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Sample records for recombination repair pathway

  1. A comparative analysis of the DNA recombination repair pathway in mycobacterial genomes.

    PubMed

    Singh, Amandeep; Bhagavat, Raghu; Vijayan, M; Chandra, Nagasuma

    2016-07-01

    In prokaryotes, repair by homologous recombination provides a major means to reinstate the genetic information lost in DNA damage. Recombination repair pathway in mycobacteria has multiple differences as compared to that in Escherichia coli. Of about 20 proteins known to be involved in the pathway, a set of 9 proteins, namely, RecF, RecO, RecR, RecA, SSBa, RuvA, RuvB and RuvC was found to be indispensable among the 43 mycobacterial strains. A domain level analysis indicated that most domains involved in recombination repair are unique to these proteins and are present as single copies in the genomes. Synteny analysis reveals that the gene order of proteins involved in the pathway is not conserved, suggesting that they may be regulated differently in different species. Sequence conservation among the same protein from different strains suggests the importance of RecO-RecA and RecFOR-RecA presynaptic pathways in the repair of double strand-breaks and single strand-breaks respectively. New annotations obtained from the analysis, include identification of a protein with a probable Holliday junction binding role present in 41 mycobacterial genomes and that of a RecB-like nuclease, containing a cas4 domain, present in 42 genomes. New insights into the binding of small molecules to the relevant proteins are provided by binding pocket analysis using three dimensional structural models. Analysis of the various features of the recombination repair pathway, presented here, is likely to provide a framework for further exploring stress response and emergence of drug resistance in mycobacteria. PMID:27450012

  2. Homologous recombination and nonhomologous end-joining repair pathways regulate fragile site stability.

    PubMed

    Schwartz, Michal; Zlotorynski, Eitan; Goldberg, Michal; Ozeri, Efrat; Rahat, Ayelet; le Sage, Carlos; Chen, Benjamin P C; Chen, David J; Agami, Reuven; Kerem, Batsheva

    2005-11-15

    Common fragile sites are specific loci that form gaps and constrictions on metaphase chromosomes exposed to replication stress, which slows DNA replication. These sites have a role in chromosomal rearrangements in tumors; however, the molecular mechanism of their expression is unclear. Here we show that replication stress leads to focus formation of Rad51 and phosphorylated DNA-PKcs, key components of the homologous recombination (HR) and nonhomologous end-joining (NHEJ), double-strand break (DSB) repair pathways, respectively. Down-regulation of Rad51, DNA-PKcs, or Ligase IV, an additional component of the NHEJ repair pathway, leads to a significant increase in fragile site expression under replication stress. Replication stress also results in focus formation of the DSB markers, MDC1 and gammaH2AX. These foci colocalized with those of Rad51 and phospho-DNA-PKcs. Furthermore, gammaH2AX and phospho-DNA-PKcs foci were localized at expressed fragile sites on metaphase chromosomes. These findings suggest that DSBs are formed at common fragile sites as a result of replication perturbation. The repair of these breaks by both HR and NHEJ pathways is essential for chromosomal stability at these sites. PMID:16291645

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

    PubMed

    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

  4. 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

  5. Nrf2 facilitates repair of radiation induced DNA damage through homologous recombination repair pathway in a ROS independent manner in cancer cells.

    PubMed

    Jayakumar, Sundarraj; Pal, Debojyoti; Sandur, Santosh K

    2015-09-01

    Nrf2 is a redox sensitive transcription factor that is involved in the co-ordinated transcription of genes involved in redox homeostasis. But the role of Nrf2 in DNA repair is not investigated in detail. We have employed A549 and MCF7 cells to study the role of Nrf2 on DNA repair by inhibiting Nrf2 using all-trans retinoic acid (ATRA) or by knock down approach prior to radiation exposure (4 Gy). DNA damage and repair analysis was studied by γH2AX foci formation and comet assay. Results suggested that the inhibition of Nrf2 in A549 or MCF7 cells led to significant slowdown in DNA repair as compared to respective radiation controls. The persistence of residual DNA damage even in the presence of free radical scavenger N-acetyl cysteine, suggested that the influence of Nrf2 on DNA repair was not linked to its antioxidant functions. Further, its influence on non-homologous end joining repair pathway was studied by inhibiting both Nrf2 and DNA-PK together. This led to synergistic reduction of survival fraction, indicating that Nrf2 may not be influencing the NHEJ pathway. To investigate the role of homologous recombination repair (HR) pathway, RAD51 foci formation was monitored. There was a significant reduction in the foci formation in cells treated with ATRA or shRNA against Nrf2 as compared to their respective radiation controls. Further, Nrf2 inhibition led to significant reduction in mRNA levels of RAD51. BLAST analysis was also performed on upstream regions of DNA repair genes to identify antioxidant response element and found that many repair genes that are involved in HR pathway may be regulated by Nrf2. Together, these results suggest the involvement of Nrf2 in DNA repair, a hitherto unknown function of Nrf2, putatively through its influence on HR pathway. PMID:26133502

  6. 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

  7. 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.

