. Second, we found that the replication processivity clamp proliferating cell nuclear antigen (PCNA) is dispensable for heteroduplex rejection, but is important for repairing mismatches formed during SSA. Third, we showed that modest overexpression of Msh6 results in a significant increase in heteroduplex rejection; this increase is due to a compromise in Msh2-Msh3 function required for the clipping of 3′ tails. Thus 3′ tail clipping during SSA is a critical regulatory step in the repair vs. rejection decision; rejection is favored before the 3′ tails are clipped. Unexpectedly, Msh6 overexpression, through interactions with PCNA, disrupted heteroduplex rejection between divergent sequences in another recombination substrate. These observations illustrate the delicate balance that exists between repair and replication factors to optimize genome stability. PMID:26680658

  4. The anaphase-promoting complex/cyclosome controls repair and recombination by ubiquitylating Rhp54 in fission yeast.

    PubMed

    Trickey, Michelle; Grimaldi, Margaret; Yamano, Hiroyuki

    2008-06-01

    Homologous recombination (HR) is important for maintaining genome integrity and for the process of meiotic chromosome segregation and the generation of variation. HR is regulated throughout the cell cycle, being prevalent in the S and G2 phases and suppressed in the G1 phase. Here we show that the anaphase-promoting complex/cyclosome (APC/C) regulates homologous recombination in the fission yeast Schizosaccharomyces pombe by ubiquitylating Rhp54 (an ortholog of Rad54). We show that Rhp54 is a novel APC/C substrate that is destroyed in G1 phase in a KEN-box- and Ste9/Fizzy-related manner. The biological consequences of failing to temporally regulate HR via Rhp54 degradation are seen in haploid cells only in the absence of antirecombinase Srs2 function and are more extensive in diploid cells, which become sensitive to a range of DNA-damaging agents, including hydroxyurea, methyl methanesulfonate, bleomycin, and UV. During meiosis, expression of nondegradable Rhp54 inhibits interhomolog recombination and stimulates sister chromatid recombination. We thus propose that it is critical to control levels of Rhp54 in G1 to suppress HR repair of double-strand breaks and during meiosis to coordinate interhomolog recombination.

  5. Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea

    PubMed Central

    Heider, Margaret R.; Burkhart, Brett W.; Santangelo, Thomas J.; Gardner, Andrew F.

    2017-01-01

    Incorporation of ribonucleotides during DNA replication has severe consequences for genome stability. Although eukaryotes possess a number of redundancies for initiating and completing repair of misincorporated ribonucleotides, archaea such as Thermococcus rely only upon RNaseH2 to initiate the pathway. Because Thermococcus DNA polymerases incorporate as many as 1,000 ribonucleotides per genome, RNaseH2 must be efficient at recognizing and nicking at embedded ribonucleotides to ensure genome integrity. Here, we show that ribonucleotides are incorporated by the hyperthermophilic archaeon Thermococcus kodakarensis both in vitro and in vivo and a robust ribonucleotide excision repair pathway is critical to keeping incorporation levels low in wild-type cells. Using pre-steady-state and steady-state kinetics experiments, we also show that archaeal RNaseH2 rapidly cleaves at embedded ribonucleotides (200-450 s−1), but exhibits an ∼1,000-fold slower turnover rate (0.06–0.17 s−1), suggesting a potential role for RNaseH2 in protecting or marking nicked sites for further processing. We found that following RNaseH2 cleavage, the combined activities of polymerase B (PolB), flap endonuclease (Fen1), and DNA ligase are required to complete ribonucleotide processing. PolB formed a ribonucleotide-containing flap by strand displacement synthesis that was cleaved by Fen1, and DNA ligase sealed the nick for complete repair. Our study reveals conservation of the overall mechanism of ribonucleotide excision repair across domains of life. The lack of redundancies in ribonucleotide repair in archaea perhaps suggests a more ancestral form of ribonucleotide excision repair compared with the eukaryotic pathway. PMID:28373277

  6. Network-based characterization and prediction of human DNA repair genes and pathways

    PubMed Central

    Li, Yan-Hui; Zhang, Gai-Gai

    2017-01-01

    Network biology is a useful strategy to understand cell’s functional organization. In this study, for the first time, we successfully introduced network approaches to study properties of human DNA repair genes. Compared with non-DNA repair genes, we found distinguishing features for DNA repair genes: (i) they tend to have higher degrees; (ii) they tend to be located at global network center; (iii) they tend to interact directly with each other. Based on these features, we developed the first algorithm to predict new DNA repair genes. We tested several machine-learning models and found that support vector machine with kernel function of radial basis function (RBF) achieve the best performance, with precision = 0.74 and area under curve (AUC) = 0.96. In the end, we applied the algorithm to predict new DNA repair genes and got 32 new candidates. Literature supporting four of the predictions was found. We believe the network approaches introduced here might open a new avenue to understand DNA repair genes and pathways. The suggested algorithm and the predicted genes might be helpful for scientists in the field. PMID:28368026

  7. A GRHL3-regulated repair pathway suppresses immune-mediated epidermal hyperplasia

    PubMed Central

    Gordon, William M.; Zeller, Michael D.; Klein, Rachel H.; Swindell, William R.; Ho, Hsiang; Espetia, Francisco; Gudjonsson, Johann E.; Baldi, Pierre F.; Andersen, Bogi

    2014-01-01

    Dermal infiltration of T cells is an important step in the onset and progression of immune-mediated skin diseases such as psoriasis; however, it is not known whether epidermal factors play a primary role in the development of these diseases. Here, we determined that the prodifferentiation transcription factor grainyhead-like 3 (GRHL3), which is essential during epidermal development, is dispensable for adult skin homeostasis, but required for barrier repair after adult epidermal injury. Consistent with activation of a GRHL3-regulated repair pathway in psoriasis, we found that GRHL3 is upregulated in lesional skin and binds known epidermal differentiation gene targets. Using an imiquimod-induced model of immune-mediated epidermal hyperplasia, we found that mice lacking GRHL3 have an exacerbated epidermal damage response, greater sensitivity to disease induction, delayed resolution of epidermal lesions, and resistance to anti–IL-22 therapy compared with WT animals. ChIP-Seq and gene expression profiling of murine skin revealed that while GRHL3 regulates differentiation pathways both during development and during repair from immune-mediated damage, it targets distinct sets of genes in the 2 processes. In particular, GRHL3 suppressed a number of alarmin and other proinflammatory genes after immune injury. This study identifies a GRHL3-regulated epidermal barrier repair pathway that suppresses disease initiation and helps resolve existing lesions in immune-mediated epidermal hyperplasia. PMID:25347468

  8. The Fanconi anemia DNA damage repair pathway in the spotlight for germline predisposition to colorectal cancer

    PubMed Central

    Esteban-Jurado, Clara; Franch-Expósito, Sebastià; Muñoz, Jenifer; Ocaña, Teresa; Carballal, Sabela; López-Cerón, Maria; Cuatrecasas, Miriam; Vila-Casadesús, Maria; Lozano, Juan José; Serra, Enric; Beltran, Sergi; Brea-Fernández, Alejandro; Ruiz-Ponte, Clara; Castells, Antoni; Bujanda, Luis; Garre, Pilar; Caldés, Trinidad; Cubiella, Joaquín; Balaguer, Francesc; Castellví-Bel, Sergi

    2016-01-01

    Colorectal cancer (CRC) is one of the most common neoplasms in the world. Fanconi anemia (FA) is a very rare genetic disease causing bone marrow failure, congenital growth abnormalities and cancer predisposition. The comprehensive FA DNA damage repair pathway requires the collaboration of 53 proteins and it is necessary to restore genome integrity by efficiently repairing damaged DNA. A link between FA genes in breast and ovarian cancer germline predisposition has been previously suggested. We selected 74 CRC patients from 40 unrelated Spanish families with strong CRC aggregation compatible with an autosomal dominant pattern of inheritance and without mutations in known hereditary CRC genes and performed germline DNA whole-exome sequencing with the aim of finding new candidate germline predisposition variants. After sequencing and data analysis, variant prioritization selected only those very rare alterations, producing a putative loss of function and located in genes with a role compatible with cancer. We detected an enrichment for variants in FA DNA damage repair pathway genes in our familial CRC cohort as 6 families carried heterozygous, rare, potentially pathogenic variants located in BRCA2/FANCD1, BRIP1/FANCJ, FANCC, FANCE and REV3L/POLZ. In conclusion, the FA DNA damage repair pathway may play an important role in the inherited predisposition to CRC. PMID:27165003

  9. A GRHL3-regulated repair pathway suppresses immune-mediated epidermal hyperplasia.

    PubMed

    Gordon, William M; Zeller, Michael D; Klein, Rachel H; Swindell, William R; Ho, Hsiang; Espetia, Francisco; Gudjonsson, Johann E; Baldi, Pierre F; Andersen, Bogi

    2014-12-01

    Dermal infiltration of T cells is an important step in the onset and progression of immune-mediated skin diseases such as psoriasis; however, it is not known whether epidermal factors play a primary role in the development of these diseases. Here, we determined that the prodifferentiation transcription factor grainyhead-like 3 (GRHL3), which is essential during epidermal development, is dispensable for adult skin homeostasis, but required for barrier repair after adult epidermal injury. Consistent with activation of a GRHL3-regulated repair pathway in psoriasis, we found that GRHL3 is upregulated in lesional skin and binds known epidermal differentiation gene targets. Using an imiquimod-induced model of immune-mediated epidermal hyperplasia, we found that mice lacking GRHL3 have an exacerbated epidermal damage response, greater sensitivity to disease induction, delayed resolution of epidermal lesions, and resistance to anti-IL-22 therapy compared with WT animals. ChIP-Seq and gene expression profiling of murine skin revealed that while GRHL3 regulates differentiation pathways both during development and during repair from immune-mediated damage, it targets distinct sets of genes in the 2 processes. In particular, GRHL3 suppressed a number of alarmin and other proinflammatory genes after immune injury. This study identifies a GRHL3-regulated epidermal barrier repair pathway that suppresses disease initiation and helps resolve existing lesions in immune-mediated epidermal hyperplasia.

  10. Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

    PubMed Central

    Chappell, William H.; Gautam, Dipendra; Ok, Suzan T.; Johnson, Bryan A.; Anacker, Daniel C.

    2015-01-01

    ABSTRACT High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. IMPORTANCE Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for

  11. Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31.

    PubMed

    Chappell, William H; Gautam, Dipendra; Ok, Suzan T; Johnson, Bryan A; Anacker, Daniel C; Moody, Cary A

    2015-12-23

    High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral

  12. Ku-dependent non-homologous end-joining as the major pathway contributes to sublethal damage repair in mammalian cells.

    PubMed

    Liu, Min; Lee, Solah; Liu, Bailong; Wang, Hongyan; Dong, Lihua; Wang, Ya

    2015-01-01

    Sublethal damage repair (SLDR) is a type of repair that occurs in split-dose irradiated cells, which was discovered more than 50 years ago. However, due to conflicting reported data, it remains unclear which DNA double-strand break (DSB) repair pathway, non-homologous end-joining (NHEJ) repair, homologous recombination repair (HRR) or both, contributes to SLDR, particularly in human cells. We were interested in clarifying this question. Mammalian cell lines, including human, mouse and Chinese hamster ovary (CHO) cell lines, wild type, deficient in NHEJ or HRR were irradiated with either single dose or two split doses at 2- or 4-h intervals. The clonogenic assay was used to evaluate these cell radiosensitivities. All wild-type or HRR-deficient cells (including human, mouse and CHO cells) showed a higher survival rate after exposure to split-dose versus single-dose radiation; however, all classical NHEJ-deficient cells (including human, mouse and hamster cells) did not show any apparent sensitivity changes between single-dose and split-dose irradiation. Classical NHEJ mainly contributes to SLDR in mammalian cells (including human cells). These results have the potential to improve radiotherapy.

  13. Evidence for an Inducible Repair-Recombination System in the Female Germ Line of Drosophila Melanogaster. III. Correlation between Reactivity Levels, Crossover Frequency and Repair Efficiency

    PubMed Central

    Laurencon, A.; Gay, F.; Ducau, J.; Bregliano, J. C.

    1997-01-01

    We previously reported evidence that the so-called reactivity level, a peculiar cellular state of oocytes that regulates the frequency of transposition of I factor, a LINE element-like retrotransposon, might be one manifestation of a DNA repair system. In this article, we report data showing that the reactivity level is correlated with the frequency of crossing over, at least on the X chromosome and on the pericentromeric region of the third chromosome. Moreover, a check for X-chromosome losses and recessive lethals produced after gamma irradiation in flies with different reactivity levels, but common genetic backgrounds, brings more precise evidence for the relationship between reactivity levels and DNA repair. Those results support the existence of a repair-recombination system whose efficiency is modulated by endogenous and environmental factors. The implications of this biological system in connecting genomic variability and environment may shed new lights on adaptative mechanisms. We propose to call it VAMOS for variability modulation system. PMID:9258678

  14. Calcium influx-mediated translocation of m-calpain induces Ku80 cleavage and enhances the Ku80-related DNA repair pathway

    PubMed Central

    Baek, Kyung Hye; Yu, Han Vit; Kim, Eosu; Na, Younghwa; Kwon, Youngjoo

    2016-01-01

    Proteomic analysis of ionomycin-treated and untreated mammary epithelial MCF10A cells elucidated differences in Ku80 cleavage. Ku80, a subunit of the Ku protein complex, is an initiator of the non-homologous, end-joining (NHEJ), double-strand breaks (DSBs) repair pathway. The nuclear Ku80 was cleaved in a calcium concentration-dependent manner by m-calpain but not by m-calpain. The cleavage of nuclear Ku80 at its α/β domain was validated by Western blotting analysis using flag-tagged expression vectors of truncated versions of Ku80 and a flag antibody and was confirmed in m-calpain knock-down cells and in vitro cell-free evaluation with recombinant proteins of calpains, Ku70, and Ku80. In addition, the cleaved Ku80 still formed a Ku heterodimer and promoted DNA DSB repair activity. Taken together, these findings indicate that translocated m-calpain enhances the NHEJ pathway through the cleavage of Ku80. Based on the present study, m-calpain in DNA repair pathways might be a novel anticancer drug target, or its mechanism might be a possible route for resistance acquisition of DNA damage-inducing chemotherapeutics. PMID:27121057

  15. RNF20-SNF2H Pathway of Chromatin Relaxation in DNA Double-Strand Break Repair.

    PubMed

    Kato, Akihiro; Komatsu, Kenshi

    2015-07-14

    Rapid progress in the study on the association of histone modifications with chromatin remodeling factors has broadened our understanding of chromatin dynamics in DNA transactions. In DNA double-strand break (DSB) repair, the well-known mark of histones is the phosphorylation of the H2A variant, H2AX, which has been used as a surrogate marker of DSBs. The ubiquitylation of histone H2B by RNF20 E3 ligase was recently found to be a DNA damage-induced histone modification. This modification is required for DSB repair and regulated by a distinctive pathway from that of histone H2AX phosphorylation. Moreover, the connection between H2B ubiquitylation and the chromatin remodeling activity of SNF2H has been elucidated. In this review, we summarize the current knowledge of RNF20-mediated processes and the molecular link to H2AX-mediated processes during DSB repair.

  16. Role of the insulin-like growth factor I/insulin receptor substrate 1 axis in Rad51 trafficking and DNA repair by homologous recombination.

    PubMed

    Trojanek, Joanna; Ho, Thu; Del Valle, Luis; Nowicki, Michal; Wang, Jin Ying; Lassak, Adam; Peruzzi, Francesca; Khalili, Kamel; Skorski, Tomasz; Reiss, Krzysztof

    2003-11-01

    The receptor for insulin-like growth factor I (IGF-IR) controls normal and pathological growth of cells. DNA repair pathways represent an unexplored target through which the IGF-IR signaling system might support pathological growth leading to cellular transformation. However, this study demonstrates that IGF-I stimulation supports homologous recombination-directed DNA repair (HRR). This effect involves an interaction between Rad51 and the major IGF-IR signaling molecule, insulin receptor substrate 1 (IRS-1). The binding occurs within the cytoplasm, engages the N-terminal domain of IRS-1, and is attenuated by IGF-I-mediated IRS-1 tyrosine phosphorylation. In the absence of IGF-I stimulation, or if mutated IGF-IR fails to phosphorylate IRS-1, localization of Rad51 to the sites of damaged DNA is diminished. These results point to a direct role of IRS-1 in HRR and suggest a novel role for the IGF-IR/IRS-1 axis in supporting the stability of the genome.

  17. Either non-homologous ends joining or homologous recombination is required to repair double-strand breaks in the genome of macrophage-internalized Mycobacterium tuberculosis.

    PubMed

    Brzostek, Anna; Szulc, Izabela; Klink, Magdalena; Brzezinska, Marta; Sulowska, Zofia; Dziadek, Jaroslaw

    2014-01-01

    The intracellular pathogen Mycobacterium tuberculosis (Mtb) is constantly exposed to a multitude of hostile conditions and is confronted by a variety of potentially DNA-damaging assaults in vivo, primarily from host-generated antimicrobial toxic radicals. Exposure to reactive nitrogen species and/or reactive oxygen species causes different types of DNA damage, including oxidation, depurination, methylation and deamination, that can result in single- or double-strand breaks (DSBs). These breaks affect the integrity of the whole genome and, when left unrepaired, can lead to cell death. Here, we investigated the role of the DSB repair pathways, homologous recombination (HR) and non-homologous ends joining (NHEJ), in the survival of Mtb inside macrophages. To this end, we constructed Mtb strains defective for HR (ΔrecA), NHEJ [Δ(ku,ligD)], or both DSB repair systems [Δ(ku,ligD,recA)]. Experiments using these strains revealed that either HR or NHEJ is sufficient for the survival and propagation of tubercle bacilli inside macrophages. Inhibition of nitric oxide or superoxide anion production with L-NIL or apocynin, respectively, enabled the Δ(ku,ligD,recA) mutant strain lacking both systems to survive intracellularly. Complementation of the Δ(ku,ligD,recA) mutant with an intact recA or ku-ligD rescued the ability of Mtb to propagate inside macrophages.