  8. Recombination and DNA Repair in Helicobacter pylori

    PubMed Central

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

    2013-01-01

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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. Homologous Recombination Assay for Interstrand Cross-Link Repair

    PubMed Central

    Nakanishi, Koji; Cavallo, Francesca; Brunet, Erika; Jasin, Maria

    2012-01-01

    DNA interstrand cross-links (ICLs) covalently link both strands of the DNA duplex, impeding cellular processes like DNA replication. Homologous recombination (HR) is considered to be a major pathway for the repair of ICLs in mammalian cells as mutants for HR components are highly sensitive to DNA-damaging agents that cause ICLs. This chapter describes GFP assays to measure HR following site-specific ICL formation with psoralen through DNA triplex technology. This approach can be used to determine the genetic requirements for ICL-induced HR in relation to those involved in HR repair of other DNA lesions such as double-strand breaks. PMID:21660700

  15. 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.

  16. Mutational analysis of the Drosophila DNA repair and recombination gene mei-9.

    PubMed Central

    Yildiz, Ozlem; Kearney, Hutton; Kramer, Benjamin C; Sekelsky, Jeff J

    2004-01-01

    Drosophila mei-9 is essential for several DNA repair and recombination pathways, including nucleotide excision repair (NER), interstrand crosslink repair, and meiotic recombination. To better understand the role of MEI-9 in these processes, we characterized 10 unique mutant alleles of mei-9. These include a P-element insertion that disrupts repair functions but not the meiotic function; three nonsense mutations, one of which has nearly wild-type levels of protein; three missense mutations, one of which disrupts the meiotic function but not repair functions; two small in-frame deletions; and one frameshift. PMID:15166153

  17. 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

  18. 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

  19. 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

  20. Heterochromatic breaks move to the nuclear periphery to continue recombinational repair

    PubMed Central

    Ryu, Taehyun; Spatola, Brett; Delabaere, Laetitia; Bowlin, Katherine; Hopp, Hannah; Kunitake, Ryan; Karpen, Gary H.; Chiolo, Irene

    2015-01-01

    Heterochromatin mostly comprises repeated sequences prone to harmful ectopic recombination during double-strand break (DSB) repair. In Drosophila cells, ‘safe’ homologous recombination (HR) repair of heterochromatic breaks relies on a specialized pathway that relocalizes damaged sequences away from the heterochromatin domain before strand invasion. Here we show that heterochromatic DSBs move to the nuclear periphery to continue HR repair. Relocalization depends on nuclear pore and inner nuclear membrane proteins (INMPs) that anchor repair sites to the nuclear periphery via the Smc5/6-interacting proteins STUbL/RENi. Both the initial block to HR progression inside the heterochromatin domain, and the targeting of repair sites to the nuclear periphery, rely on SUMO and SUMO E3 ligases. This study reveals a critical role for SUMOylation in the spatial and temporal regulation of HR repair in heterochromatin, and identifies the nuclear periphery as a specialized site for heterochromatin repair in a multicellular eukaryote. PMID:26502056

  1. Heterochromatic breaks move to the nuclear periphery to continue recombinational repair.

    PubMed

    Ryu, Taehyun; Spatola, Brett; Delabaere, Laetitia; Bowlin, Katherine; Hopp, Hannah; Kunitake, Ryan; Karpen, Gary H; Chiolo, Irene

    2015-11-01

    Heterochromatin mostly comprises repeated sequences prone to harmful ectopic recombination during double-strand break (DSB) repair. In Drosophila cells, 'safe' homologous recombination (HR) repair of heterochromatic breaks relies on a specialized pathway that relocalizes damaged sequences away from the heterochromatin domain before strand invasion. Here we show that heterochromatic DSBs move to the nuclear periphery to continue HR repair. Relocalization depends on nuclear pores and inner nuclear membrane proteins (INMPs) that anchor repair sites to the nuclear periphery through the Smc5/6-interacting proteins STUbL/RENi. Both the initial block to HR progression inside the heterochromatin domain, and the targeting of repair sites to the nuclear periphery, rely on SUMO and SUMO E3 ligases. This study reveals a critical role for SUMOylation in the spatial and temporal regulation of HR repair in heterochromatin, and identifies the nuclear periphery as a specialized site for heterochromatin repair in a multicellular eukaryote. PMID:26502056

  2. Regulators of homologous recombination repair as novel targets for cancer treatment

    PubMed Central

    Krajewska, Małgorzata; Fehrmann, Rudolf S. N.; de Vries, Elisabeth G. E.; van Vugt, Marcel A. T. M.