  18. Inhibition of Both EGFR and IGF1R Sensitized Prostate Cancer Cells to Radiation by Synergistic Suppression of DNA Homologous Recombination Repair

    PubMed Central

    Ma, Jian Jun; Xu, Peng; Zhang, Wei; Li, Yu Mei; Fu, Qiang; Zhu, Guang Feng; Xue, Wei; Lei, Yong Hua; Gao, Jing Yu; Wang, Juan Ying; Shao, Chen; Yi, Cheng Gang; Wang, He

    2013-01-01

    Reduced sensitivity of prostate cancer (PC) cells to radiation therapy poses a significant challenge in the clinic. Activation of epidermal growth factor receptor (EGFR), type 1 insulin-like growth factor receptor (IGF1R), and crosstalk between these two signaling pathways have been implicated in the development of radiation resistance in PC. This study assessed the effects of targeting both receptors on the regulation of radio-sensitivity in PC cells. Specific inhibitors of EGFR and IGF1R, Erlotinib and AG1024, as well as siRNA targeting EGFR and IGF1R, were used to radio-sensitize PC cells. Our results showed that co-inhibiting both receptors significantly dampened cellular growth and DNA damage repair, and increased radio-sensitivity in PC cells. These effects were carried out through synergistic inhibition of homologous recombination-directed DNA repair (HRR), but not via inhibition of non-homologous end joining (NHEJ). Furthermore, the compromised HRR capacity was caused by reduced phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent interaction with Rad51. The synergistic effect of the EGFR and IGF1R inhibitors was also confirmed in nude mouse xenograft assay. This is the first study testing co-inhibiting EGFR and IGF1R signaling in the context of radio-sensitivity in PC and it may provide a promising adjuvant therapeutic approach to improve the outcome of PC patients to radiation treatment. PMID:23950876

  19. The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation.

    PubMed

    Holmes, Amie L; Joyce, Kellie; Xie, Hong; Falank, Carolyne; Hinz, John M; Wise, John Pierce

    2014-04-01

    Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation.

  20. Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system.

    PubMed Central

    Jessberger, R; Berg, P

    1991-01-01

    We have designed an in vitro system using mammalian nuclear extracts, or fractions derived from them, that can restore the sequences missing at double-strand breaks (gaps) or in deletions. The recombination substrates consist of (i) recipient DNA, pSV2neo with gaps or deletions ranging from 70 to 390 bp in the neo sequence, and (ii) donor DNAs with either complete homology to the recipient (pSV2neo) or plasmids whose homology with pSV2neo is limited to a 1.0- to 1.3-kbp neo segment spanning the gaps or deletions. Incubation of these substrates with various enzyme fractions results in repair of the recipient DNA's disrupted neo gene. The recombinational repair was monitored by transforming recA Escherichia coli to kanamycin resistance and by a new assay which measures the extent of DNA strand transfer from the donor substrate to the recipient DNA. Thus, either streptavidin- or antidigoxigenin-tagged beads are used to separate the biotinylated or digoxigeninylated recipient DNA, respectively, after incubation with the isotopically labeled donor DNA. In contrast to the transfection assay, the DNA strand transfer measurements are direct, quantitative, rapid, and easy, and they provide starting material for the characterization of the recombination products and intermediates. Accordingly, DNA bound to beads serves as a suitable template for the polymerase chain reaction. With appropriate pairs of oligonucleotide primers, we have confirmed that both gaps and deletions are fully repaired, that deletions can be transferred from the recipient DNA to the donor's intact neo sequence, and that cointegrant molecules containing donor and recipient DNA sequences are formed. Images PMID:1986239

  1. RAD50 Is Required for Efficient Initiation of Resection and Recombinational Repair at Random, γ-Induced Double-Strand Break Ends

    PubMed Central

    Westmoreland, Jim; Ma, Wenjian; Yan, Yan; Van Hulle, Kelly; Malkova, Anna; Resnick, Michael A.

    2009-01-01

    Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent. PMID:19763170

  2. Association study of genetic variation in DNA repair pathway genes and risk of basal cell carcinoma.

    PubMed

    Lin, Yuan; Chahal, Harvind S; Wu, Wenting; Cho, Hyunje G; Ransohoff, Katherine J; Song, Fengju; Tang, Jean Y; Sarin, Kavita Y; Han, Jiali

    2017-09-01

    DNA repair plays a critical role in protecting the genome from ultraviolet radiation and maintaining the genomic integrity of cells. Genetic variants in DNA repair-related genes can influence an individual's DNA repair capacity, which may be related to the risk of developing basal cell carcinoma (BCC). We comprehensively assessed the associations of 2,965 independent single-nucleotide polymorphisms (SNPs) across 165 DNA repair pathway genes with BCC risk in a genome-wide association meta-analysis totaling 17,187 BCC cases and 287,054 controls from two data sets. After multiple testing corrections, we identified three SNPs (rs2805831 upstream of XPA: OR = 0.93, P = 1.35 × 10(-6) ; rs659857 in exon of MUS81: OR = 1.06, P = 3.09 × 10(-6) and rs57343616 in 3' UTR of NABP2: OR = 1.11, P = 6.47 × 10(-6) ) as significantly associated with BCC risk in meta-analysis, and all of them were nominally significant in both data sets. Furthermore, rs659857 [T] was significantly associated with decreased expression of MUS81 mRNA in the expression quantitative trait locus (eQTL) analysis. Our findings suggest that the inherited common variation in three DNA repair genes-XPA, MUS81 and NABP2-may be involved in the development of BCC. To our knowledge, our study is the first report thoroughly examining the effects of SNPs across DNA repair pathway genes on BCC risk based on a genome-wide association meta-analysis. © 2017 UICC.

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

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

  5. The Fanconi anemia pathway promotes replication-dependent DNA interstrand cross-link repair.

    PubMed

    Knipscheer, Puck; Räschle, Markus; Smogorzewska, Agata; Enoiu, Milica; Ho, The Vinh; Schärer, Orlando D; Elledge, Stephen J; Walter, Johannes C

    2009-12-18

    Fanconi anemia is a human cancer predisposition syndrome caused by mutations in 13 Fanc genes. The disorder is characterized by genomic instability and cellular hypersensitivity to chemicals that generate DNA interstrand cross-links (ICLs). A central event in the activation of the Fanconi anemia pathway is the mono-ubiquitylation of the FANCI-FANCD2 complex, but how this complex confers ICL resistance remains enigmatic. Using a cell-free system, we showed that FANCI-FANCD2 is required for replication-coupled ICL repair in S phase. Removal of FANCD2 from extracts inhibits both nucleolytic incisions near the ICL and translesion DNA synthesis past the lesion. Reversal of these defects requires ubiquitylated FANCI-FANCD2. Our results show that multiple steps of the essential S-phase ICL repair mechanism fail when the Fanconi anemia pathway is compromised.

  6. Genetic polymorphisms in DNA repair and oxidative stress pathways associated with malignant melanoma susceptibility.

    PubMed

    Ibarrola-Villava, Maider; Peña-Chilet, Maria; Fernandez, Lara P; Aviles, Jose A; Mayor, Matias; Martin-Gonzalez, Manuel; Gomez-Fernandez, Cristina; Casado, Beatriz; Lazaro, Pablo; Lluch, Ana; Benitez, Javier; Lozoya, Rafael; Boldo, Enrique; Pizarro, Angel; Martinez-Cadenas, Conrado; Ribas, Gloria

    2011-11-01

    Base excision repair (BER) and nucleotide excision repair (NER) pathways eliminate a wide variety of DNA damage, including UV photoproducts. The ability of each individual to repair DNA damage following different causes might explain at least in part the variability in cancer susceptibility. Moreover, inflammatory response to UV exposure may further contribute to skin carcinogenesis by oxidative stress mechanisms. Single nucleotide polymorphisms in genes encoding various DNA-repair enzymes and oxidative stress factors may be candidate low-penetrance variants with a role in susceptibility to different cancers, particularly in those with aetiologies linked to environmental exposure, such as malignant melanoma (MM). In this case-control study, 684 Spanish sporadic MM patients and 406 cancer-free control subjects were included and the role of 46 polymorphisms belonging to 16 BER and NER genes as well as 11 genes involved in oxidative stress processes were investigated. One polymorphism was identified to be individually associated with MM in the Spanish population. The variant was found in the NOS1 oxidative stress gene (rs2682826; p-value=0.01). These results suggest a putative role of oxidative stress processes in the genetic predisposition to melanoma. To the authors' knowledge, this is the largest DNA repair-related SNP study in melanoma risk conducted in the Spanish population up to now. Furthermore, it also represents a comprehensive genetic study of several oxidative stress polymorphisms tested in relation to MM susceptibility. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Exposure of Human Lung Cells to Tobacco Smoke Condensate Inhibits the Nucleotide Excision Repair Pathway

    PubMed Central

    Holcomb, Nathaniel; Goswami, Mamta; Han, Sung Gu; Clark, Samuel; Orren, David K.; Gairola, C. Gary; Mellon, Isabel

    2016-01-01

    Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER) pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC), a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6–4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6–4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke. PMID:27391141

  8. Recombination Pathways in Green InGaN/GaN Multiple Quantum Wells

    NASA Astrophysics Data System (ADS)

    Lin, Tao; Kuo, Hao Chung; Jiang, Xiao Dong; Feng, Zhe Chuan

    2017-02-01

    This paper reports the transient photoluminescence (PL) properties of an InGaN/GaN multiple quantum well (MQW) light-emitting diode (LED) with green emission. Recombination of localized excitons was proved to be the main microscopic mechanism of green emission in the sample. The PL dynamics were ascribed to two pathways of the exciton recombination, corresponding to the fast decay and the slow decay, respectively. The origins of slow decay and fast decay were assigned to local compositional fluctuations of indium and thickness variations of InGaN layers, respectively. Furthermore, the contributions of two decay pathways to the green PL were found to vary at different emission photon energy. The fraction of fast decay pathway decreased with decreasing photon energy. The slow radiative PL from deep localized exciton recombination suffered less suppression from non-radiative delocalization process, for the higher requested activation energy. All these results supported a clear microscopy mechanism of excitation-emission process of the green MQW LED structure.

  9. Recombination Pathways in Green InGaN/GaN Multiple Quantum Wells.

    PubMed

    Lin, Tao; Kuo, Hao Chung; Jiang, Xiao Dong; Feng, Zhe Chuan

    2017-12-01

    This paper reports the transient photoluminescence (PL) properties of an InGaN/GaN multiple quantum well (MQW) light-emitting diode (LED) with green emission. Recombination of localized excitons was proved to be the main microscopic mechanism of green emission in the sample. The PL dynamics were ascribed to two pathways of the exciton recombination, corresponding to the fast decay and the slow decay, respectively. The origins of slow decay and fast decay were assigned to local compositional fluctuations of indium and thickness variations of InGaN layers, respectively. Furthermore, the contributions of two decay pathways to the green PL were found to vary at different emission photon energy. The fraction of fast decay pathway decreased with decreasing photon energy. The slow radiative PL from deep localized exciton recombination suffered less suppression from non-radiative delocalization process, for the higher requested activation energy. All these results supported a clear microscopy mechanism of excitation-emission process of the green MQW LED structure.

  10. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

    PubMed Central

    2012-01-01

    Βackground The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. Results A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production. Conclusions Co

  11. Mutations in the yeast SRB2 general transcription factor suppress hpr1-induced recombination and show defects in DNA repair.

    PubMed

    Piruat, J I; Aguilera, A

    1996-08-01

    We have obtained genetic and molecular evidence that the hrs2-1 mutation, isolated as a suppressor of the hyperrecombination phenotype of hpr1 delta, is in the SRB2 gene, which encodes a component of the RNA polII holoenzyme. A newly constructed srb2 delta allele restores the wild-type levels of deletions in hpr1 delta cells, indicating that the lack of a functional SRB2 transcription factor suppresses recombination between direct repeats. These results suggest a direct connection between transcription and recombination between DNA repeats. On the other hand, the hrs2-1 mutation (renamed srb2-101), in which Gly150 has been changed to Asp, makes cells sensitive to long MMS treatments, a phenotype observed for the srb2 delta null allele only in a hpr1 delta background. This indicates that mutations in the basal transcription factor SRB2 impair DNA repair of MMS-induced damage, which adds a new connection between transcription and DNA repair. We discuss the possibility that hpr1-induced deletions occurred as a consequence of a SRB2-dependent stalled or blocked transcription complex.

  12. Mutations in the Yeast Srb2 General Transcription Factor Suppress Hpr1-Induced Recombination and Show Defects in DNA Repair

    PubMed Central

    Piruat, J. L.; Aguilera, A.

    1996-01-01

    We have obtained genetic and molecular evidence that the hrs2-1 mutation, isolated as a suppressor of the hyperrecombination phenotype of hpr1Δ, is in the SRB2 gene, which encodes a component of the RNA polII holoenzyme. A newly constructed srb2Δ allele restores the wild-type levels of deletions in hpr1Δ cells, indicating that the lack of a functional SRB2 transcription factor suppresses recombination between direct repeats. These results suggest a direct connection between transcription and recombination between DNA repeats. On the other hand, the hrs2-1 mutation (renamed srb2-101), in which Gly(150) has been changed to Asp, makes cells sensitive to long MMS treatments, a phenotype observed for the srb2Δ null allele only in a hpr1Δ background. This indicates that mutations in the basal transcription factor SRB2 impair DNA repair of MMS-induced damage, which adds a new connection between transcription and DNA repair. We discuss the possibility that hpr1-induced deletions occurred as a consequence of a SRB2-dependent stalled or blocked transcription complex. PMID:8844143

  13. Combining Heavy Ion Radiation and Artificial MicroRNAs to Target the Homologous Recombination Repair Gene Efficiently Kills Human Tumor Cells

    SciTech Connect

    Zheng Zhiming; Wang Ping; Wang Hongyan; Zhang Xiangming; Wang Minli; Cucinotta, Francis A.; Wang Ya

    2013-02-01

    Purpose: Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Methods and Materials: Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Results: Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Conclusions: Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death.

  14. New Genes Originated via Multiple Recombinational Pathways in the β-Globin Gene Family of Rodents

    PubMed Central

    Hoffmann, Federico G.; Opazo, Juan C.; Storz, Jay F.

    2008-01-01

    Species differences in the size or membership composition of multigene families can be attributed to lineage-specific additions of new genes via duplication, losses of genes via deletion or inactivation, and the creation of chimeric genes via domain shuffling or gene fusion. In principle, it should be possible to infer the recombinational pathways responsible for each of these different types of genomic change by conducting detailed comparative analyses of genomic sequence data. Here, we report an attempt to unravel the complex evolutionary history of the β-globin gene family in a taxonomically diverse set of rodent species. The main objectives were: 1) to characterize the genomic structure of the β-globin gene cluster of rodents; 2) to assign orthologous and paralogous relationships among duplicate copies of β-like globin genes; and 3) to infer the specific recombinational pathways responsible for gene duplications, gene deletions, and the creation of chimeric fusion genes. Results of our comparative genomic analyses revealed that variation in gene family size among rodent species is mainly attributable to the differential gain and loss of later expressed β-globin genes via unequal crossing-over. However, two distinct recombinational mechanisms were implicated in the creation of chimeric fusion genes. In muroid rodents, a chimeric γ/ε fusion gene was created by unequal crossing-over between the embryonic ε- and γ-globin genes. Interestingly, this γ/ε fusion gene was generated in the same fashion as the “anti-Lepore” 5′-δ-(β/δ)-β-3′ duplication mutant in humans (the reciprocal exchange product of the pathological hemoglobin Lepore deletion mutant). By contrast, in the house mouse, Mus musculus, a chimeric β/δ fusion pseudogene was created by a β-globin → δ-globin gene conversion event. Although the γ/ε and β/δ fusion genes share a similar chimeric gene structure, they originated via completely different recombinational pathways. PMID

  15. Inhibition of Wnt/β-catenin pathway promotes regenerative repair of cutaneous and cartilage injury

    PubMed Central

    Bastakoty, Dikshya; Saraswati, Sarika; Cates, Justin; Lee, Ethan; Nanney, Lillian B.; Young, Pampee P.