    2015-01-01

    To cope with DNA damage, cells possess a complex signaling network called the ‘DNA damage response’, which coordinates cell cycle control with DNA repair. The importance of this network is underscored by the cancer predisposition that frequently goes along with hereditary mutations in DNA repair genes. One especially important DNA repair pathway in this respect is homologous recombination (HR) repair. Defects in HR repair are observed in various cancers, including hereditary breast, and ovarian cancer. Intriguingly, tumor cells with defective HR repair show increased sensitivity to chemotherapeutic reagents, including platinum-containing agents. These observations suggest that HR-proficient tumor cells might be sensitized to chemotherapeutics if HR repair could be therapeutically inactivated. HR repair is an extensively regulated process, which depends strongly on the activity of various other pathways, including cell cycle pathways, protein-control pathways, and growth factor-activated receptor signaling pathways. In this review, we discuss how the mechanistic wiring of HR is controlled by cell-intrinsic or extracellular pathways. Furthermore, we have performed a meta-analysis on available genome-wide RNA interference studies to identify additional pathways that control HR repair. Finally, we discuss how these HR-regulatory pathways may provide therapeutic targets in the context of radio/chemosensitization. PMID:25852742

  3. Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

    PubMed Central

    Symington, Lorraine S.

    2002-01-01

    The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination. PMID:12456786

  4. 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

  5. Single-strand gap repair involves both RecF and RecBCD pathways.

    PubMed

    Pagès, Vincent

    2016-08-01

    Homologous recombination repairs discontinuities in DNA including single-strand gaps (SSGs) and double-strand breaks (DSBs). This commentary describes how the RecBCD and RecF pathways might be exchangeable for the repair of their respective DSB and SSG canonical substrates. In particular, I will discuss how the RecBCD pathway could engage in the repair of an SSG even when the latter is not associated with a DSB. PMID:26874520

  6. DNA Repair Pathway Gene Expression Score Correlates with Repair Proficiency and Tumor Sensitivity to Chemotherapy

    PubMed Central

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

    2016-01-01

    Homologous recombination (HR) and non-homologous end joining (NHEJ) are alternative pathways of double-strand DNA break repair. We developed a method to quantify the efficiency of DNA repair pathways in the context of cancer therapy. The Recombination Proficiency Score (RPS) utilizes the expression levels for four genes involved in DNA repair pathway preference (RIF1, PARI, RAD51, and Ku80), such that high expression of these genes yields a low RPS. Carcinoma cells with low RPS exhibit HR suppression and frequent DNA copy number alterations, which are characteristic of error-prone repair processes that arise in HR-deficient backgrounds. The RPS system was clinically validated in patients with breast or non-small cell lung carcinomas (NSCLC). Tumors with low RPS were associated with greater mutagenesis, adverse clinical features, and inferior patient survival rates, suggesting that HR suppression plays a central role in promoting the genomic instability that fuels malignant progression. This adverse prognosis associated with low RPS was diminished if NSCLC patients received adjuvant chemotherapy, suggesting that HR suppression and associated sensitivity to platinum-based drugs counteracts the adverse prognosis associated with low RPS. Therefore, RPS may predict which therapies will be effective for individual patients, thereby enabling more personalized oncology care. PMID:24670686

  7. TOPBP1 takes RADical command in recombinational DNA repair.

    PubMed

    Liu, Yi; Smolka, Marcus B

    2016-02-01

    TOPBP1 is a key player in DNA replication and DNA damage signaling. In this issue, Moudry et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201507042) uncover a crucial role for TOPBP1 in DNA repair by revealing its requirement for RAD51 loading during repair of double strand breaks by homologous recombination. PMID:26811424

  8. Rad52 forms DNA repair and recombination centers during S phase

    PubMed Central

    Lisby, Michael; Rothstein, Rodney; Mortensen, Uffe H.