    2015-01-01

    Wound healing in mammals is a fibrotic process. The mechanisms driving fibrotic (as opposed to regenerative) repair are poorly understood. Herein we report that therapeutic Wnt inhibition with topical application of small-molecule Wnt inhibitors can reduce fibrosis and promote regenerative cutaneous wound repair. In the naturally stented model of ear punch injury, we found that Wnt/β-catenin pathway is activated most notably in the dermis of the wound bed early (d 2) after injury and subsides to baseline levels by d10. Topical application of either of 2 mechanistically distinct small-molecule Wnt pathway inhibitors (a tankyrase inhibitor, XAV-939, and the U.S. Food and Drug Administration–approved casein kinase activator, pyrvinium) in C57Bl/6J mice resulted in significantly increased rates of wound closure (72.3 ± 14.7% with XAV-939; and 52.1 ± 20.9% with pyrvinium) compared with contralateral controls (38.1 ± 23.0 and 40.4.±16.7%, respectively). Histologically, Wnt inhibition reduced fibrosis as measured by α-smooth muscle actin positive myofibroblasts and collagen type I α1 synthesis. Wnt inhibition also restored skin architecture including adnexal structures in ear wounds and dermal–epidermal junction with rete pegs in excisional wounds. Additionally, in ear punch injury Wnt inhibitor treatment enabled regeneration of auricular cartilage. Our study shows that pharmacologic Wnt inhibition holds therapeutic utility for regenerative repair of cutaneous wounds.—Bastakoty, D., Saraswati, S., Cates, J., Lee, E., Nanney, L. B., Young, P. P. Inhibition of Wnt/β-catenin pathway promotes regenerative repair of cutaneous and cartilage injury. PMID:26268926

  16. Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway

    PubMed Central

    Bettiga, Maurizio; Bengtsson, Oskar; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

    2009-01-01

    Background Sustainable and economically viable manufacturing of bioethanol from lignocellulose raw material is dependent on the availability of a robust ethanol producing microorganism, able to ferment all sugars present in the feedstock, including the pentose sugars L-arabinose and D-xylose. Saccharomyces cerevisiae is a robust ethanol producer, but needs to be engineered to achieve pentose sugar fermentation. Results A new recombinant S. cerevisiae strain expressing an improved fungal pathway for the utilization of L-arabinose and D-xylose was constructed and characterized. The new strain grew aerobically on L-arabinose and D-xylose as sole carbon sources. The activities of the enzymes constituting the pentose utilization pathway(s) and product formation during anaerobic mixed sugar fermentation were characterized. Conclusion Pentose fermenting recombinant S. cerevisiae strains were obtained by the expression of a pentose utilization pathway of entirely fungal origin. During anaerobic fermentation the strain produced biomass and ethanol. L-arabitol yield was 0.48 g per gram of consumed pentose sugar, which is considerably less than previously reported for D-xylose reductase expressing strains co-fermenting L-arabinose and D-xylose, and the xylitol yield was 0.07 g per gram of consumed pentose sugar. PMID:19630951

  17. Connecting by breaking and repairing: mechanisms of DNA strand exchange in meiotic recombination

    PubMed Central

    Sansam, Christopher L; Pezza, Roberto J

    2015-01-01

    During prophase of meiosis I, homologous chromosomes interact and undergo recombination. Successful completion of these processes is required in order for the homologous chromosomes to mount the meiotic spindle as a pair. The organization of the chromosomes into pairs ensures orderly segregation to opposite poles of the dividing cell, such that each gamete receives one copy of each chromosome. Chiasmata, the cytological manifestation of crossover products of recombination, physically connect the homologs in pairs, providing a linkage that facilitates their segregation. Consequently, mutations that reduce the level of recombination are invariably associated with increased errors in meiotic chromosome segregation. In this review, we focus on recent biochemical and genetic advances in elucidating the mechanisms of meiotic DNA strand exchange catalyzed by the Dmc1 protein. We also discuss the mode by which two recombination mediators, Hop2 and Mnd1, facilitate rate-limiting steps of DNA strand exchange catalyzed by Dmc1. PMID:25953379

  18. Connecting by breaking and repairing: mechanisms of DNA strand exchange in meiotic recombination.

    PubMed

    Sansam, Christopher L; Pezza, Roberto J

    2015-07-01

    During prophase of meiosis I, homologous chromosomes interact and undergo recombination. Successful completion of these processes is required in order for the homologous chromosomes to mount the meiotic spindle as a pair. The organization of the chromosomes into pairs ensures orderly segregation to opposite poles of the dividing cell, such that each gamete receives one copy of each chromosome. Chiasmata, the cytological manifestation of crossover products of recombination, physically connect the homologs in pairs, providing a linkage that facilitates their segregation. Consequently, mutations that reduce the level of recombination are invariably associated with increased errors in meiotic chromosome segregation. In this review, we focus on recent biochemical and genetic advances in elucidating the mechanisms of meiotic DNA strand exchange catalyzed by the Dmc1 protein. We also discuss the mode by which two recombination mediators, Hop2 and Mnd1, facilitate rate-limiting steps of DNA strand exchange catalyzed by Dmc1.

  19. Evidence for the role of Mycobacterium tuberculosis RecG helicase in DNA repair and recombination.

    PubMed

    Thakur, Roshan S; Basavaraju, Shivakumar; Somyajit, Kumar; Jain, Akshatha; Subramanya, Shreelakshmi; Muniyappa, Kalappa; Nagaraju, Ganesh

    2013-04-01

    In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacterium tuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M. tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichia coli ∆recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions. © 2013 The Authors Journal compilation © 2013 FEBS.

  20. The role of BRCA1 in homologous recombination repair in response to replication stress: significance in tumorigenesis and cancer therapy

    PubMed Central

    2013-01-01

    Germ line mutations in breast cancer gene 1 (BRCA1) predispose women to breast and ovarian cancers. Although BRCA1 is involved in many important biological processes, the function of BRCA1 in homologous recombination (HR) mediated repair is considered one of the major mechanisms contributing to its tumor suppression activity, and the cause of hypersensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors when BRCA1 is defective. Mounting evidence suggests that the mechanism of repairing DNA double strand breaks (DSBs) by HR is different than the mechanism operating when DNA replication is blocked. Although BRCA1 has been recognized as a central component in HR, the precise role of BRCA1 in HR, particularly under replication stress, has remained largely unknown. Given the fact that DNA lesions caused by replication blockages are the primary substrates for HR in mitotic cells, functional analysis of BRCA1 in HR repair in the context of replication stress should benefit our understanding of the molecular mechanisms underlying tumorigenesis associated with BRCA1 deficiencies, as well as the development of therapeutic approaches for cancer patients carrying BRCA1 mutations or reduced BRCA1 expression. This review focuses on the current advances in this setting and also discusses the significance in tumorigenesis and cancer therapy. PMID:23388117

  1. Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway.

    PubMed

    Jang, Seok-Won; Jung, Jin Ki; Kim, Jung Min

    2016-09-01

    The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance.

  2. Polynucleotide kinase-phosphatase enables neurogenesis via multiple DNA repair pathways to maintain genome stability.

    PubMed

    Shimada, Mikio; Dumitrache, Lavinia C; Russell, Helen R; McKinnon, Peter J

    2015-10-01

    Polynucleotide kinase-phosphatase (PNKP) is a DNA repair factor possessing both 5'-kinase and 3'-phosphatase activities to modify ends of a DNA break prior to ligation. Recently, decreased PNKP levels were identified as the cause of severe neuropathology present in the human microcephaly with seizures (MCSZ) syndrome. Utilizing novel murine Pnkp alleles that attenuate expression and a T424GfsX48 frame-shift allele identified in MCSZ individuals, we determined how PNKP inactivation impacts neurogenesis. Mice with PNKP inactivation in neural progenitors manifest neurodevelopmental abnormalities and postnatal death. This severe phenotype involved defective base excision repair and non-homologous end-joining, pathways required for repair of both DNA single- and double-strand breaks. Although mice homozygous for the T424GfsX48 allele were lethal embryonically, attenuated PNKP levels (akin to MCSZ) showed general neurodevelopmental defects, including microcephaly, indicating a critical developmental PNKP threshold. Directed postnatal neural inactivation of PNKP affected specific subpopulations including oligodendrocytes, indicating a broad requirement for genome maintenance, both during and after neurogenesis. These data illuminate the basis for selective neural vulnerability in DNA repair deficiency disease. © 2015 The Authors.

  3. Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase Recombinational Assembly (SIRA).

    PubMed

    Merrick, C A; Wardrope, C; Paget, J E; Colloms, S D; Rosser, S J

    2016-01-01

    Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of commodity compounds and fine chemicals offers a cheaper, greener, and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. Serine integrase recombinational assembly (SIRA) is a rapid DNA assembly method that utilizes serine integrases, and is particularly applicable to rapid optimization of engineered metabolic pathways. Using six pairs of orthogonal attP and attB sites with different central dinucleotide sequences that follow SIRA design principles, we have demonstrated that ΦC31 integrase can be used to (1) insert a single piece of DNA into a substrate plasmid; (2) assemble three, four, and five DNA parts encoding the enzymes for functional metabolic pathways in a one-pot reaction; (3) generate combinatorial libraries of metabolic pathway constructs with varied ribosome binding site strengths or gene orders in a one-pot reaction; and (4) replace and add DNA parts within a construct through targeted postassembly modification. We explain the mechanism of SIRA and the principles behind designing a SIRA reaction. We also provide protocols for making SIRA reaction components and practical methods for applying SIRA to rapid optimization of metabolic pathways.

  4. Red meat and poultry intake, polymorphisms in the nucleotide excision repair and mismatch repair pathways and colorectal cancer risk.

    PubMed

    Joshi, Amit D; Corral, Román; Siegmund, Kimberly D; Haile, Robert W; Le Marchand, Loïc; Martínez, Maria Elena; Ahnen, Dennis J; Sandler, Robert S; Lance, Peter; Stern, Mariana C

    2009-03-01

    Diets high in red meat have been consistently associated with colorectal cancer (CRC) risk and may result in exposure to carcinogens that cause DNA damage [i.e polycyclic aromatic hydrocarbons, heterocyclic amines (HCAs) and N-nitroso compounds]. Using a family-based study, we investigated whether polymorphisms in the nucleotide excision repair (NER) (ERCC1 3' untranslated region (UTR) G/T, XPD Asp312Asn and Lys751Gln, XPC intron 11 C/A, XPA 5' UTR C/T, XPF Arg415Gln and XPG Asp1104His) and mismatch repair (MLH1 Ile219Val and MSH2 Gly322Asp) pathways modified the association with red meat and poultry intake. We tested for gene-environment interactions using case-only analyses (n = 577) and compared the results using case-unaffected sibling comparisons (n = 307 sibships). Increased risk of CRC was observed for intake of more than or equal to three servings per week of red meat [odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.3-2.5)] or high-temperature cooked red meat (OR = 1.6, 95% CI = 1.1-2.2). Intake of red meat heavily brown on the outside or inside increased CRC risk only among subjects who carried the XPD codon 751 Lys/Lys genotype (case-only interaction P = 0.006 and P = 0.001, respectively, for doneness outside or inside) or the XPD codon 312 Asp/Asp genotype (case-only interaction P = 0.090 and P < 0.001, respectively). These interactions were stronger for rectal cancer cases (heterogeneity test P = 0.002 for XPD Asp312Asn and P = 0.03 for XPD Lys751Gln) and remained statistically significant after accounting for multiple testing. Case-unaffected sibling analyses were generally supportive of the case-only results. These findings highlight the possible contribution of diets high in red meat to the formation of lesions that elicit the NER pathway, such as carcinogen-induced bulky adducts.

  5. Red meat and poultry intake, polymorphisms in the nucleotide excision repair and mismatch repair pathways and colorectal cancer risk

    PubMed Central

    Joshi, Amit D.; Corral, Román; Siegmund, Kimberly D.; Haile, Robert W.; Le Marchand, Loïc; Martínez, Maria Elena; Ahnen, Dennis J.; Sandler, Robert S.; Lance, Peter; Stern, Mariana C.

    2009-01-01

    Diets high in red meat have been consistently associated with colorectal cancer (CRC) risk and may result in exposure to carcinogens that cause DNA damage [i.e polycyclic aromatic hydrocarbons, heterocyclic amines (HCAs) and N-nitroso compounds]. Using a family-based study, we investigated whether polymorphisms in the nucleotide excision repair (NER) (ERCC1 3′ untranslated region (UTR) G/T, XPD Asp312Asn and Lys751Gln, XPC intron 11 C/A, XPA 5′ UTR C/T, XPF Arg415Gln and XPG Asp1104His) and mismatch repair (MLH1 Ile219Val and MSH2 Gly322Asp) pathways modified the association with red meat and poultry intake. We tested for gene–environment interactions using case-only analyses (n = 577) and compared the results using case-unaffected sibling comparisons (n = 307 sibships). Increased risk of CRC was observed for intake of more than or equal to three servings per week of red meat [odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.3–2.5)] or high-temperature cooked red meat (OR = 1.6, 95% CI = 1.1–2.2). Intake of red meat heavily brown on the outside or inside increased CRC risk only among subjects who carried the XPD codon 751 Lys/Lys genotype (case-only interaction P = 0.006 and P = 0.001, respectively, for doneness outside or inside) or the XPD codon 312 Asp/Asp genotype (case-only interaction P = 0.090 and P < 0.001, respectively). These interactions were stronger for rectal cancer cases (heterogeneity test P = 0.002 for XPD Asp312Asn and P = 0.03 for XPD Lys751Gln) and remained statistically significant after accounting for multiple testing. Case-unaffected sibling analyses were generally supportive of the case-only results. These findings highlight the possible contribution of diets high in red meat to the formation of lesions that elicit the NER pathway, such as carcinogen-induced bulky adducts. PMID:19029193

  6. MutS homologue hMSH5: recombinational DSB repair and non-synonymous polymorphic variants.

    PubMed

    Wu, Xiling; Xu, Yang; Feng, Katey; Tompkins, Joshua D; Her, Chengtao

    2013-01-01

    Double-strand breaks (DSBs) constitute the most deleterious form of DNA lesions that can lead to genome alterations and cell death, and the vast majority of DSBs arise pathologically in response to DNA damaging agents such as ionizing radiation (IR) and chemotherapeutic agents. Recent studies have implicated a role for the human MutS homologue hMSH5 in homologous recombination (HR)-mediated DSB repair and the DNA damage response. In the present study, we show that hMSH5 promotes HR-based DSB repair, and this property resides in the carboxyl-terminal portion of the protein. Our results demonstrate that DSB-triggered hMSH5 chromatin association peaks at the proximal regions of the DSB and decreases gradually with increased distance from the break. Furthermore, the DSB-triggered hMSH5 chromatin association is preceded by and relies on the assembly of hMRE11 and hRad51 at the proximal regions of the DSB. Lastly, the potential effects of hMSH5 non-synonymous variants (L85F, Y202C, V206F, R351G, L377F, and P786S) on HR and cell survival in response to DSB-inducing anticancer agents have been analyzed. These experiments show that the expression of hMSH5 variants elicits different survival responses to anticancer drugs cisplatin, bleomycin, doxorubicin and camptothecin. However, the effects of hMSH5 variants on survival responses to DSB-inducing agents are not directly correlated to their effects exerted on HR-mediated DSB repair, suggesting that the roles of hMSH5 variants in the processes of DNA damage response and repair are multifaceted.

  7. Acute ischemic stroke after cardiac catheterization: the protamine low-dose recombinant tissue plasminogen activator pathway.

    PubMed

    Guevara, Carlos; Quijada, Alonso; Rosas, Carolina; Bulatova, Katya; Lara, Hugo; Nieto, Elena; Morales, Marcelo

    2016-05-20

    Intravenous thrombolysis is the preferred treatment for acute ischemic stroke; however, it remains unestablished in the area of cardiac catheterization. We report three patients with acute ischemic stroke after cardiac catheterization. After reversing the anticoagulant effect of unfractionated heparin with protamine, all of the patients were successfully off-label thrombolyzed with reduced doses of intravenous recombinant tissue plasminogen activator (0.6 mg/kg). This dose was preferred to reduce the risk of symptomatic cerebral or systemic bleeding. The sequential pathway of protamine recombinant tissue plasminogen activator at reduced doses may be safer for reducing intracranial or systemic bleeding events, whereas remaining efficacious for the treatment of acute ischemic stroke after cardiac catheterization.

  8. Helicobacter pylori infection modulates the expression of miRNAs associated with DNA mismatch repair pathway.

    PubMed

    Santos, Juliana C; Brianti, Mitsue T; Almeida, Victor R; Ortega, Manoela M; Fischer, Wolfgang; Haas, Rainer; Matheu, Ander; Ribeiro, Marcelo L

    2017-04-01

    Genetic and epigenetic inactivation of DNA mismatch repair (MMR) genes might lead to modifications in cancer-related gene expression and cancer development. Recently, it has been shown that the infection by Helicobacter pylori, the major causative agent of gastric cancer, induces DNA damage and inhibits MMR DNA repair. Also, it has been reported that microRNAs (miRs) have an important role in regulating genomic stability and MMR DNA repair. Thus, the aim of this study was to identify miRs regulating MMR pathway in H. pylori-associated gastric carcinogenesis. To address this question, a gastric epithelial cell line and AGS cancer gastric cells were infected with several H. pylori strains. MMR gene expression and miRs correlating with H. pylori strain infection were evaluated. The results showed that H. pylori infection significantly down-regulated the expression of all selected MMR genes. Also, H. pylori infection modulated the expression of several miRs (including miR-150-5p, miR-155-5p, and miR-3163), after 4, 8, and 12 h of infection. Computational prediction of candidate miRs and their predicted MMR targeting sites were obtained from TargetScan, mirDB, and MetaCore. The generated data indicated that the selected miRs (miR-150-5p, miR-155-5p, and miR-3163) could possibly target and modulate MMR genes (POLD3, MSH2, and MSH3, respectively). The target validation was performed using mimics and luciferase gene reporter assays. Briefly, this study shows that H. pylori impairs MMR DNA repair pathway and identifies miRs that regulate MMR gene expression in gastric cancer. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

    SciTech Connect

    Mulari, Mika T.K. Nars, Martin; Laitala-Leinonen, Tiina; Kaisto, Tuula; Metsikkoe, Kalervo; Sun Yi; Vaeaenaenen, H. Kalervo

    2008-05-01

    Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-{beta}-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption.

  10. Identification of Pathways in Liver Repair Potentially Targeted by Secretory Proteins from Human Mesenchymal Stem Cells

    PubMed Central

    Winkler, Sandra; Hempel, Madlen; Brückner, Sandra; Tautenhahn, Hans-Michael; Kaufmann, Roland; Christ, Bruno

    2016-01-01

    Background: The beneficial impact of mesenchymal stem cells (MSC) on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1) increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM) decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT) and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor) signalling pathways as relevant networks. Relationships to transforming growth factor β (TGF-β) and hypoxia-inducible factor 1-α (HIF1-α) signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration. PMID:27409608

  11. DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases

    PubMed Central

    Bettencourt, Conceição; Hensman‐Moss, Davina; Flower, Michael; Wiethoff, Sarah; Brice, Alexis; Goizet, Cyril; Stevanin, Giovanni; Koutsis, Georgios; Karadima, Georgia; Panas, Marios; Yescas‐Gómez, Petra; García‐Velázquez, Lizbeth Esmeralda; Alonso‐Vilatela, María Elisa; Lima, Manuela; Raposo, Mafalda; Traynor, Bryan; Sweeney, Mary; Wood, Nicholas; Giunti, Paola; Durr, Alexandra; Holmans, Peter; Houlden, Henry; Tabrizi, Sarah J.