    2001-01-01

    Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases. In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands. In this study, we find that S. cerevisiae Rad52 fused to green fluorescent protein (GFP) is fully functional in DNA repair and recombination. After induction of DNA double-strand breaks by γ-irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci. Interestingly, Rad52 foci are formed almost exclusively during the S phase of mitotic cells, consistent with coordination between recombinational repair and DNA replication. This notion is further strengthened by the dramatic increase in the frequency of Rad52 focus formation observed in a pol12-100 replication mutant and a mec1 DNA damage checkpoint mutant. Furthermore, our data indicate that each Rad52 focus represents a center of recombinational repair capable of processing multiple DNA lesions. PMID:11459964

  9. 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

  10. Molecular Pathways: Targeting DNA Repair Pathway Defects Enriched in Metastasis.

    PubMed

    Corcoran, Niall M; Clarkson, Michael J; Stuchbery, Ryan; Hovens, Christopher M

    2016-07-01

    The maintenance of a pristine genome, free from errors, is necessary to prevent cellular transformation and degeneration. When errors in DNA are detected, DNA damage repair (DDR) genes and their regulators are activated to effect repair. When these DDR pathways are themselves mutated or aberrantly downregulated, cancer and neurodegenerative disorders can ensue. Multiple lines of evidence now indicate, however, that defects in key regulators of DNA repair pathways are highly enriched in human metastasis specimens and hence may be a key step in the acquisition of metastasis and the ability of localized disease to disseminate. Some of the key regulators of checkpoints in the DNA damage response are the TP53 protein and the PARP enzyme family. Targeting of these pathways, especially through PARP inhibition, is now being exploited therapeutically to effect significant clinical responses in subsets of individuals, particularly in patients with ovarian cancer or prostate cancer, including cancers with a marked metastatic burden. Targeting DNA repair-deficient tumors with drugs that take advantage of the fundamental differences between normal repair-proficient cells and repair-deficient tumors offers new avenues for treating advanced disease in the future. Clin Cancer Res; 22(13); 3132-7. ©2016 AACR. PMID:27169997

  11. Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair.

    PubMed

    Ceccaldi, Raphael; Liu, Jessica C; Amunugama, Ravindra; Hajdu, Ildiko; Primack, Benjamin; Petalcorin, Mark I R; O'Connor, Kevin W; Konstantinopoulos, Panagiotis A; Elledge, Stephen J; Boulton, Simon J; Yusufzai, Timur; D'Andrea, Alan D

    2015-02-12

    Large-scale genomic studies have shown that half of epithelial ovarian cancers (EOCs) have alterations in genes regulating homologous recombination (HR) repair. Loss of HR accounts for the genomic instability of EOCs and for their cellular hyper-dependence on alternative poly-ADP ribose polymerase (PARP)-mediated DNA repair mechanisms. Previous studies have implicated the DNA polymerase θ (Polθ also known as POLQ, encoded by POLQ) in a pathway required for the repair of DNA double-strand breaks, referred to as the error-prone microhomology-mediated end-joining (MMEJ) pathway. Whether Polθ interacts with canonical DNA repair pathways to prevent genomic instability remains unknown. Here we report an inverse correlation between HR activity and Polθ expression in EOCs. Knockdown of Polθ in HR-proficient cells upregulates HR activity and RAD51 nucleofilament assembly, while knockdown of Polθ in HR-deficient EOCs enhances cell death. Consistent with these results, genetic inactivation of an HR gene (Fancd2) and Polq in mice results in embryonic lethality. Moreover, Polθ contains RAD51 binding motifs and it blocks RAD51-mediated recombination. Our results reveal a synthetic lethal relationship between the HR pathway and Polθ-mediated repair in EOCs, and identify Polθ as a novel druggable target for cancer therapy. PMID:25642963

  12. Interplay between Fanconi anemia and homologous recombination pathways in genome integrity.

    PubMed

    Michl, Johanna; Zimmer, Jutta; Tarsounas, Madalena

    2016-05-01

    The Fanconi anemia (FA) pathway plays a central role in the repair of DNA interstrand crosslinks (ICLs) and regulates cellular responses to replication stress. Homologous recombination (HR), the error-free pathway for double-strand break (DSB) repair, is required during physiological cell cycle progression for the repair of replication-associated DNA damage and protection of stalled replication forks. Substantial crosstalk between the two pathways has recently been unravelled, in that key HR proteins such as the RAD51 recombinase and the tumour suppressors BRCA1 and BRCA2 also play important roles in ICL repair. Consistent with this, rare patient mutations in these HR genes cause FA pathologies and have been assigned FA complementation groups. Here, we focus on the clinical and mechanistic implications of the connection between these two cancer susceptibility syndromes and on how these two molecular pathways of DNA replication and repair interact functionally to prevent genomic instability. PMID:27037238

  13. Recombinational repair and restart of damaged replication forks.

    PubMed

    McGlynn, Peter; Lloyd, Robert G

    2002-11-01

    Genome duplication necessarily involves the replication of imperfect DNA templates and, if left to their own devices, replication complexes regularly run into problems. The details of how cells overcome these replicative 'hiccups' are beginning to emerge, revealing a complex interplay between DNA replication, recombination and repair that ensures faithful passage of the genetic material from one generation to the next. PMID:12415303

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

    PubMed Central

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

    2015-01-01

    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. PMID:25646469

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

    PubMed

    Kowalczykowski, Stephen C

    2015-11-01

    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. PMID:26525148

  16. Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway

    PubMed Central

    Kim, Hyungjin; D'Andrea, Alan D.