    2016-01-01

    Objective The polyglutamine diseases, including Huntington's disease (HD) and multiple spinocerebellar ataxias (SCAs), are among the commonest hereditary neurodegenerative diseases. They are caused by expanded CAG tracts, encoding glutamine, in different genes. Longer CAG repeat tracts are associated with earlier ages at onset, but this does not account for all of the difference, and the existence of additional genetic modifying factors has been suggested in these diseases. A recent genome‐wide association study (GWAS) in HD found association between age at onset and genetic variants in DNA repair pathways, and we therefore tested whether the modifying effects of variants in DNA repair genes have wider effects in the polyglutamine diseases. Methods We assembled an independent cohort of 1,462 subjects with HD and polyglutamine SCAs, and genotyped single‐nucleotide polymorphisms (SNPs) selected from the most significant hits in the HD study. Results In the analysis of DNA repair genes as a group, we found the most significant association with age at onset when grouping all polyglutamine diseases (HD+SCAs; p = 1.43 × 10–5). In individual SNP analysis, we found significant associations for rs3512 in FAN1 with HD+SCAs (p = 1.52 × 10–5) and all SCAs (p = 2.22 × 10–4) and rs1805323 in PMS2 with HD+SCAs (p = 3.14 × 10–5), all in the same direction as in the HD GWAS. Interpretation We show that DNA repair genes significantly modify age at onset in HD and SCAs, suggesting a common pathogenic mechanism, which could operate through the observed somatic expansion of repeats that can be modulated by genetic manipulation of DNA repair in disease models. This offers novel therapeutic opportunities in multiple diseases. Ann Neurol 2016;79:983–990 PMID:27044000

  12. The relationship between mTOR signalling pathway and recombinant antibody productivity in CHO cell lines

    PubMed Central

    2014-01-01

    Background High recombinant protein productivity in mammalian cell lines is often associated with phenotypic changes in protein content, energy metabolism, and cell growth, but the key determinants that regulate productivity are still not clearly understood. The mammalian target of rapamycin (mTOR) signalling pathway has emerged as a central regulator for many cellular processes including cell growth, apoptosis, metabolism, and protein synthesis. This role of this pathway changes in response to diverse environmental cues and allows the upstream proteins that respond directly to extracellular signals (such as nutrient availability, energy status, and physical stresses) to communicate with downstream effectors which, in turn, regulate various essential cellular processes. Results In this study, we have performed a transcriptomic analysis using a pathway-focused polymerase chain reaction (PCR) array to compare the expression of 84 target genes related to the mTOR signalling in two recombinant CHO cell lines with a 17.4-fold difference in specific monoclonal antibody productivity (q p ). Eight differentially expressed genes that exhibited more than a 1.5-fold change were identified. Pik3cd (encoding the Class 1A catalytic subunit of phosphatidylinositol 3-kinase [PI3K]) was the most differentially expressed gene having a 71.3-fold higher level of expression in the high producer cell line than in the low producer. The difference in the gene’s transcription levels was confirmed at the protein level by examining expression of p110δ. Conclusion Expression of p110δ correlated with specific productivity (q p ) across six different CHO cell lines, with a range of expression levels from 3 to 51 pg/cell/day, suggesting that p110δ may be a key factor in regulating productivity in recombinant cell lines. PMID:24533650

  13. DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells

    SciTech Connect

    Urushihara, Yusuke; Kobayashi, Junya; Matsumoto, Yoshihisa; Komatsu, Kenshi; Oda, Shoji; Mitani, Hiroshi

    2012-12-14

    Highlights: Black-Right-Pointing-Pointer We investigated the effect of DNA-PK inhibition on DSB repair using fish cells. Black-Right-Pointing-Pointer A radiation sensitive mutant RIC1 strain showed a low level of DNA-PK activity. Black-Right-Pointing-Pointer DNA-PK dysfunction leads defects in HR repair and DNA-PKcs autophosphorylation. Black-Right-Pointing-Pointer DNA-PK dysfunction leads a slight increase in the number of 53BP1 foci after DSBs. Black-Right-Pointing-Pointer DNA-PK dysfunction leads an alternative NHEJ that depends on 53BP1. -- Abstract: Nonhomologous end joining (NHEJ) and homologous recombination (HR) are known as DNA double-strand break (DSB) repair pathways. It has been reported that DNA-PK, a member of PI3 kinase family, promotes NHEJ and aberrant DNA-PK causes NHEJ deficiency. However, in this study, we demonstrate that a wild-type cell line treated with DNA-PK inhibitor and a mutant cell line with dysfunctional DNA-PK showed decreased HR efficiency in fish cells (Medaka, Oryzias latipes). Previously, we reported that the radiation-sensitive mutant RIC1 strain has a defect in the Histone H2AX phosphorylation after {gamma}-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of {gamma}H2AX foci after {gamma}-irradiation in wild-type cells, but had no significant effect in RIC1 cells. In addition, RIC1 cells showed significantly lower levels of DNA-PK kinase activity compared with wild-type cells. We investigated NHEJ and HR efficiency after induction of DSBs. Wild-type cells treated with NU7026 and RIC1 cells showed decreased HR efficiency. These results indicated that aberrant DNA-PK causes the reduction in the number of {gamma}H2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after {gamma}-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the

  14. Electron Tunneling Pathways and Role of Adenine in Repair of Cyclobutane Pyrimidine Dimer by DNA Photolyase

    PubMed Central

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

    2012-01-01

    Electron tunneling pathways in enzymes are critical to their catalytic efficiency. Through electron tunneling, photolyase, a photoenzyme, splits UV-induced cyclobutane pyrimidine dimer into two normal bases. Here, we report our systematic characterization and analyses of photo-initiated three electron transfer processes and cyclobutane ring splitting by following the entire dynamical evolution during enzymatic repair with femtosecond resolution. We observed the complete dynamics of the reactants, all intermediates and final products, and determined their reaction time scales. Using (deoxy)uracil and thymine as dimer substrates, we unambiguously determined the electron tunneling pathways for the forward electron transfer to initiate repair and for the final electron return to restore the active cofactor and complete the catalytic photocycle. Significantly, we found that the adenine moiety of the unusual bent flavin cofactor is essential to mediating all electron-transfer dynamics through a super-exchange mechanism, leading to a delicate balance of time scales. The cyclobutane ring splitting takes tens of picoseconds while electron-transfer dynamics all occur on a longer time scale. The active-site structural integrity, unique electron tunneling pathways and the critical role of adenine assure the synergy of these elementary steps in this complex photorepair machinery to achieve maximum repair efficiency which is close to unity. Finally, we used the Marcus electron-transfer theory to evaluate all three electron transfer processes and thus obtained their reaction driving forces (free energies), reorganization energies, and electronic coupling constants, concluding the forward and futile back electron transfer in the normal region and that the final electron return of the catalytic cycle is in the inverted region. PMID:22533849

  15. Electron tunneling pathways and role of adenine in repair of cyclobutane pyrimidine dimer by DNA photolyase.

    PubMed

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

    2012-05-16

    Electron tunneling pathways in enzymes are critical to their catalytic efficiency. Through electron tunneling, photolyase, a photoenzyme, splits UV-induced cyclobutane pyrimidine dimer into two normal bases. Here, we report our systematic characterization and analyses of photoinitiated three electron transfer processes and cyclobutane ring splitting by following the entire dynamical evolution during enzymatic repair with femtosecond resolution. We observed the complete dynamics of the reactants, all intermediates and final products, and determined their reaction time scales. Using (deoxy)uracil and thymine as dimer substrates, we unambiguously determined the electron tunneling pathways for the forward electron transfer to initiate repair and for the final electron return to restore the active cofactor and complete the catalytic photocycle. Significantly, we found that the adenine moiety of the unusual bent flavin cofactor is essential to mediating all electron-transfer dynamics through a superexchange mechanism, leading to a delicate balance of time scales. The cyclobutane ring splitting takes tens of picoseconds, while electron-transfer dynamics all occur on a longer time scale. The active-site structural integrity, unique electron tunneling pathways, and the critical role of adenine ensure the synergy of these elementary steps in this complex photorepair machinery to achieve maximum repair efficiency which is close to unity. Finally, we used the Marcus electron-transfer theory to evaluate all three electron-transfer processes and thus obtained their reaction driving forces (free energies), reorganization energies, and electronic coupling constants, concluding that the forward and futile back-electron transfer is in the normal region and that the final electron return of the catalytic cycle is in the inverted region.

  16. Analysis of strand transfer and template switching mechanisms of DNA gap repair by homologous recombination in Escherichia coli: predominance of strand transfer.

    PubMed

    Izhar, Lior; Goldsmith, Moshe; Dahan, Ronny; Geacintov, Nicholas; Lloyd, Robert G; Livneh, Zvi

    2008-09-12

    Daughter strand gaps formed upon interruption of replication at DNA lesions in Escherichia coli can be repaired by either translesion DNA synthesis or homologous recombination (HR) repair. Using a plasmid-based assay system that enables discrimination between strand transfer and template switching (information copying) modes of HR gap repair, we found that approximately 80% of strand gaps were repaired by physical strand transfer from the donor, whereas approximately 20% appear to be repaired by template switching. HR gap repair operated on both small and bulky lesions and largely depended on RecA and RecF but not on the RecBCD nuclease. In addition, we found that HR was mildly reduced in cells lacking the RuvABC and RecG proteins involved in resolution of Holliday junctions. These results, obtained for the first time under conditions that detect the two HR gap repair mechanisms, provide in vivo high-resolution molecular evidence for the predominance of the strand transfer mechanism in HR gap repair. A small but significant portion of HR gap repair appears to occur via a template switching mechanism.

  17. Generation and CRISPR/Cas9 editing of transformed progenitor B cells as a pseudo-physiological system to study DNA repair gene function in V(D)J recombination.

    PubMed

    Lenden Hasse, Hélène; Lescale, Chloé; Bianchi, Joy J; Yu, Wei; Bedora-Faure, Marie; Deriano, Ludovic

    2017-09-04

    Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions. Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  18. The Roles of the BLM Helicase in Homologous Recombination and DNA Repair

    DTIC Science & Technology

    2005-05-01

    repair of DNA double strand breaks (DSBs), harmful lesions that can lead to genomic instability, cell death, or tumorigenesis. There is an emerging role...autosomal recessive disease in which patients are ,predisposed to cancer, including breast cancers. Cells isolated from these patients display genomic...mutagenic chemicals that result in DNA damage. Among the most lethal DNA lesions are double -strand breaks (DSBs), which if left unrepaired can lead to

  19. Initiation of the ATM-Chk2 DNA damage response through the base excision repair pathway.

    PubMed

    Chou, Wen-Cheng; Hu, Ling-Yueh; Hsiung, Chia-Ni; Shen, Chen-Yang

    2015-08-01

    The DNA damage response (DDR) is activated by various genotoxic stresses. Base lesions, which are structurally simple and predominantly fixed by base excision repair (BER), can trigger the ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2) pathway, a DDR component. How these lesions trigger DDR remains unclear. Here we show that, for alkylation damage, methylpurine-DNA glycosylase (MPG) and apurinic/apyrimidinic endonuclease 1, both of which function early in BER, are required for ATM-Chk2-dependent DDR. In addition, other DNA glycosylases, including uracil-DNA glycosylase and 8-oxoguanine glycosylase, which are involved in repairing deaminated bases and oxidative damage, also induced DDR. The early steps of BER therefore play a vital role in modulating the ATM-Chk2 DDR in response to base lesions, facilitating downstream BER processing for repair, in which the formation of a single-strand break was shown to play a critical role. Moreover, MPG knockdown rescued cell lethality, its overexpression led to cell death triggered by DNA damage and, more interestingly, higher MPG expression in breast and ovarian cancers corresponded with a greater probability of relapse-free survival after chemotherapy, underscoring the importance of glycosylase-dependent DDR. This study highlights the crosstalk between BER and DDR that contributes to maintaining genomic integrity and may have clinical applications in cancer therapy.

  20. Homologous recombination-dependent repair of telomeric DSBs in proliferating human cells

    PubMed Central

    Mao, Pingsu; Liu, Jingfan; Zhang, Zepeng; Zhang, Hong; Liu, Haiying; Gao, Song; Rong, Yikang S.; Zhao, Yong

    2016-01-01

    Telomeres prevent chromosome ends from being recognized as double-stranded breaks (DSBs). Meanwhile, G/C-rich repetitive telomeric DNA is susceptible to attack by DNA-damaging agents. How cells balance the need to protect DNA ends and the need to repair DNA lesions in telomeres is unknown. Here we show that telomeric DSBs are efficiently repaired in proliferating cells, but are irreparable in stress-induced and replicatively senescent cells. Using the CRISPR-Cas9 technique, we specifically induce DSBs at telomeric or subtelomeric regions. We find that DSB repair (DSBR) at subtelomeres occurs in an error-prone manner resulting in small deletions, suggestive of NHEJ. However, DSBR in telomeres involves ‘telomere-clustering', 3′-protruding C-rich telomeric ssDNA, and HR between sister-chromatid or interchromosomal telomeres. DSBR in telomeres is suppressed by deletion or inhibition of Rad51. These findings reveal proliferation-dependent DSBR in telomeres and suggest that telomeric HR, which is normally constitutively suppressed, is activated in the context of DSBR. PMID:27396625

  1. The plant-specific CDKB1-CYCB1 complex mediates homologous recombination repair in Arabidopsis.

    PubMed

    Weimer, Annika K; Biedermann, Sascha; Harashima, Hirofumi; Roodbarkelari, Farshad; Takahashi, Naoki; Foreman, Julia; Guan, Yonsheng; Pochon, Gaëtan; Heese, Maren; Van Damme, Daniël; Sugimoto, Keiko; Koncz, Csaba; Doerner, Peter; Umeda, Masaaki; Schnittger, Arp

    2016-10-04

    Upon DNA damage, cyclin-dependent kinases (CDKs) are typically inhibited to block cell division. In many organisms, however, it has been found that CDK activity is required for DNA repair, especially for homology-dependent repair (HR), resulting in the conundrum how mitotic arrest and repair can be reconciled. Here, we show that Arabidopsis thaliana solves this dilemma by a division of labor strategy. We identify the plant-specific B1-type CDKs (CDKB1s) and the class of B1-type cyclins (CYCB1s) as major regulators of HR in plants. We find that RADIATION SENSITIVE 51 (RAD51), a core mediator of HR, is a substrate of CDKB1-CYCB1 complexes. Conversely, mutants in CDKB1 and CYCB1 fail to recruit RAD51 to damaged DNA CYCB1;1 is specifically activated after DNA damage and we show that this activation is directly controlled by SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a transcription factor that acts similarly to p53 in animals. Thus, while the major mitotic cell-cycle activity is blocked after DNA damage, CDKB1-CYCB1 complexes are specifically activated to mediate HR. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  2. Real-time analysis of double-strand DNA break repair by homologous recombination

    PubMed Central

    Hicks, Wade M.; Yamaguchi, Miyuki; Haber, James E.

    2011-01-01

    The ability to induce synchronously a single site-specific double-strand break (DSB) in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during DSB repair. Special attention has been paid to the switching of mating-type genes in Saccharomyces cerevisiae, a process initiated by the HO endonuclease by cleaving the MAT locus. A DSB in MATa is repaired by homologous recombination—specifically, by gene conversion—using a heterochromatic donor, HMLα. Repair results in the replacement of the a-specific sequences (Ya) by Yα and switching from MATa to MATα. We report that MAT switching requires the DNA replication factor Dpb11, although it does not require the Cdc7-Dbf4 kinase or the Mcm and Cdc45 helicase components. Using Southern blot, PCR, and ChIP analysis of samples collected every 10 min, we extend previous studies of this process to identify the times for the loading of Rad51 recombinase protein onto the DSB ends at MAT, the subsequent strand invasion by the Rad51 nucleoprotein filament into the donor sequences, the initiation of new DNA synthesis, and the removal of the nonhomologous Y sequences. In addition we report evidence for the transient displacement of well-positioned nucleosomes in the HML donor locus during strand invasion. PMID:21292986

  3. Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

    PubMed Central

    Vriend, Lianne E.M.; Prakash, Rohit; Chen, Chun-Chin; Vanoli, Fabio; Cavallo, Francesca; Zhang, Yu; Jasin, Maria; Krawczyk, Przemek M.

    2016-01-01

    DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR (nickHR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided nuclease Cas9. However, the mechanisms of nickHR are largely unexplored. Here, we applied Cas9 nickases to study nickHR in mammalian cells. We find that nickHR is unaffected by inhibition of major damage signaling kinases and that it is not suppressed by nonhomologous end-joining (NHEJ) components, arguing that nick processing does not require a DSB intermediate to trigger HR. Relative to a single nick, nicking both strands enhances HR, consistent with a DSB intermediate, even when nicks are induced up to ∼1kb apart. Accordingly, HR and NHEJ compete for repair of these paired nicks, but, surprisingly, only when 5' overhangs or blunt ends can be generated. Our study advances the understanding of molecular mechanisms driving nick and paired-nick repair in mammalian cells and clarify phenomena associated with Cas9-mediated genome editing. PMID:27001513

  4. APE1 is dispensable for S-region cleavage but required for its repair in class switch recombination.

    PubMed

    Xu, Jianliang; Husain, Afzal; Hu, Wenjun; Honjo, Tasuku; Kobayashi, Maki

    2014-12-02

    Activation-induced cytidine deaminase (AID) is essential for antibody diversification, namely somatic hypermutation (SHM) and class switch recombination (CSR). The deficiency of apurinic/apyrimidinic endonuclease 1 (Ape1) in CH12F3-2A B cells reduces CSR to ∼20% of wild-type cells, whereas the effect of APE1 loss on SHM has not been examined. Here we show that, although APE1's endonuclease activity is important for CSR, it is dispensable for SHM as well as IgH/c-myc translocation. Importantly, APE1 deficiency did not show any defect in AID-induced S-region break formation, but blocked both the recruitment of repair protein Ku80 to the S region and the synapse formation between Sμ and Sα. Knockdown of end-processing factors such as meiotic recombination 11 homolog (MRE11) and carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) further reduced the remaining CSR in Ape1-null CH12F3-2A cells. Together, our results show that APE1 is dispensable for SHM and AID-induced DNA breaks and may function as a DNA end-processing enzyme to facilitate the joining of broken ends during CSR.

  5. APE1 is dispensable for S-region cleavage but required for its repair in class switch recombination

    PubMed Central

    Xu, Jianliang; Husain, Afzal; Hu, Wenjun; Honjo, Tasuku; Kobayashi, Maki

    2014-01-01

    Activation-induced cytidine deaminase (AID) is essential for antibody diversification, namely somatic hypermutation (SHM) and class switch recombination (CSR). The deficiency of apurinic/apyrimidinic endonuclease 1 (Ape1) in CH12F3-2A B cells reduces CSR to ∼20% of wild-type cells, whereas the effect of APE1 loss on SHM has not been examined. Here we show that, although APE1’s endonuclease activity is important for CSR, it is dispensable for SHM as well as IgH/c-myc translocation. Importantly, APE1 deficiency did not show any defect in AID-induced S-region break formation, but blocked both the recruitment of repair protein Ku80 to the S region and the synapse formation between Sμ and Sα. Knockdown of end-processing factors such as meiotic recombination 11 homolog (MRE11) and carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) further reduced the remaining CSR in Ape1-null CH12F3-2A cells. Together, our results show that APE1 is dispensable for SHM and AID-induced DNA breaks and may function as a DNA end-processing enzyme to facilitate the joining of broken ends during CSR. PMID:25404348

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

    PubMed

    Johnson, Nicholas M; Lemmens, Bennie B L G; Tijsterman, Marcel

    2013-01-01

    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.

  7. Dual inhibition of ATR and ATM potentiates the activity of trabectedin and lurbinectedin by perturbing the DNA damage response and homologous recombination repair

    PubMed Central

    Soares, Daniele G.; Selle, Frédéric; Morel, Claire; Galmarini, Carlos M.; Henriques, João A. P.; Larsen, Annette K.; Escargueil, Alexandre E.

    2016-01-01

    Trabectedin (Yondelis®, ecteinascidin-743, ET-743) is a marine-derived natural product approved for treatment of advanced soft tissue sarcoma and relapsed platinum-sensitive ovarian cancer. Lurbinectedin is a novel anticancer agent structurally related to trabectedin. Both ecteinascidins generate DNA double-strand breaks that are processed through homologous recombination repair (HRR), thereby rendering HRR-deficient cells particularly sensitive. We here characterize the DNA damage response (DDR) to trabectedin and lurbinectedin in HeLa cells. Our results show that both compounds activate the ATM/Chk2 (ataxia-telangiectasia mutated/checkpoint kinase 2) and ATR/Chk1 (ATM and RAD3-related/checkpoint kinase 1) pathways. Interestingly, pharmacological inhibition of Chk1/2, ATR or ATM is not accompanied by any significant improvement of the cytotoxic activity of the ecteinascidins while dual inhibition of ATM and ATR strongly potentiates it. Accordingly, concomitant inhibition of both ATR and ATM is an absolute requirement to efficiently block the formation of γ-H2AX, MDC1, BRCA1 and Rad51 foci following exposure to the ecteinascidins. These results are not restricted to HeLa cells, but are shared by cisplatin-sensitive and -resistant ovarian carcinoma cells. Together, our data identify ATR and ATM as central coordinators of the DDR to ecteinascidins and provide a mechanistic rationale for combining these compounds with ATR and ATM inhibitors. PMID:27029031

  8. Dual inhibition of ATR and ATM potentiates the activity of trabectedin and lurbinectedin by perturbing the DNA damage response and homologous recombination repair.

    PubMed

    Lima, Michelle; Bouzid, Hana; Soares, Daniele G; Selle, Frédéric; Morel, Claire; Galmarini, Carlos M; Henriques, João A P; Larsen, Annette K; Escargueil, Alexandre E

    2016-05-03

    Trabectedin (Yondelis®, ecteinascidin-743, ET-743) is a marine-derived natural product approved for treatment of advanced soft tissue sarcoma and relapsed platinum-sensitive ovarian cancer. Lurbinectedin is a novel anticancer agent structurally related to trabectedin. Both ecteinascidins generate DNA double-strand breaks that are processed through homologous recombination repair (HRR), thereby rendering HRR-deficient cells particularly sensitive. We here characterize the DNA damage response (DDR) to trabectedin and lurbinectedin in HeLa cells. Our results show that both compounds activate the ATM/Chk2 (ataxia-telangiectasia mutated/checkpoint kinase 2) and ATR/Chk1 (ATM and RAD3-related/checkpoint kinase 1) pathways. Interestingly, pharmacological inhibition of Chk1/2, ATR or ATM is not accompanied by any significant improvement of the cytotoxic activity of the ecteinascidins while dual inhibition of ATM and ATR strongly potentiates it. Accordingly, concomitant inhibition of both ATR and ATM is an absolute requirement to efficiently block the formation of γ-H2AX, MDC1, BRCA1 and Rad51 foci following exposure to the ecteinascidins. These results are not restricted to HeLa cells, but are shared by cisplatin-sensitive and -resistant ovarian carcinoma cells. Together, our data identify ATR and ATM as central coordinators of the DDR to ecteinascidins and provide a mechanistic rationale for combining these compounds with ATR and ATM inhibitors.

  9. The purine scaffold Hsp90 inhibitor PU-H71 sensitizes cancer cells to heavy ion radiation by inhibiting DNA repair by homologous recombination and non-homologous end joining.

    PubMed

    Lee, Younghyun; Li, Huizi Keiko; Masaoka, Aya; Sunada, Shigeaki; Hirakawa, Hirokazu; Fujimori, Akira; Nickoloff, Jac A; Okayasu, Ryuichi

    2016-10-01

    PU-H71 is a purine-scaffold Hsp90 inhibitor developed to overcome limitations of conventional Hsp90 inhibitors. This study was designed to investigate the combined effect of PU-H71 and heavy ion irradiation on human tumor and normal cells. The effects of PU-H71 were determined by monitoring cell survival by colony formation, and DNA double-strand break (DSB) repair by γ-H2AX foci and immuno-blotting DSB repair proteins. The mode of cell death was evaluated by sub-G1 DNA content (as an indicator for apoptosis), and mitotic catastrophe. PU-H71 enhanced heavy ion irradiation-induced cell death in three human cancer cell lines, but the drug did not radiosensitize normal human fibroblasts. In irradiated tumor cells, PU-H71 increased the persistence of γ-H2AX foci, and it reduced RAD51 foci and phosphorylated DNA-PKcs, key DSB repair proteins involved in homologous recombination (HR) and non-homologous end joining (NHEJ). In some tumor cell lines, PU-H71 altered the sub-G1 cell fraction and mitotic catastrophe following carbon ion irradiation. Our results demonstrate that PU-H71 sensitizes human cancer cells to heavy ion irradiation by inhibiting both HR and NHEJ DSB repair pathways. PU-H71 holds promise as a radiosensitizer for enhancing the efficacy of heavy ion radiotherapy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. Identification of two novel BRCA1-partner genes in the DNA double-strand break repair pathway.

    PubMed

    Guglielmi, Chiara; Cerri, Iacopo; Evangelista, Monica; Collavoli, Anita; Tancredi, Mariella; Aretini, Paolo; Caligo, Maria Adelaide

    2013-10-01

    M1775R and A1789T are two missense variants located within the BRCT domains of BRCA1 gene. The M1775R is a known deleterious variant, while the A1789T is an unclassified variant that has been analyzed and classified as probably deleterious for the first time by our group. In a previous study, we described the expression profile of HeLa G1 cells transfected with the two variants and we found that they altered molecular mechanisms critical for cell proliferation and genome integrity. Considering that the mutations in the BRCA1 C terminus (BRCT) domains are associated to a phenotype with an altered ability in the DNA double-strand break repair, we chose three of the genes previously identified, EEF1E1, MRE11A, and OBFC2B, to be tested for an homologous recombination (HR) in vitro assay. For our purpose, we performed a gene expression knockdown by siRNA transfection in HeLa cells, containing an integrated recombination substrate (hprtDRGFP), for each of the target genes included BRCA1. The knockdown of BRCA1, OBFC2B, MRE11A, and EEF1E1 reduces the HR rate, respectively, of 97.6, 28.6, 41.8, and 42.3 % compared to cells transfected with a scrambled negative control duplex and all these differences are statistically significant (P < 0.05; Kruskal-Wallis test). Finally, we analyzed the effect of target gene depletion both on HR that on cell survival after DNA-damage induction by ionizing radiation. The clonogenic assay showed that the down-regulation of the target genes reduced the cell survival, but the effect on the HR, is not evident. Only the BRCA1-siRNA confirmed the inhibition effect on HR. Overall these results confirmed the involvement of MRE11A in the HR pathway and identified two new genes, OBFC2B and EEF1E1, which according to these data and the knowledge obtained from literature, might be involved in BRCA1-pathway.

  11. Chitosan prevents adhesion during rabbit flexor tendon repair via the sirtuin 1 signaling pathway.

    PubMed

    Chen, Qiang; Lu, Hui; Yang, Hu

    2015-09-01

    Chitosan has been demonstrated to exert potent anti-adhesive activity during tendon repair; however, the underlying molecular mechanisms remain unclear. The present study aimed to investigate the preventive effects of chitosan on adhesion in rabbit tendon repair, and to investigate the role of the sirtuin (SIRT)1 signaling pathway in this process. A total of 30 rabbits were divided randomly into three equal groups: Group 1, saline treatment; group 2, chitosan treatment; and group 3, chitosan + nicotinamide treatment. The flexor tendon of each of the rabbits was injured, and subsequently each rabbit was injected with the one of the reagents. Six weeks post‑surgery, all of the rabbits were sacrificed and their flexor tendons were harvested for subsequent evaluation of adhesion. Western blotting was used to determine the protein expression levels of specific signaling molecules. An MTT assay was conducted to evaluate the viability of human tenocytes and flow cytometry was used to analyze the apoptotic rate of the cells. The present study demonstrated that treatment with chitosan relieved adhesion in the rabbits with flexor tendon injuries. In addition, chitosan treatment increased SIRT1 expression, and reduced acetylated p65 and p53 expression in the tendons. The effects of chitosan on the tendons were attenuated by treatment with nicotinamide (a SIRT1 inhibitor). In the human tenocytes, pretreatment with chitosan resulted in an inhibition of interleukin (IL)‑1β‑induced apoptosis. Furthermore, chitosan reversed the IL‑1β‑induced downregulation of SIRT1 and upregulation of acetylated p65 and p53. Furthermore, downregulation of Sirt1 by RNA interference abrogated the effects of chitosan on the levels of p65 and p53 acetylation, and the rate of tenocyte apoptosis. In conclusion, chitosan treatment prevented adhesion via the SIRT1 signaling pathway during rabbit flexor tendon repair. These results indicate that SIRT1 may be targeted for therapeutic

  12. Caenorhabditis elegans POLQ-1 and HEL-308 function in two distinct DNA interstrand cross-link repair pathways.

    PubMed

    Muzzini, Diego M; Plevani, Paolo; Boulton, Simon J; Cassata, Giuseppe; Marini, Federica

    2008-06-01

    DNA interstrand cross-links (ICLs) are highly cytotoxic DNA lesions hindering DNA replication and transcription. Whereas in bacteria and yeast the molecular mechanisms involved in ICL repair are genetically well dissected, the scenario in multicellular organisms remains unclear. Here, we report that the two new mus308 genes, polq-1 and hel-308 are involved in ICL repair in Caenorhabditis elegans. After treatment with ICL agents, a decrease in survival and an increase in checkpoint-induced cell-cycle arrest and apoptosis of germ cells is observed in mutants of both genes. Although sensitive to ICL agents and to a minor extent to IR, cytological and epistatic analyses suggest that polq-1 and hel-308 are involved in different DNA repair pathways. While hel-308 functions in a Fanconi anemia-dependent pathway, polq-1 has a role in a novel distinct and brc-1 (CeBRCA1)-dependent ICL repair process in metazoans.

  13. Heteroduplex Chain Polarity in Recombination of Phage λ by the Red, Recbcd, Recbc(d(-)) and Recf Pathways

    PubMed Central

    Siddiqi, I.; Stahl, M. M.; Stahl, F. W.

    1991-01-01

    We have examined the chain polarity of heteroduplex DNA in unreplicated, bacteriophage λ splice recombinants when recombination was by the RecBCD, RecBC(D(-)), or RecF pathway of Escherichia coli or the Red pathway of λ. For each of these pathways, recombination is activated by the cutting of cos that accompanies chromosome packaging, and is effected by recombination enzymes acting at the right end created by that cutting. For exchanges occurring near cos, one parent makes a lesser physical and genetic contribution than does the other. For each pathway, when the phage carried standard cos, this minority contribution was predominantly on the r chain, ending 5' at the right end of λ. When standard cos was replaced by a cloned inverted cos located centrally on the standard λ genetic map, minority contribution was predominantly on the l chain. In each case, the polarity of the overlap was usually that formed by 3' overhangs of parental information and material. These results are discussed in the context of current models of recombination for the different pathways. PMID:1829428

  14. Cumulative defects in DNA repair pathways drive the PARP inhibitor response in high-grade serous epithelial ovarian cancer cell lines

    PubMed Central

    Fleury, Hubert; Carmona, Euridice; Morin, Vincent G.; Meunier, Liliane; Masson, Jean-Yves; Tonin, Patricia N.; Provencher, Diane; Mes-Masson, Anne-Marie

    2017-01-01

    PARP inhibitors (PARPi), such as Olaparib, have shown promising results in high-grade serous (HGS) epithelial ovarian cancer (EOC) treatment. PARPi sensitivity has been mainly associated with homologous recombination (HR) deficiency, but clinical trials have shown that predicting actual patient response is complex. Here, we investigated gene expression microarray, HR functionality and Olaparib sensitivity of 18 different HGS EOC cell lines and demonstrate that PARPi sensitivity is not only associated with HR defects. Gene target validation show that down regulation of genes in the nucleotide excision repair (NER) and mismatch repair (MMR) pathways (ERCC8 and MLH1, respectively) increases PARPi response. The highest sensitivity was observed when genes in both the HR and either NER or MMR pathways were concomitantly down regulated. Using clinical samples, patients with these concurrent down regulations could be identified. Based on these results, a novel model to predict PARPi sensitivity is herein proposed. This model implies that the extreme responders identified in clinical trials have deficiencies in HR and either NER or MMR. PMID:27374179

  15. Cloning and sequencing of the PIF gene involved in repair and recombination of yeast mitochondrial DNA.

    PubMed Central

    Foury, F; Lahaye, A

    1987-01-01

    The nuclear gene PIF of Saccharomyces cerevisiae is required for both repair of mitochondrial DNA (mtDNA) and recognition of a recombinogenic signal characterized by a 26-bp palindromic AT sequence in the ery region of mtDNA. This gene has been cloned in yeast by genetic complementation of pif mutants. Its chromosomal disruption does not destroy the genetic function of mitochondria. The nucleotide sequence of the 3.5-kb insert from a complementing plasmid reveals an open reading frame encoding a potential protein of 857 amino acids and Mr = 97,500. An ATP-binding domain is present in the central part of the gene and in the carboxy-terminal region a putative DNA-binding site is present. Its alpha helix-turn-alpha helix motif is found in DNA-binding proteins such as lambda and lactose repressors which recognize symmetric sequences. Significant amino acid homology is observed with yeast RAD3 and E. coli UvrD (helicase II) proteins which are required for excision repair of damaged DNA. Images Fig. 1. Fig. 2. PMID:3038524

  16. Genetic controls of meiotic recombination and somatic DNA metabolism in Drosophila melanogaster.

    PubMed Central

    Baker, B S; Boyd, J B; Carpenter, A T; Green, M M; Nguyen, T D; Ripoll, P; Smith, P D

    1976-01-01

    Recombination-defective meiotic mutants and mutagen-sensitive mutants of D. melanogaster have been examined for their effects on meiotic chromosome behavior, sensitivity to killing by mutagens, somatic chromosome integrity, and DNA repair processes. Several loci have been identified that specify functions that are necessary for both meiotic recombination and DNA repair processes, whereas mutants at combination and DNA repair processes, whereas mutants at other loci appear to be defective in only one pathway of DNA processing. PMID:825857

  17. Lambda red mediated gap repair utilizes a novel replicative intermediate in Escherichia coli.

    PubMed

    Reddy, Thimma R; Fevat, Léna M S; Munson, Sarah E; Stewart, A Francis; Cowley, Shaun M

    2015-01-01

    The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction

  18. Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering.

    PubMed

    Zhai, Yafei; Han, Donglei; Pan, Ying; Wang, Shuaishuai; Fang, Junqiang; Wang, Peng; Liu, Xian-wei

    2015-02-01

    Guanosine 5'-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5'-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue.