    2012-01-01

    The maintenance of genome stability is critical for survival, and its failure is often associated with tumorigenesis. The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links (ICLs), and a germline defect in the pathway results in FA, a cancer predisposition syndrome driven by genome instability. Central to this pathway is the monoubiquitination of FANCD2, which coordinates multiple DNA repair activities required for the resolution of ICLs. Recent studies have demonstrated how the FA pathway coordinates three critical DNA repair processes, including nucleolytic incision, translesion DNA synthesis (TLS), and homologous recombination (HR). Here, we review recent advances in our understanding of the downstream ICL repair steps initiated by ubiquitin-mediated FA pathway activation. PMID:22751496

  17. 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

  18. 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

  19. 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

  20. Genetic evidence that synaptonemal complex axial elements govern recombination pathway choice in mice.

    PubMed

    Li, Xin Chenglin; Bolcun-Filas, Ewelina; Schimenti, John C

    2011-09-01

    Chiasmata resulting from interhomolog recombination are critical for proper chromosome segregation at meiotic metaphase I, thus preventing aneuploidy and consequent deleterious effects. Recombination in meiosis is driven by programmed induction of double strand breaks (DSBs), and the repair of these breaks occurs primarily by recombination between homologous chromosomes, not sister chromatids. Almost nothing is known about the basis for recombination partner choice in mammals. We addressed this problem using a genetic approach. Since meiotic recombination is coupled with synaptonemal complex (SC) morphogenesis, we explored the role of axial elements--precursors to the lateral element in the mature SC--in recombination partner choice, DSB repair pathways, and checkpoint control. Female mice lacking the SC axial element protein SYCP3 produce viable, but often aneuploid, oocytes. We describe genetic studies indicating that while DSB-containing Sycp3-/- oocytes can be eliminated efficiently, those that survive have completed repair before the execution of an intact DNA damage checkpoint. We find that the requirement for DMC1 and TRIP13, proteins normally essential for recombination repair of meiotic DSBs, is substantially bypassed in Sycp3 and Sycp2 mutants. This bypass requires RAD54, a functionally conserved protein that promotes intersister recombination in yeast meiosis and mammalian mitotic cells. Immunocytological and genetic studies indicated that the bypass in Sycp3-/- Dmc1-/- oocytes was linked to increased DSB repair. These experiments lead us to hypothesize that axial elements mediate the activities of recombination proteins to favor interhomolog, rather than intersister recombinational repair of genetically programmed DSBs in mice. The elimination of this activity in SYCP3- or SYCP2-deficient oocytes may underlie the aneuploidy in derivative mouse embryos and spontaneous abortions in women. PMID:21750255

  1. Homology-directed Fanconi anemia pathway crosslink repair is dependent on DNA replication

    PubMed Central

    Nakanishi, Koji; Cavallo, Francesca; Perrouault, Loïc; Giovannangeli, Carine; Moynahan, Mary Ellen; Barchi, Marco; Brunet, Erika; Jasin, Maria

    2012-01-01

    Homologous recombination (also termed homology-directed repair, HDR) is a major pathway for the repair of DNA interstrand crosslinks (ICLs) in mammalian cells. Cells from Fanconi anemia (FA) patients are characterized by extreme ICL sensitivity, but their reported defect in HDR is mild. Here, we examined ICL-induced HDR using a GFP reporter and observed a profound defect in ICL-induced HDR in FA cells, but only when the reporter could replicate. PMID:21423196

  2. Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways

    PubMed Central

    Gupta, Richa; Barkan, Daniel; Redelman-Sidi, Gil; Shuman, Stewart; Glickman, Michael S.

    2013-01-01

    Bacterial pathogens rely on their DNA repair pathways to resist genomic damage inflicted by the host. DNA double-strand breaks (DSBs) are especially threatening to bacterial viability. DSB repair by homologous recombination (HR) requires nucleases that resect DSB ends and a strand exchange protein that facilitates homology search. RecBCD and RecA perform these functions in E. coli and constitute the major pathway of error free DSB repair. Mycobacteria, including the human pathogen M. tuberculosis, elaborate an additional error-prone pathway of DSB repair via nonhomologous end-joining (NHEJ) catalyzed by Ku and DNA ligase D (LigD). Little is known about the relative contributions of HR and NHEJ to mycobacterial chromosome repair, the factors that dictate pathway choice, or the existence of additional DSB repair pathways. Here we demonstrate that Mycobacterium smegmatis has three DSB repair pathway options: HR, NHEJ, and a novel mechanism of single-strand annealing (SSA). Inactivation of NHEJ or SSA is compensated by elevated HR. We find that mycobacterial RecBCD does not participate in HR or confer resistance to ionizing radiation (IR), but is required for the RecA-independent SSA pathway. In contrast, the mycobacterial helicase-nuclease AdnAB participates in the RecA-dependent HR pathway, and is a major determinant of resistance to IR and oxidative DNA damage. These findings reveal distinctive features of mycobacterial DSB repair, most notably the dedication of the RecBCD and AdnAB helicase-nuclease machines to distinct repair pathways. PMID:21219454

  3. Prolonged Particulate Hexavalent Chromium Exposure Suppresses Homologous Recombination Repair in Human Lung Cells.

    PubMed

    Browning, Cynthia L; Qin, Qin; Kelly, Deborah F; Prakash, Rohit; Vanoli, Fabio; Jasin, Maria; Wise, John Pierce

    2016-09-01

    Genomic instability is one of the primary models of carcinogenesis and a feature of almost all cancers. Homologous recombination (HR) repair protects against genomic instability by maintaining high genomic fidelity during the repair of DNA double strand breaks. The defining step of HR repair is the formation of the Rad51 nucleofilament, which facilitates the search for a homologous sequence and invasion of the template DNA strand. Particulate hexavalent chromium (Cr(VI)), a human lung carcinogen, induces DNA double strand breaks and chromosome instability. Since the loss of HR repair increases Cr(VI)-induced chromosome instability, we investigated the effect of extended Cr(VI) exposure on HR repair. We show acute (24 h) Cr(VI) exposure induces a normal HR repair response. In contrast, prolonged (120 h) exposure to particulate Cr(VI) inhibited HR repair and Rad51 nucleofilament formation. Prolonged Cr(VI) exposure had a profound effect on Rad51, evidenced by reduced protein levels and Rad51 mislocalization to the cytoplasm. The response of proteins involved in Rad51 nuclear import and nucleofilament formation displayed varying responses to prolonged Cr(VI) exposure. BRCA2 formed nuclear foci after prolonged Cr(VI) exposure, while Rad51C foci formation was suppressed. These results suggest that particulate Cr(VI), a major chemical carcinogen, inhibits HR repair by targeting Rad51, causing DNA double strand breaks to be repaired by a low fidelity, Rad51-independent repair pathway. These results further enhance our understanding of the underlying mechanism of Cr(VI)-induced chromosome instability and thus, carcinogenesis. PMID:27449664

  4. Multiple host-cell recombination pathways act in Agrobacterium-mediated transformation of plant cells.

    PubMed

    Mestiri, Imen; Norre, Frédéric; Gallego, Maria E; White, Charles I

    2014-02-01

    Using floral-dip, tumorigenesis and root callus transformation assays of both germline and somatic cells, we present here results implicating the four major non-homologous and homologous recombination pathways in Agrobacterium-mediated transformation of Arabidopsis thaliana. All four single mutant lines showed similar mild reductions in transformability, but knocking out three of four pathways severely compromised Agrobacterium-mediated transformation. Although integration of T-DNA into the plant genome is severely compromised in the absence of known DNA double-strand break repair pathways, it does still occur, suggesting the existence of other pathways involved in T-DNA integration. Our results highlight the functional redundancy of the four major plant recombination pathways in transformation, and provide an explanation for the lack of strong effects observed in previous studies on the roles of plant recombination functions in transformation. PMID:24299074

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

    PubMed Central

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

    2014-01-01

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

  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. Malaria parasites utilize both homologous recombination and alternative end joining pathways to maintain genome integrity

    PubMed Central

    Kirkman, Laura A.; Lawrence, Elizabeth A.; Deitsch, Kirk W.