  19. Inhibition of homologous recombination repair with Pentoxifylline targets G2 cells generated by radiotherapy and induces major enhancements of the toxicity of cisplatin and melphalan given after irradiation

    PubMed Central

    Bohm, Lothar

    2006-01-01

    The presentation reviews the modus operandi of the dose modifying drug Pentoxifylline and the dose enhancement factors which can be achieved in different cell types. Preclinical and clinical data show that Pentoxifylline improves the oxygenation of hypoxic tumours and enhances tumour control by irradiation. In vitro experiments demonstrate that Pentoxifylline also operates when oxygen is not limiting and produces dose modifying factors in the region of 1.2 – 2.0. This oxygen independent effect is poorly understood. In p53 mutant cells irradiation induces a G2 block which is abrogated by Pentoxifylline. The enhancement of cell kill observed when Pentoxifylline and irradiation are given together could arise from rapid entry of damaged tumour cells into mitosis and propagation of DNA lesions as the result of curtailment of repair time. Recovery ratios and repair experiments using CFGE after high dose irradiation demonstrate that Pentoxifylline inhibits repair directly and that curtailment of repair time is not the explanation. Use of the repair defective xrs1 and the parental repair competent CHO-K1 cell line shows that Pentoxifylline inhibits homologous recombination repair which operates predominantly in the G2 phase of the cell cycle. When irradiated cells residing in G2 phase are exposed to very low doses of cisplatin at a toxic dose of 5 %. (TC: 0.05) massive toxicity enhancements up to a factor of 80 are observed in melanoma, squamous carcinoma and prostate tumour cell lines. Enhancements of radiotoxicity seen when Pentoxifylline and radiation are applied together are small and do not exceed a factor of 2.0. The capacity of Pentoxifyline to inhibit homologous recombination repair has not as yet been clinically utilized. A suitable application could be in the treatment of cervical carcinoma where irradiation and cisplatin are standard modality. In vitro data also strongly suggest that regimes where irradiation is used in combination with alkylating drugs may

  20. Inhibition of homologous recombination repair with Pentoxifylline targets G2 cells generated by radiotherapy and induces major enhancements of the toxicity of cisplatin and melphalan given after irradiation.

    PubMed

    Bohm, Lothar

    2006-05-03

    The presentation reviews the modus operandi of the dose modifying drug Pentoxifylline and the dose enhancement factors which can be achieved in different cell types. Preclinical and clinical data show that Pentoxifylline improves the oxygenation of hypoxic tumours and enhances tumour control by irradiation. In vitro experiments demonstrate that Pentoxifylline also operates when oxygen is not limiting and produces dose modifying factors in the region of 1.2-2.0. This oxygen independent effect is poorly understood. In p53 mutant cells irradiation induces a G2 block which is abrogated by Pentoxifylline. The enhancement of cell kill observed when Pentoxifylline and irradiation are given together could arise from rapid entry of damaged tumour cells into mitosis and propagation of DNA lesions as the result of curtailment of repair time. Recovery ratios and repair experiments using CFGE after high dose irradiation demonstrate that Pentoxifylline inhibits repair directly and that curtailment of repair time is not the explanation. Use of the repair defective xrs1 and the parental repair competent CHO-K1 cell line shows that Pentoxifylline inhibits homologous recombination repair which operates predominantly in the G2 phase of the cell cycle. When irradiated cells residing in G2 phase are exposed to very low doses of cisplatin at a toxic dose of 5%. (TC: 0.05) massive toxicity enhancements up to a factor of 80 are observed in melanoma, squamous carcinoma and prostate tumour cell lines. Enhancements of radiotoxicity seen when Pentoxifylline and radiation are applied together are small and do not exceed a factor of 2.0. The capacity of Pentoxifyline to inhibit homologous recombination repair has not as yet been clinically utilized. A suitable application could be in the treatment of cervical carcinoma where irradiation and cisplatin are standard modality. In vitro data also strongly suggest that regimes where irradiation is used in combination with alkylating drugs may also

  1. A recombinant DNA method for understanding the mechanism of Salmonella reverse mutations: Role of repair

    SciTech Connect

    Felton, J.S.; Wu, R.; Shen, N.H.; Healy, S.K.; Fuscoe, J.C.

    1988-10-01

    This is a report of an investigation of the specific changes in the DNA of Salmonella revertants induced by environmental mutagens and their metabolites. These compounds were chosen because they specifically induce frameshift lesions in strains TA1538 and TA98. DNA lesions in these strains are analyzed in the hisD gene and can lead to reversion of histidine dependence by either deletions, insertions, or suppressor mutations. The understanding of the types of DNA-base changes induced by both a mutagen and its metabolites can lead to a better understanding of not only the mutational process and repair mechanisms, but also the potency and mode of action of specific metabolites and their corresponding DNA adduct(s). The detailed developed of the methods used for this research and the original findings with benzo(a)pyrene have been recently published. 4 refs., 2 figs.

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

    PubMed Central

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

    2014-01-01

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

  3. UV stimulation of chromosomal marker exchange in Sulfolobus acidocaldarius: implications for DNA repair, conjugation and homologous recombination at extremely high temperatures.

    PubMed Central

    Schmidt, K J; Beck, K E; Grogan, D W

    1999-01-01

    The hyperthermophilic archaeon Sulfolobus acidocaldarius exchanges and recombines chromosomal markers by a conjugational mechanism, and the overall yield of recombinants is greatly increased by previous exposure to UV light. This stimulation was studied in an effort to clarify its mechanism and that of marker exchange itself. A variety of experiments failed to identify a significant effect of UV irradiation on the frequency of cell pairing, indicating that subsequent steps are primarily affected, i.e., transfer of DNA between cells or homologous recombination. The UV-induced stimulation decayed rather quickly in parental cells during preincubation at 75 degrees, and the rate of decay depended on the incubation temperature. Preincubation at 75 degrees decreased the yield of recombinants neither from unirradiated parental cells nor from parental suspensions subsequently irradiated. We interpret these results as evidence that marker exchange is stimulated by recombinogenic DNA lesions formed as intermediates in the process of repairing UV photoproducts in the S. acidocaldarius chromosome. PMID:10430571

  4. 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

    PubMed Central

    Domagala, Pawel; Jakubowska, Anna; Jaworska-Bieniek, Katarzyna; Kaczmarek, Katarzyna; Durda, Katarzyna; Kurlapska, Agnieszka; Cybulski, Cezary; Lubinski, Jan

    2015-01-01

    Purpose 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. Methods 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. Results 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. Conclusions 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. PMID:26083025

  5. 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.

    PubMed

    Domagala, Pawel; Jakubowska, Anna; Jaworska-Bieniek, Katarzyna; Kaczmarek, Katarzyna; Durda, Katarzyna; Kurlapska, Agnieszka; Cybulski, Cezary; Lubinski, Jan

    2015-01-01

    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.

  6. Functions of Human Rad51 and Other Recombination Factors in DNA Double-Strand Break Repair

    DTIC Science & Technology

    2002-06-01

    cells express five Rad5l-like proteins - XRCC2, XRCC3 , Rad51B, Rad51C and Rad51D. These Rad51 paralogs are important for homologous recombination and...complex with 42-kD Rad5 IC species was detected. The observed sizes XRCC3 (Schild et al. 2000; Kurumizaka et al. 2001; Mas- A kIa a-Rad51B kDa a-RadSIC...RadS1C XRCC3 or that the XRCC3 -Rad51C complex was not A x-Rad5l B retained on the Q column. 07a 0- The results above indicated that Rad5lB and Rad5lC

  7. Gene and pathway level analyses of germline DNA-repair gene variants and prostate cancer susceptibility using the iCOGS-genotyping array.

    PubMed

    Saunders, Edward J; Dadaev, Tokhir; Leongamornlert, Daniel A; Al Olama, Ali Amin; Benlloch, Sara; Giles, Graham G; Wiklund, Fredrik; Gronberg, Henrik; Haiman, Christopher A; Schleutker, Johanna; Nordestgaard, Borge G; Travis, Ruth C; Neal, David; Pasayan, Nora; Khaw, Kay-Tee; Stanford, Janet L; Blot, William J; Thibodeau, Stephen N; Maier, Christiane; Kibel, Adam S; Cybulski, Cezary; Cannon-Albright, Lisa; Brenner, Hermann; Park, Jong Y; Kaneva, Radka; Batra, Jyotsna; Teixeira, Manuel R; Pandha, Hardev; Govindasami, Koveela; Muir, Ken; Easton, Douglas F; Eeles, Rosalind A; Kote-Jarai, Zsofia

    2016-04-12

    Germline mutations within DNA-repair genes are implicated in susceptibility to multiple forms of cancer. For prostate cancer (PrCa), rare mutations in BRCA2 and BRCA1 give rise to moderately elevated risk, whereas two of B100 common, low-penetrance PrCa susceptibility variants identified so far by genome-wide association studies implicate RAD51B and RAD23B. Genotype data from the iCOGS array were imputed to the 1000 genomes phase 3 reference panel for 21 780 PrCa cases and 21 727 controls from the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (PRACTICAL) consortium. We subsequently performed single variant, gene and pathway-level analyses using 81 303 SNPs within 20 Kb of a panel of 179 DNA-repair genes. Single SNP analyses identified only the previously reported association with RAD51B. Gene-level analyses using the SKAT-C test from the SNP-set (Sequence) Kernel Association Test (SKAT) identified a significant association with PrCa for MSH5. Pathway-level analyses suggested a possible role for the translesion synthesis pathway in PrCa risk and Homologous recombination/Fanconi Anaemia pathway for PrCa aggressiveness, even though after adjustment for multiple testing these did not remain significant. MSH5 is a novel candidate gene warranting additional follow-up as a prospective PrCa-risk locus. MSH5 has previously been reported as a pleiotropic susceptibility locus for lung, colorectal and serous ovarian cancers.

  8. Gene and pathway level analyses of germline DNA-repair gene variants and prostate cancer susceptibility using the iCOGS-genotyping array