    2014-01-01

    Malaria parasites replicate asexually within their mammalian hosts as haploid cells and are subject to DNA damage from the immune response and chemotherapeutic agents that can significantly disrupt genomic integrity. Examination of the annotated genome of the parasite Plasmodium falciparum identified genes encoding core proteins required for the homologous recombination (HR) pathway for repairing DNA double-strand breaks (DSBs), but surprisingly none of the components of the canonical non-homologous end joining (C-NHEJ) pathway were identified. To better understand how malaria parasites repair DSBs and maintain genome integrity, we modified the yeast I-SceI endonuclease system to generate inducible, site-specific DSBs within the parasite’s genome. Analysis of repaired genomic DNA showed that parasites possess both a typical HR pathway resulting in gene conversion events as well as an end joining (EJ) pathway for repair of DSBs when no homologous sequence is available. The products of EJ were limited in number and identical products were observed in multiple independent experiments. The repair junctions frequently contained short insertions also found in the surrounding sequences, suggesting the possibility of a templated repair process. We propose that an alternative end-joining pathway rather than C-NHEJ, serves as a primary method for repairing DSBs in malaria parasites. PMID:24089143

  8. Complexities of the DNA Base Excision Repair Pathway for Repair of Oxidative DNA Damage

    PubMed Central

    Mitra, Sankar; Boldogh, Istvan; Izumi, Tadahide; Hazra, Tapas K.

    2016-01-01

    Oxidative damage represents the most significant insult to organisms because of continuous production of the reactive oxygen species (ROS) in vivo. Oxidative damage in DNA, a critical target of ROS, is repaired primarily via the base excision repair (BER) pathway which appears to be the simplest among the three excision repair pathways. However, it is now evident that although BER can be carried with four or five enzymes in vitro, a large number of proteins, including some required for nucleotide excision repair (NER), are needed for in vivo repair of oxidative damage. Furthermore, BER in transcribed vs. nontranscribed DNA regions requires distinct sets of proteins, as in the case of NER. We propose an additional complexity in repair of replicating vs. nonreplicating DNA. Unlike DNA bulky adducts, the oxidized base lesions could be incorporated in the nascent DNA strand, repair of which may share components of the mismatch repair process. Distinct enzyme specificities are thus warranted for repair of lesions in the parental vs. nascent DNA strand. Repair synthesis may be carried out by DNA polymerase β or replicative polymerases δ and ε. Thus, multiple subpathways are needed for repairing oxidative DNA damage, and the pathway decision may require coordination of the successive steps in repair. Such coordination includes transfer of the product of a DNA glycosylase to AP-endonuclease, the next enzyme in the pathway. Interactions among proteins in the pathway may also reflect such coordination, characterization of which should help elucidate these subpathways and their in vivo regulation. PMID:11746753

  9. ALKBH1 is dispensable for abasic site cleavage during base excision repair and class switch recombination.

    PubMed

    Müller, Tina A; Yu, Kefei; Hausinger, Robert P; Meek, Katheryn

    2013-01-01

    Potential roles of the abasic site lyase activity associated with AlkB homolog 1 (ALKBH1) were assessed by studies focusing on the two cellular processes that create abasic sites as intermediates: base excision repair and class switch recombination. Alkbh1(-/-) pups (lacking exon 3) were born at a lower than expected frequency from heterozygous parents, suggesting a reduced survival rate and non-Mendelian inheritance, and they exhibited a gender bias in favor of males (70% males and 30% females). To study ALKBH1's potential involvement in DNA repair, fibroblasts were isolated from Alkbh1(-/-) mice, spontaneously immortalized and tested for resistance to DNA damaging agents. Alkbh1(-/-) and isogenic cells expressing hALKBH1 showed no difference in survival to the DNA damaging agents methyl-methionine sulfate or H2O2. This result indicates that ALKBH1 does not play a major role in the base excision repair pathway. To assess ALKBH1's role in class switch recombination, splenic B cells were isolated from Alkbh1(-/-) and Alkbh1(+/+) mice and subjected to switching from IgM to IgG1. No differences were found in IgG1 switching, suggesting that Alkbh1 is not involved in class switch recombination of the immunoglobulin heavy chain during B lymphocyte activation. PMID:23825659

  10. 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.

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

    PubMed Central

    Kakarougkas, A

    2014-01-01

    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. PMID:24363387

  12. 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. PMID:25117625

  13. Fanconi Anemia: A Signal Transduction and DNA Repair Pathway

    PubMed Central

    Kupfer, Gary M.

    2013-01-01

    Fanconi anemia (FA) is a fascinating, rare genetic disorder marked by congenital defects, bone marrow failure, and cancer susceptibility. Research in recent years has led to the elucidation of FA as a DNA repair disorder and involved multiple pathways as well as having wide applicability to common cancers, including breast, ovarian, and head and neck. This review will describe the clinical aspects of FA as well as the current state of its molecular pathophysiology. In particular, work from the Kupfer laboratory will be described that demonstrates how the FA pathway interacts with multiple DNA repair pathways, including the mismatch repair system and signal transduction pathway of the DNA damage response. PMID:24348213