    PubMed Central

    Saunders, Edward J; Dadaev, Tokhir; Leongamornlert, Daniel A; Olama, Ali Amin Al; Benlloch, Sara; Giles, Graham G; Wiklund, Fredrik; Grönberg, Henrik; Haiman, Christopher A; Schleutker, Johanna; Nordestgaard, Børge G; Travis, Ruth C; Neal, David; Pasayan, Nora; Khaw, Kay-Tee; Stanford, Janet L; Blot, William J; Thibodeau, Stephen N; Maier, Christiane; Kibel, Adam S; Cybulski, Cezary; Cannon-Albright, Lisa; Brenner, Hermann; Park, Jong Y; Kaneva, Radka; Batra, Jyotsna; Teixeira, Manuel R; Pandha, Hardev; Govindasami, Koveela; Muir, Ken; Abbasi, Z; Abdul-Hamid, M Akhlil; Abel, Paul D; Abrams, Paul H; Adab, Fawzi A; Adamson, Andrew; Adeyoju, A; Afzal, Naveed; Ahiaku, Ernest K N; Ahmed, Munir; Al Sudani, Mohammed L; Alcock, Christopher; Ali, Zulfiqar; Almond, David J; Alonzi, Roberto; Al-Samarraie, Amir S M; Al-Samerraie; Al-Singary, Waleed; Al-Sudani; Anderson, John; Andrews, Steven; Andrews, Henry; Anjum, Iqbal; Anson, Ken; Anyamene, Nicola A; Apakama, Ike; Aparcia, F; Archbold, J A A; Ash, D; Ashford, Richard F U; Azzabi, A; Badenoch, David; Bahl, Amit; Bailey, M J; Bailey, Karen; Ball, Andrew J; Banerjee, G; Barber, N; Barber, Jim; Baria; Barnes, Douglas G; Bashir, J; Basu, Pradip; Bates, Christopher A; Bax, N A; Baxter-Smith, D; Bdesha, Amar; Beacock, Christopher J M; Beaney, Ronald P; Beard, Ralph; Beatty, John D; Beck, Rupert; Beese, Gail; Beesley, Sharon; Bell, C Richard W; Bellringer, James; Benson, Richard; Beresford; Bevis, Christopher R A; Bhana, Rajanee; Bhanot, S; Bhatnagar, A; Bhatt, R I; Birch, Brian; Birtle, Alison; Bishop, M; Biyani, C Shekhar; Blacklock, A R E; Blades, Rosemary; Bliss, Peter; Bloomfield, David J; Boddy, S; Booth, C M; Bose, Pradeep; Bott, Michael C; Bottomley, David; Boucher, Nigel R; Bowen, J; Bower, Mark; Bowsher, W G; Boyd, P J R; Bramble, F James; Brewster, Simon F; Briggs, Tim; Brock, Cathryn; Brock, Sue; Bromage, Stephen; Brough, Richard; Brown, Richard; Brown, Stephen; Brown, Richard; Browning, Tony J; Bryan, N; Burgess, Neil A; Burns-Cox, Nicholas; Butterworth, Paul C; Cahill, D; Callaghan, P S; Calleary, John; Calleja, M; Calman, Frances; Camilleri, Philip; Campbell, Alister; Cannon, Andrea; Carnell, Dawn M; Carr, T W; Carter, Simon; Carter, Charles J M; Carter, Adam C; Castle, Bruce M; Chadwick, David; Chahal, Rohit; Chakraborti, P; Chappell; Charig, C; Chetiyawardana, Anula D; Chilton, Christopher; Chinegwundoh, F I; Chong, Irene; Choudhury, Ananya; Chow, Wai-Man; Christmas, Timothy J; Churn, Mark J; Clarke, Noel W; Clavijo-Eisele, Jorge; Coe, M; Cohen, N P; Coker, C; Cole, Trevor; Cole, David J; Cole, O; Collins, Gerald; Collinson, Matthew; Conn, I; Connell, C; Cook, Audrey; Cooke, Peter; Cooksey, Graeme; Coombs, L; Copland, Robert F; Cornaby, Andrew J; Cornford, P A; Corolis; Corr, John; Costello, C B; Coull, N; Cowan, Richard; Cox, Robert; Coyle, C; Crew, Jeremy; Crisp, John C; Cross, W; Cross, W; Cruger, Dorthe; Crundwell, Malcolm; Cummings; Dahar, Nazeer; Daniel, Francis N; Darrad, J; Daruwala, Pallon; Das, Gautam; Datta, Shibendra; Davidson, S; Davies, Joseph; Davison, Owen W; Dawkins, Guy; Dawson, Chris; Bolla, Alan R De; Dearnaley, David; Desai, Ken M; Deutsch, George P; Dick, John; Dickinson, Andrew J; Dickson, Jeanette; Dinneen, Michael; Dixit, Sanjay; Dobbs, H Jane; Doble, A; Dodds, David; Doherty, Alan; Donaldson, P; Dooldeniya, M; Douglas, S Fiona; Drake; Duchesne, Gill M; Duffy, Peter; Dunn, Michael; Dunsmuir, W D; Durrani, Sajid K; Eaton, Alan C; Eccles, Diane; Eddy, B; Eden, C D; Edwards, J; Elkabir, Jeremy; Elliott, P Tony; Ellis, B W; Ellis, R; El-Modir, A; Elves, Andrew W S; Elwell, Christine; Emberton, Mark; Emmerson, Louise; England, Roland C D; Errington, R D; Evans, D Gareth; Falconer, Alison; Fawcett, Derek; Featherston, C; Featherstone, Carolyn J; Feggetter, Jeremy; Ferguson, C; Fermont, D; Ferro, Michael; Fletcher, Matthew; Folkes, A; Ford, Trevor F; Foster, Paul W; Franks, Kevin N; Frim, Olivera; Gale, Joanna; Gallegos, Christopher; Gelister, James S; Ghana; Gibbs, Stephanie; Gilbert, Hugh; Gillatt, David; Glaholm, John; Glass, Jonathan M; Glenister, James; Goode, Thomas D; Gordon, E M; Gower, Richard L; Graham, John; Green, Damian; Greenland, Jonathan; Grieve, Robert; Griffiths, Thomas R L; Gujral, Sandy; Gupta, Nishi; Gurun, Riza Murat; Guy, Peter J; Haldar, Neil; Halder, N; Hamdy, F C; Hamilton, C; Hammonds, John; Hampson, S J; Hanbury, Damien C; Hardman, P D John; Harland, Stephen J; Harney, John M; Harper, Peter; Harris, Sarah; Harris, D; Harrison, G S M; Harriss, D R; Harvey-Hills, N; Hawkyard, Simon; Heath, Catherine M; Hehir, Michael; Hellawell, Giles O; Hendry, David; Henley, Mike; Henry, Ann; Hetherington, John; Hickish, Tamas; Hicks, James A; Hilman, Serena; Hindley, Richard; Hindmarsh, John R; Hines, John; Hingorani, M; Ho, Edwin T S; Hodgson, Shirley; Hoffman, U; Holden, David; Hollingdale, A; Hollins, Graham W; Holmes, Simon A V; Horan, Gail; Horwich, Alan; Hoskin, Peter; Howell, Graham P; Hrouda, D; Huddart, Robert; Hudson, Liz; Hughes, Rob; Hughes, Michael; Hughes, Owen; Humber, Caroline; Iacovou, John W; Ibrahim, A; Inglis, John A; Irving, Stuart; Irwin, C; Izatt, Louise; Izegbu, Victor; Jameel, Basharat; James, Michael J; James, N; James, R Lester; Javle, Pradip; Jenkins, P; Jhavar, Sameer; Jones, Gareth; Jones, Chris R; Jones, David A; Joseph, J; Joss, Shelagh; Kaisary, Amir; Kaliski, Alexandre L; Kapur, G; Karim, O; Karp, Stephen J; Keeley, F X; Kelkar, Anand R; Kelleher, J P; Kelly, John; Kenwrick, Sue; Khan, F; Khoo, Vincent; Kimber, Rachel M; Kinder, R; Kirby, Roger S; Kirk, David; Kirkbride, Peter; Kirollos, Magdi M; Kockelbergh, Roger; Koenig, Philip C W C; Kooiman, Gordon G; Koreich, O; Koupparis, Anthony; Kourah, Mohamed; Kraus, Sigurd; Kujawa, Magda L; Kulkarni, Ravi; Kumar, M; Kunkler, Ian H; Kynaston, H; Lachlan, Katherine L; Laing, Robert; Lalloo, Fiona; Lancashire, M; Langley, Stephen E M; Laniado, Marc; Larner, T R; Lau, Maurice W; Lawrence, W T; Lawson, Anne; Le Roux, Pieter J; Leader, Mary; Lee, J O; Lee, L; Lee, A; Lemburger, R John; Leone, Priscilla; Lester, Jason; Leung, Hing; Lewis, J; Lewis, D Christopher; Liston, Thomas; Livsey, Jacqueline; Lloyd, S; Locke, Imogen; Lodge, Richard; Logue, John; Longmuir, Mark; Lucas, Malcolm G; Luscombe, C J; Lydon, Anna; Lynch, Michael; Lynn, Naing N K; MacDermott, James P A; Macdonagh, Ruaraidh P; Macdonald; Madaan, Sanjeev; Madhava, Kudingila R; Maguire, Joseph; Maher, E R; Mahmood, Rana; Mair, Graeme H M; Malone, Peter R; Mangar, Stephen A; Mantle, Mark; Mark, I; Mason, Robert; Mason, M D; Matanhelia; Matenhelia, Shyam; Matthews, Philip N; McAleese, J; McBride, Donna; McFarlane, Jonathan; McGrath; McIlhenny, Craig; McInerney, Paul; McIntosh, Gregor; McKinna, F; McLaren, Duncan; McLarty, Esther; McMenemin, Rhona; McNeill, Alan; McNicholas, T A; Meddings, Robert N; Mee, A David; Melcher, Lucinda; Memon; Menzes, Pravin; Miller, Marek; Mills, Robert; Mitchell, S; Mithal, Natasha; Mitra, Anita; Mobb, Gillian E; Moffat, Leslie E F; Mokete; Money-Kyrle, Julian; Montgomery, Bruce; Moody, Martin P; Morley, Roland; Morris, Sean B; Morrison, Patrick; Mort, Diana; Mostafid, Amir H; Motiwala, Hanif; Mufti, Gulzar; Muir, Gordon; Mumtaz, Faiz; Murphy, Michael; Murray, Keith W; Murray, Alexandra; Murrell, Shirley; Muthukumar, D; Naerger, Harry; Namasivayam, Siva; Nargund, Vinod; Nawrocki; Neilson, Donald; Nethersell, A; Barwell, Julian; Newby, Jacqueline C; Newman, Hugh; Newton, R; Oakley, Neil; O'Boyle, P J; O'Brien, J; O'Brien, Tim S; O'Donnell, H; O'Donoghue, Neil; O'Donoghue, E; Ogden, Chris; Ohja, Hemant; Oliver, Tim; Ong, Eng K; O'Reilly, P; O'Rourke, J S; Osborn, David; Ostler, Peter; O'Sullivan, Joe; Owen, J; Palfrey, Edward; Panades, Miguel; Panakis, Niki; Pancharatnam, M; Pantelides, Michalakis L; Panwar, U; Parikh, Omi; Parker, Chris; Parker, Christopher H; Parys, Bohdan T; Pascoe, Sarah; Patel, Anup; Paterson, Joan; Pathack, S; Pati, Jhumur; Patterson, Helen; Pattu; Paul, A; Payne, Heather; Peake, David; Pedley, I; Pengelly, A; Peracha, Amjad M; Perry, Matthew; Persad, Raj; Peters, John; Philp, N H; Philp, T; Pickering, Lisa M; Pigott, Katharine; Plail, R; Plowman, P Nicholas; Pocock, Richard D; Pope, A J; Popert, Rick; Porter, Tim; Potter, John M; Powell, Christopher; Powles, Thomas B; Prasad, Krishna; Prasad, Seshadri Sri; Prejbisz, J W; Prescott, Stephen; Protheroe, Andrew; Qureshi, Khaver N; Raby, Nigel; Ragavan, Narasimhan; Raju, Palaniappa G S; Ramachandra, Prakash B; Raman, R; Rane, Abhay; Rankin, Julia; Rao, Y; Ratan, Hari L; Ravi, Ramachandran; Ravishankar, K; Read; Reddy, Paul J; Rimington, Peter R; Ritchie, Peter A; Roberts, J Trevor; Robertson, Andrew; Robinson, Angus; Robinson, Anne C; Robinson, Lee Q; Rochester, Mark A; Rogers, P B; Rosenbaum, Tomas P; Rothwell, Neil; Rowbotham, Carl; Rowe; Rowley, Kathryn; Ruddy, Deborah; Rundle, John; Russell, John M; Ryan, P G; Sabharwal, A; Saggar, Anand K; Samanci, Ali; Sangar, Vijay K; Saxby, M F; Schwaibold, Hartwig; Scoble, John E; Scrase, Christopher; Selim; Sells, Henry; Sethia, Krishna K; Shackley, David C; Shaffer; Shah, Nihil; Shakespeare, D; Shanley, Sue; Sharma, Neerah K; Sheehan, Denise J; Sherwin, Elizabeth; Shum, Poh Lin; LucySide; Sidek, Norma; Sikora, Karol; Simcock, R; Sinclair, Andrew M; Singh, Pravin; Siva, M; Smith, Michael F; Smith, James; Sokal, Michael; Sole, Graham M; Speakman, Mark J; Spiers, Alexander; Sreenivasan, Thiagarajan; Srihari, Narayanan N; Srinivasan; Sriram, Rajagopalan; Staffurth, John N; Stewart, D; Stockdale, Andrew; Stott, Mark A; Stower, M J; Strachan, John R; Stuart, Nicholas S A; Sugden, Elaine; Summerton, Duncan; Sundar, Santhanam; Sundaram, S K; Suresh, Gokarakonda; Susnerwala, Shabbir; Swami, Kuchibhotla S; Symons, Stephanie J; Syndikus, Isabel; Tahir, Saad; Tanquay, J; Taylor, John W; Taylor, J W; Terry, T; Thomas, Robert J; Thomas, Stephen A; Thompson, Alan; Thomson, Alastair H; Thurston, A; Tilsley, Owen; Tindall, Stuart F; Tipples, K; Tong; Toussi, Hamid; Toy, Elizabeth W; Trembath, Richard C; Tulloch, David N; Turner, Kevin J; Tweedle, James; Tyrell, C J; Umez-Eronini, N; Urwin, Graeme H; Vale, Justin A; Van As; Van As, Nicholas; Vasanthan, Subramaniam; Vesey, Sean; Vilarino-Varela, Maria; Violet, John; Virdi, Jaspal; Wade, Robert; Waite, Katherine; Walker, E M; Walker, Roger; Wallace, David M A; Watkin, Nicholas A; Watson, M E; Waxman, J H; Waymont, Brian; Weaver, Andrew; Webb, Ralph J; Wedderburn, Andrew; Wells, Paula; Wemyss-Holden, G D; Weston, P M T; Wheatley, Duncan; Whelan, P; Whillis, D; Wilde, Adam D; Wiles, Vicki; Wilkins, Marie; Williams, John H; Williams, Simon; Willis, Michael; Wills, Michael I; Wilson, Richard; Wilson, J R; Winkler, Mathias H; Wise, Marcus; Woodhams, Simon; Woodhouse, C; Woodward, Cathryn; Woolf; Woolfenden, K A; Worlding, Jane; Wright, Mark; WYLIE; Wylie, James P; Wynne, Chris; Zang, Angelika; Zarkar, A; Cox, Angela; Brown, Paul M.; George, Anne; Marsden, Gemma; Lane, Athene; Davis, Michael; Bollina, Prasad; Bonnington, Sue; Bradshaw, Lynne; Catto, James; Cooper, Debbie; Down, Liz; Doble, Andrew; Doherty, Alan; Durkan, Garrett; Elliott, Emma; Gillatt, David; Herbert, Pippa; Holding, Peter; Howson, Joanne; Jones, Mandy; Kockelbergh, Roger; Kumar, Rajeev; Kynaston, Howard; Lane, Athene; Lennon, Teresa; Lyons, Norma; Leung, Hing; Mason, Malcolm; Moody, Hilary; Powell, Philip; Paul, Alan; Prescott, Stephen; Rosario, Derek; OSullivan, Patricia; Thompson, Pauline; Tidball, Sarah; Cook, Margaret; Morgan, Angela; Lophatananon, Artitaya; Fisher, Cyril; Tymrakiewicz, Malgorzata; Guy, Michelle; Wilkinson, Rosemary; Jugurnauth-Little, Sara; Hazel, Steve; Southey, Melissa C; Fitzgerald, Liesel M; Pedersen, John; Hopper, John; Karlsson, Ami; Cavalli-Bjoerkman, Carin; Johansson, Jan-Erik; Adolfson, Jan; Aly, Markus; Broms, Michael; Stattin, Paer; Henderson, Brian E; Schumacher, Fredrick; Auvinen, Anssi; Taari, Kimmo; Maeaettaenen, Liisa; Kujala, Paula; Murtola, Teemu; Tammela, Teuvo LJ; Wahlfors, Tiina; Roder, Andreas; Iversen, Peter; Klarskov, Peter; Nielsen, Sune F; Key, Tim J; Wallinder, Hans; Gustafsson, Sven; Donovan, Jenny L; Hamdy, Freddie; Cox, Angela; George, Anne; Lane, Athene; Marsden, Gemma; Davis, Michael; Brown, Paul; Pharoah, Paul; Signorello, Lisa B; Zheng, Wei; McDonnell, Shannon K; Schaid, Daniel J; Wang, Liang; Tillmans, Lori; Riska, Shaun; Schnoeller, Thomas; Herkommer, Kathleen; Luedeke, Manuel; Vogel, Walther; Wokolorczyk, Dominika; Lubiski, Jan; Kluzniak, Wojciech; Butterbach, Katja; Stegmaier, Christa; Holleczek, Bernd; Lin, Hui-Yi; Park, Hyun; Pow-Sang, Julio; Sellers, Thomas; Slavov, Chavdar; Vlahova, Aleksandrina; Mitkova, Atanaska; Kachakova, Darina; Popov, Elenko; Christova, Svetlana; Dikov, Tihomir; Mitev, Vanio; Eckert, Allison; Spurdle, Amanda; Collins, Angus; Wood, Glenn; Malone, Greg; Clements, Judith A; Kerr, Kris; Turner, Megan; Saunders, Pamela; Heathcote, Peter; Srinivasan, Srilakshmi; Moya, Leire; Yeadon, Trina; Santos, Joana; Jerónimo, Carmen; Paulo, Paula; Pinto, Pedro; Henrique, Rui; Maia, Sofia; Michael, Agnieszka; Kierzek, Andrzej; Wu, Huihai; Easton, Douglas F; Eeles, Rosalind A; Kote-Jarai, Zsofia

    2016-01-01

    Background: Germline mutations within DNA-repair genes are implicated in susceptibility to multiple forms of cancer. For prostate cancer (PrCa), rare mutations in BRCA2 and BRCA1 give rise to moderately elevated risk, whereas two of ∼100 common, low-penetrance PrCa susceptibility variants identified so far by genome-wide association studies implicate RAD51B and RAD23B. Methods: Genotype data from the iCOGS array were imputed to the 1000 genomes phase 3 reference panel for 21 780 PrCa cases and 21 727 controls from the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (PRACTICAL) consortium. We subsequently performed single variant, gene and pathway-level analyses using 81 303 SNPs within 20 Kb of a panel of 179 DNA-repair genes. Results: Single SNP analyses identified only the previously reported association with RAD51B. Gene-level analyses using the SKAT-C test from the SNP-set (Sequence) Kernel Association Test (SKAT) identified a significant association with PrCa for MSH5. Pathway-level analyses suggested a possible role for the translesion synthesis pathway in PrCa risk and Homologous recombination/Fanconi Anaemia pathway for PrCa aggressiveness, even though after adjustment for multiple testing these did not remain significant. Conclusions: MSH5 is a novel candidate gene warranting additional follow-up as a prospective PrCa-risk locus. MSH5 has previously been reported as a pleiotropic susceptibility locus for lung, colorectal and serous ovarian cancers. PMID:26964030

  9. Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-L-fucose production in recombinant Escherichia coli.

    PubMed

    Lee, Won-Heong; Shin, So-Yeon; Kim, Myoung-Dong; Han, Nam Soo; Seo, Jin-Ho

    2012-03-01

    Guanosine 5'-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5'-diphosphate (GDP)-L-fucose. In this study, improvement of GDP-L-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-L-fucose. The effects of overexpression of inosine 5'-monophosphate (IMP) dehydrogenase, guanosine 5'-monophosphate (GMP) synthetase (GuaB and GuaA), GMP reductase (GuaC) and guanosine-inosine kinase (Gsk) on GDP-L-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk led to a significant improvement of GDP-L-fucose production. Maximum GDP-L-fucose concentration of 305.5 ± 5.3 mg l(-1) was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes. Such an enhancement of GDP-L-fucose production could be due to the increase in the intracellular level of GMP.

  10. The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair.

    PubMed

    Amundsen, S K; Taylor, A F; Smith, G R

    2000-06-20

    The RecBCD enzyme is required for homologous recombination and DNA repair in Escherichia coli. The structure and function of RecBCD enzyme is altered on its interaction with the recombination hotspot Chi (5'-GCTGGTGG-3'). It has been hypothesized that the RecD subunit plays a role in Chi-dependent regulation of enzyme activity [Thaler, D. S., Sampson, E., Siddiqi, I., Rosenberg, S. M., Stahl, F. W. & Stahl, M. (1988) in Mechanisms and Consequences of DNA Damage Processing, eds. Friedberg, E. & Hanawalt, P. (Liss, New York), pp. 413-422; Churchill, J. J., Anderson, D. G. & Kowalczykowski, S. C. (1999) Genes Dev. 13, 901-911]. We tested the hypothesis that the RecD subunit inhibits recombination by deleting recD from the nuclease- and recombination-deficient mutant recB(D1080A)CD. We report here that the resulting strain, recB(D1080A)C, was proficient for recombination and DNA repair. Recombination proficiency was accompanied by a change in enzyme activity: RecB(D1080A)C enzyme loaded RecA protein onto DNA during DNA unwinding whereas RecB(D1080A)CD enzyme did not. Together, these genetic and biochemical results demonstrate that RecA loading by RecBCD enzyme is required for recombination in E. coli cells and suggest that RecD interferes with the enzyme domain required for its loading. A nuclease-dependent signal appears to be required for a change in RecD that allows RecA loading. Because RecA loading is not observed with wild-type RecBCD enzyme until it acts at a Chi site, our observations support the view that RecD inhibits recombination until the enzyme acts at Chi.

  11. The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair

    PubMed Central

    Amundsen, Susan K.; Taylor, Andrew F.; Smith, Gerald R.

    2000-01-01

    The RecBCD enzyme is required for homologous recombination and DNA repair in Escherichia coli. The structure and function of RecBCD enzyme is altered on its interaction with the recombination hotspot Chi (5′-GCTGGTGG-3′). It has been hypothesized that the RecD subunit plays a role in Chi-dependent regulation of enzyme activity [Thaler, D. S., Sampson, E., Siddiqi, I., Rosenberg, S. M., Stahl, F. W. & Stahl, M. (1988) in Mechanisms and Consequences of DNA Damage Processing, eds. Friedberg, E. & Hanawalt, P. (Liss, New York), pp. 413–422; Churchill, J. J., Anderson, D. G. & Kowalczykowski, S. C. (1999) Genes Dev. 13, 901–911]. We tested the hypothesis that the RecD subunit inhibits recombination by deleting recD from the nuclease- and recombination-deficient mutant recBD1080ACD. We report here that the resulting strain, recBD1080AC, was proficient for recombination and DNA repair. Recombination proficiency was accompanied by a change in enzyme activity: RecBD1080AC enzyme loaded RecA protein onto DNA during DNA unwinding whereas RecBD1080ACD enzyme did not. Together, these genetic and biochemical results demonstrate that RecA loading by RecBCD enzyme is required for recombination in E. coli cells and suggest that RecD interferes with the enzyme domain required for its loading. A nuclease-dependent signal appears to be required for a change in RecD that allows RecA loading. Because RecA loading is not observed with wild-type RecBCD enzyme until it acts at a Chi site, our observations support the view that RecD inhibits recombination until the enzyme acts at Chi. PMID:10840065

  12. Nuclear Dynamics of Heterochromatin Repair.

    PubMed

    Amaral, Nuno; Ryu, Taehyun; Li, Xiao; Chiolo, Irene

    2017-02-01

    Repairing double-strand breaks (DSBs) is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination and chromosome rearrangements. Recent studies in Drosophila cells revealed that faithful homologous recombination (HR) repair of heterochromatic DSBs relies on the relocalization of DSBs to the nuclear periphery before Rad51 recruitment. We summarize here the exciting progress in understanding this pathway, including conserved responses in mammalian cells and surprising similarities with mechanisms in yeast that deal with DSBs in distinct sites that are difficult to repair, including other repeated sequences. We will also point out some of the most important open questions in the field and emerging evidence suggesting that deregulating these pathways might have dramatic consequences for human health.

  13. Regulation and disregulation of mammalian nucleotide excision repair: A pathway to nongermline breast carcinogenesis

    DOE PAGES

    Latimer, Jean J.; Majekwana, Vongai J.; Pabon-Padin, Yashira R.; ...

    2014-12-19

    Nucleotide excision repair (NER) is important as a modulator of disease, especially in constitutive deficiencies, such as the cancer predisposition syndrome Xeroderma pigmentosum. We have found profound variation of NER capacity among normal individuals, between cell-types and during carcinogenesis. NER is a repair system for many types of DNA damage, and therefore many types of genotoxic carcinogenic exposures, including ultraviolet light, products of organic combustion, metals, oxidative stress, etc. Since NER is intimately related to cellular metabolism, requiring components of both the DNA replicative and transcription machinery, it has a narrow range of functional viability. Thus, genes in the NERmore » pathway are expressed at the low levels manifested by, for example, nuclear transcription factors. Since NER activity and gene expression vary by cell-type, it is inherently epigenetically regulated. Furthermore, this epigenetic regulation is disregulated during sporadic breast carcinogenesis. Loss of NER is one basis of genomic instability, a required element in cellular transformation, and one that potentially modulates response to therapy. In this article, we demonstrate differences in NER capacity in eight adult mouse tissues, and place this result into the context of our previous work on mouse extraembryonic tissues, normal human tissues and sporadic early stage human breast cancer.« less

  14. Regulation and disregulation of mammalian nucleotide excision repair: A pathway to nongermline breast carcinogenesis

    SciTech Connect

    Latimer, Jean J.; Majekwana, Vongai J.; Pabon-Padin, Yashira R.; Pimpley, Manasi R.; Grant, Stephen G.

    2014-12-19

    Nucleotide excision repair (NER) is important as a modulator of disease, especially in constitutive deficiencies, such as the cancer predisposition syndrome Xeroderma pigmentosum. We have found profound variation of NER capacity among normal individuals, between cell-types and during carcinogenesis. NER is a repair system for many types of DNA damage, and therefore many types of genotoxic carcinogenic exposures, including ultraviolet light, products of organic combustion, metals, oxidative stress, etc. Since NER is intimately related to cellular metabolism, requiring components of both the DNA replicative and transcription machinery, it has a narrow range of functional viability. Thus, genes in the NER pathway are expressed at the low levels manifested by, for example, nuclear transcription factors. Since NER activity and gene expression vary by cell-type, it is inherently epigenetically regulated. Furthermore, this epigenetic regulation is disregulated during sporadic breast carcinogenesis. Loss of NER is one basis of genomic instability, a required element in cellular transformation, and one that potentially modulates response to therapy. In this article, we demonstrate differences in NER capacity in eight adult mouse tissues, and place this result into the context of our previous work on mouse extraembryonic tissues, normal human tissues and sporadic early stage human breast cancer.

  15. Meiotic Recombination: The Essence of Heredity.

    PubMed

    Hunter, Neil

    2015-10-28

    The study of homologous recombination has its historical roots in meiosis. In this context, recombination occurs as a programmed event that culminates in the formation of crossovers, which are essential for accurate chromosome segregation and create new combinations of parental alleles. Thus, meiotic recombination underlies both the independent assortment of parental chromosomes and genetic linkage. This review highlights the features of meiotic recombination that distinguish it from recombinational repair in somatic cells, and how the molecular processes of meiotic recombination are embedded and interdependent with the chromosome structures that characterize meiotic prophase. A more in-depth review presents our understanding of how crossover and noncrossover pathways of meiotic recombination are differentiated and regulated. The final section of this review summarizes the studies that have defined defective recombination as a leading cause of pregnancy loss and congenital disease in humans.

  16. LRF maintains genome integrity by regulating the non-homologous end joining pathway of DNA repair

    PubMed Central

    Liu, Xue-Song; Chandramouly, Gurushankar; Rass, Emilie; Guan, Yinghua; Wang, Guocan; Hobbs, Robin M.; Rajendran, Anbazhagan; Xie, Anyong; Shah, Jagesh V.; Davis, Anthony J.; Scully, Ralph; Lunardi, Andrea; Pandolfi, Pier Paolo

    2015-01-01

    Leukemia/lymphoma-related factor (LRF) is a POZ/BTB and Krüppel (POK) transcriptional repressor characterized by context-dependent key roles in cell fate decision and tumorigenesis. Here we demonstrate an unexpected transcription-independent function for LRF in the classical non-homologous end joining (cNHEJ) pathway of double-strand break (DSB) repair. We find that LRF loss in cell lines and mouse tissues results in defective cNHEJ, genomic instability and hypersensitivity to ionizing radiation. Mechanistically, we show that LRF binds and stabilizes DNA-PKcs on DSBs, in turn favouring DNA-PK activity. Importantly, LRF loss restores ionizing radiation sensitivity to p53 null cells, making LRF an attractive biomarker to direct p53-null LRF-deficient tumours towards therapeutic treatments based on genotoxic agents or PARP inhibitors following a synthetic lethal strategy. PMID:26446488

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

    SciTech Connect

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

    2011-08-29

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

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

    SciTech Connect

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

    2011-12-31

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

  19. Structure of the FANCI-FANCD2 complex: insights into the Fanconi anemia DNA repair pathway.

    PubMed

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

    2011-07-15

    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 ~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.

  20. LRF maintains genome integrity by regulating the non-homologous end joining pathway of DNA repair.

    PubMed

    Liu, Xue-Song; Chandramouly, Gurushankar; Rass, Emilie; Guan, Yinghua; Wang, Guocan; Hobbs, Robin M; Rajendran, Anbazhagan; Xie, Anyong; Shah, Jagesh V; Davis, Anthony J; Scully, Ralph; Lunardi, Andrea; Pandolfi, Pier Paolo

    2015-10-08

    Leukemia/lymphoma-related factor (LRF) is a POZ/BTB and Krüppel (POK) transcriptional repressor characterized by context-dependent key roles in cell fate decision and tumorigenesis. Here we demonstrate an unexpected transcription-independent function for LRF in the classical non-homologous end joining (cNHEJ) pathway of double-strand break (DSB) repair. We find that LRF loss in cell lines and mouse tissues results in defective cNHEJ, genomic instability and hypersensitivity to ionizing radiation. Mechanistically, we show that LRF binds and stabilizes DNA-PKcs on DSBs, in turn favouring DNA-PK activity. Importantly, LRF loss restores ionizing radiation sensitivity to p53 null cells, making LRF an attractive biomarker to direct p53-null LRF-deficient tumours towards therapeutic treatments based on genotoxic agents or PARP inhibitors following a synthetic lethal strategy.

  1. Selenium-Mediated Dehalogenation of Halogenated Nucleosides and its Relevance to the DNA Repair Pathway.

    PubMed

    Mondal, Santanu; Manna, Debasish; Mugesh, Govindasamy

    2015-08-03

    Halogenated nucleosides can be incorporated into the newly synthesized DNA of replicating cells and therefore are commonly used in the detection of proliferating cells in living tissues. Dehalogenation of these modified nucleosides is one of the key pathways involved in DNA repair mediated by the uracil-DNA glycosylase. Herein, we report the first example of a selenium-mediated dehalogenation of halogenated nucleosides. We also show that the mechanism for the debromination is remarkably different from that of deiodination and that the presence of a ribose or deoxyribose moiety in the nucleosides facilitates the deiodination. The results described herein should help in understanding the metabolism of halogenated nucleosides in DNA and RNA. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Spermatogenesis, DNA damage and DNA repair mechanisms in male infertility.

    PubMed

    Gunes, Sezgin; Al-Sadaan, Maha; Agarwal, Ashok

    2015-09-01

    Spermatogenesis is a complex process of proliferation and differentiation during male germ cell development involving mitosis, meiosis and spermiogenesis. Endogenous and exogenous physical, chemical and biological sources modify the genome of spermatozoa. The genomic integrity and stability of the sperm is protected by DNA repair mechanisms. In the male germline cells, DNA repair mechanisms include nucleotide excision repair, base excision repair, DNA mismatch repair, double strand break repair and post-replication repair. Defects in repair mechanisms cause arrest of spermatogenesis and abnormal recombination, ultimately resulting in male infertility. This review focuses on molecular mechanisms of the DNA repair pathways, DNA repair defects and male infertility. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  3. The study of the relation of DNA repair pathway genes SNPs and the sensitivity to radiotherapy and chemotherapy of NSCLC

    PubMed Central

    Wang, Chunbo; Nie, Huan; Li, Yiqun; Liu, Guiyou; Wang, Xu; Xing, Shijie; Zhang, Liping; Chen, Xin; Chen, Yue; Li, Yu

    2016-01-01

    To analyze the relation between SNPs in DNA repair pathway-related genes and sensitivity of tumor radio-chemotherapy, 26 SNPs in 20 DNA repair genes were genotyped on 176 patients of NSCLC undertaking radio-chemotherapy treatment. In squamous cell carcinoma (SCC), as the rs2228000, rs2228001 (XPC), rs2273953 (TP73), rs2279744 (MDM2), rs2299939 (PTEN) and rs8178085, rs12334811 (DNA-PKcs) affected the sensitivity to chemotherapy, so did the rs8178085, rs12334811 to radiotherapy. Moreover rs344781, rs2273953 and rs12334811 were related with the survival time of SCC. In general, the “good” genotype GG (rs12334811) showed greater efficacy of radio-chemotherapy and MSF (24 months) on SCC. In adenocarcinoma, as the rs2699887 (PIK3), rs12334811 (DNA-PKcs) influenced the sensitivity to chemotherapy, so did the rs2299939, rs2735343 (PTEN) to radiotherapy. And rs402710, rs80270, rs2279744 and rs2909430 impacted the survival time of the adenocarcinoma patients. Both GG (rs2279744) and AG (rs2909430) showed a shorter survival time (MFS = 6). Additionally, some SNPs such as rs2228000, rs2228001 and rs344781 were found to regulate the expression of DNA repair pathway genes through eQTLs dataset analysis. These results indicate that SNPs in DNA repair pathway genes might regulate the expression and affect the DNA damage repair, and thereby impact the efficacy of radio-chemotherapy and the survival time of NSCLC. PMID:27246533

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

    SciTech Connect

    Hadden, C.T.

    1981-01-01

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

  5. Production of Glucaric Acid from a Synthetic Pathway in Recombinant Escherichia coli▿ †

    PubMed Central

    Moon, Tae Seok; Yoon, Sang-Hwal; Lanza, Amanda M.; Roy-Mayhew, Joseph D.; Prather, Kristala L. Jones

    2009-01-01

    A synthetic pathway has been constructed for the production of glucuronic and glucaric acids from glucose in Escherichia coli. Coexpression of the genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae and myo-inositol oxygenase (MIOX) from mice led to production of glucuronic acid through the intermediate myo-inositol. Glucuronic acid concentrations up to 0.3 g/liter were measured in the culture broth. The activity of MIOX was rate limiting, resulting in the accumulation of both myo-inositol and glucuronic acid as final products, in approximately equal concentrations. Inclusion of a third enzyme, uronate dehydrogenase (Udh) from Pseudomonas syringae, facilitated the conversion of glucuronic acid to glucaric acid. The activity of this recombinant enzyme was more than 2 orders of magnitude higher than that of Ino1 and MIOX and increased overall flux through the pathway such that glucaric acid concentrations in excess of 1 g/liter were observed. This represents a novel microbial system for the biological production of glucaric acid, a “top value-added chemical” from biomass. PMID:19060162

  6. An essential gene, ESR1, is required for mitotic cell growth, DNA repair and meiotic recombination in Saccharomyces cerevisiae.

    PubMed Central

    Kato, R; Ogawa, H

    1994-01-01

    A new mutant, which was sensitive to both methyl-methanesulfonate (MMS) and ultra-violet light (UV) and defective in meiotic recombination, was isolated from Saccharomyces cerevisiae. The gene, ESR1, was cloned by complementation of the MMS sensitivity of the mutant and found to be essential for cell growth, as the deleted haploid strain was lethal. The ESR1 gene was adjacent to the CKS1 gene on chromosome II and encoded a putative 2368-amino acid protein with a molecular weight of 273 k. The ESR1 transcript was 8.0 kb long and was induced during meiosis. The predicted Esr1 protein had a mosaic structure composed of homologous regions and showed amino acid sequence similarities to Schizosaccharomyces pombe rad3+ protein, which monitors completion of DNA repair synthesis, and cut1+ protein, which is required for spindle pole body (SPB) duplication. The Esr1 protein was also similar to phosphatidylinositol (PI) 3-kinases, including Saccharomyces cerevisiae TOR2 (and DRR1), which are involved in G1 progression. These results suggest that ESR1 is multi-functional throughout mitosis and meiosis. Images PMID:8065923

  7. XRCC3 is essential for proper double-strand break repair and homologous recombination in rice meiosis.

    PubMed

    Zhang, Bingwei; Wang, Mo; Tang, Ding; Li, Yafei; Xu, Meng; Gu, Minghong; Cheng, Zhukuan; Yu, Hengxiu

    2015-09-01

    RAD51 paralogues play important roles in the assembly and stabilization of RAD51 nucleoprotein filaments, which promote homologous pairing and strand exchange reactions in organisms ranging from yeast to vertebrates. XRCC3, a RAD51 paralogue, has been characterized in budding yeast, mouse, and Arabidopsis. In the present study, XRCC3 in rice was identified and characterized. The rice xrcc3 mutant exhibited normal vegetative growth but complete male and female sterility. Cytological investigations revealed that homologous pairing and synapsis were severely disrupted in the mutant. Meiotic chromosomes were frequently entangled from diplotene to metaphase I, resulting in chromosome fragmentation at anaphase I. The immunostaining signals from γH2AX were regular, implying that double-strand break (DSB) formation was normal in xrcc3 meiocytes. However, COM1 was not detected on early prophase I chromosomes, suggesting that the DSB end-processing system was destroyed in the mutant. Moreover, abnormal chromosome localization of RAD51C, DMC1, ZEP1, ZIP4, and MER3 was observed in xrcc3. Taken together, the results suggest that XRCC3 plays critical roles in both DSB repair and homologous chromosome recombination during rice meiosis.

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

    PubMed Central

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

    2015-01-01

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

  9. Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers

    PubMed Central

    Fonseca, A.S.; Campos, V.M.A.; Magalhães, L.A.G.; Paoli, F.

    2015-01-01

    Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers. PMID:26445337

  10. Conservation of the nucleotide excision repair pathway: characterization of hydra Xeroderma Pigmentosum group F homolog.

    PubMed

    Barve, Apurva; Ghaskadbi, Saroj; Ghaskadbi, Surendra

    2013-01-01

    Hydra, one of the earliest metazoans with tissue grade organization and nervous system, is an animal with a remarkable regeneration capacity and shows no signs of organismal aging. We have for the first time identified genes of the nucleotide excision repair (NER) pathway from hydra. Here we report cloning and characterization of hydra homolog of xeroderma pigmentosum group F (XPF) gene that encodes a structure-specific 5' endonuclease which is a crucial component of NER. In silico analysis shows that hydra XPF amino acid sequence is very similar to its counterparts from other animals, especially vertebrates, and shows all features essential for its function. By in situ hybridization, we show that hydra XPF is expressed prominently in the multipotent stem cell niche in the central region of the body column. Ectoderm of the diploblastic hydra was shown to express higher levels of XPF as compared to the endoderm by semi-quantitative RT-PCR. Semi-quantitative RT-PCR analysis also demonstrated that interstitial cells, a multipotent and rapidly cycling stem cell lineage of hydra, express higher levels of XPF mRNA than other cell types. Our data show that XPF and by extension, the NER pathway is highly conserved during evolution. The prominent expression of an NER gene in interstitial cells may have implications for the lack of senescence in hydra.

  11. Conservation of the Nucleotide Excision Repair Pathway: Characterization of Hydra Xeroderma Pigmentosum Group F Homolog

    PubMed Central

    Barve, Apurva; Ghaskadbi, Saroj; Ghaskadbi, Surendra

    2013-01-01

    Hydra, one of the earliest metazoans with tissue grade organization and nervous system, is an animal with a remarkable regeneration capacity and shows no signs of organismal aging. We have for the first time identified genes of the nucleotide excision repair (NER) pathway from hydra. Here we report cloning and characterization of hydra homolog of xeroderma pigmentosum group F (XPF) gene that encodes a structure-specific 5′ endonuclease which is a crucial component of NER. In silico analysis shows that hydra XPF amino acid sequence is very similar to its counterparts from other animals, especially vertebrates, and shows all features essential for its function. By in situ hybridization, we show that hydra XPF is expressed prominently in the multipotent stem cell niche in the central region of the body column. Ectoderm of the diploblastic hydra was shown to express higher levels of XPF as compared to the endoderm by semi-quantitative RT-PCR. Semi-quantitative RT-PCR analysis also demonstrated that interstitial cells, a multipotent and rapidly cycling stem cell lineage of hydra, express higher levels of XPF mRNA than other cell types. Our data show that XPF and by extension, the NER pathway is highly conserved during evolution. The prominent expression of an NER gene in interstitial cells may have implications for the lack of senescence in hydra. PMID:23577191

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

    PubMed

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

    2015-12-09

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

  13. Common genetic variations in cell cycle and DNA repair pathways associated with pediatric brain tumor susceptibility

    PubMed Central

    Fahmideh, Maral Adel; Lavebratt, Catharina; Schüz, Joachim; Röösli, Martin; Tynes, Tore; Grotzer, Michael A.; Johansen, Christoffer; Kuehni, Claudia E; Lannering, Birgitta; Prochazka, Michaela; Schmidt, Lisbeth S; Feychting, Maria

    2016-01-01

    Knowledge on the role of genetic polymorphisms in the etiology of pediatric brain tumors (PBTs) is limited. Therefore, we investigated the association between single nucleotide polymorphisms (SNPs), identified by candidate gene-association studies on adult brain tumors, and PBT risk. The study is based on the largest series of PBT cases to date. Saliva DNA from 245 cases and 489 controls, aged 7–19 years at diagnosis/reference date, was genotyped for 68 SNPs. Data were analyzed using unconditional logistic regression. The results showed EGFRrs730437 and EGFRrs11506105 may decrease susceptibility to PBTs, whereas ERCC1rs3212986 may increase risk of these tumors. Moreover, stratified analyses indicated CHAF1Ars243341, CHAF1Ars2992, and XRCC1rs25487 were associated with a decreased risk of astrocytoma subtype. Furthermore, an increased risk of non-astrocytoma subtype associated with EGFRrs9642393, EME1rs12450550, ATMrs170548, and GLTSCRrs1035938 as well as a decreased risk of this subtype associated with XRCC4rs7721416 and XRCC4rs2662242 were detected. This study indicates SNPs in EGFR, ERCC1, CHAF1A, XRCC1, EME1, ATM, GLTSCR1, and XRCC4 may be associated with the risk of PBTs. Therefore, cell cycle and DNA repa