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

    PubMed Central

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

    2014-01-01

    Disruptions in DNA repair pathways predispose cells to accumulating DNA damage. A growing body of evidence indicates that tumors accumulate progressively more mutations in DNA repair proteins as cancers progress. DNA repair mechanisms greatly affect the response to cytotoxic treatments, so understanding those mechanisms and finding ways to turn dysregulated repair processes against themselves to induce tumor death is the goal of all DNA repair inhibition efforts. Inhibition may be direct or indirect. This burgeoning field of research is replete with promise and challenge, as more intricacies of each repair pathway are discovered. In an era of increasing concern about healthcare costs, use of DNA repair inhibitors can prove to be highly effective stewardship of R&D resources and patient expenses. PMID:24947262

  15. BRCA1 functions independently of homologous recombination in DNA interstrand cross-link repair

    PubMed Central

    Bunting, Samuel F; Callen, Elsa; Kozak, Marina L; Kim, Jung-Min; Wong, Nancy; Lopez-Contreras, Andres J; Ludwig, Thomas; Baer, Richard; Faryabi, Robert B; Malhowski, Amy; Chen, Hua-Tang; Fernandez-Capetillo, Oscar; D’Andrea, Alan; Nussenzweig, Andre

    2012-01-01

    Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice, and rescues HR deficiency, as measured by hypersensitivity to PARP (polyADP-ribose polymerase) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand cross-links (ICLs), indicating that BRCA1 has an additional role in DNA cross-link repair that is distinct from HR. Disruption of the non-homologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi Anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair, whereas FANCD2 provides a key activity that can not be bypassed by ablation of 53BP1 or Ku. PMID:22445484

  16. Fanconi Anemia Proteins, DNA Interstrand Crosslink Repair Pathways, and Cancer Therapy

    PubMed Central

    Andreassen, Paul R.; Ren, Keqin

    2016-01-01

    DNA interstrand crosslinkers, a chemically diverse group of compounds which also induce alkylation of bases and DNA intrastrand crosslinks, are extensively utilized for cancer therapy. Understanding the cellular response to DNA damage induced by these agents is critical for more effective utilization of these compounds and for the identification of novel therapeutic targets. Importantly, the repair of DNA interstrand crosslinks (ICLs) involves many distinct DNA repair pathways, including nucleotide excision repair, translesion synthesis (TLS), and homologous recombination (HR). Additionally, proteins implicated in the pathophysiology of the multigenic disease Fanconi anemia (FA) have a role in the repair of ICLs that is not well understood. Cells from FA patients are hypersensitive to agents that induce ICLs, therefore FA proteins are potentially novel therapeutic targets. Here we will review current research directed at identifying FA genes and understanding the function of FA proteins in DNA damage responses. We will also examine interactions of FA proteins with other repair proteins and pathways, including signaling networks, which are potentially involved in ICL repair. Potential approaches to the modulation of FA protein function to enhance therapeutic outcome will be discussed. Also, mutation of many genes that encode proteins involved in ICL repair, including FA genes, increases susceptibility to cancer. A better understanding of these pathways is therefore critical for the design of individualized therapies tailored to the genetic profile of a particular malignancy. For this purpose, we will also review evidence for the association of mutation of FA genes with cancer in non-FA patients. PMID:19200054

  17. 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.

  18. 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

  19. Sibling rivalry: competition between Pol X family members in V(D)J recombination and general double strand break repair.

    PubMed

    Nick McElhinny, Stephanie A; Ramsden, Dale A

    2004-08-01

    The nonhomologous end-joining pathway is a major means for repairing double-strand breaks (DSBs) in all mitotic cell types. This repair pathway is also the only efficient means for resolving DSB intermediates in V(D)J recombination, a lymphocyte-specific genome rearrangement required for assembly of antigen receptors. A role for polymerases in end-joining has been well established. They are a major factor in determining the character of repair junctions but, in contrast to 'core' end-joining factors, typically appear to have a subtle impact on the efficiency of end-joining. Recent work implicates several members of the Pol X family in end-joining and suggests surprising complexity in the control of how these different polymerases are employed in this pathway. PMID:15242403

  20. 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

  1. 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. PMID:23475971

  2. Recombinant gelatin microspheres: novel formulations for tissue repair?

    PubMed

    Tuin, Annemarie; Kluijtmans, Sebastiaan G; Bouwstra, Jan B; Harmsen, Martin C; Van Luyn, Marja J A

    2010-06-01

    Microspheres (MSs) can function as multifunctional scaffolds in different approaches of tissue repair (TR), as a filler, a slow-release depot for growth factors, or a delivery vehicle for cells. Natural cell adhesion-supporting extracellular matrix components like gelatin are good materials for these purposes. Recombinant production of gelatin allows for on-demand design of gelatins, which is why we aim at developing recombinant gelatin (RG) MSs for TR. Two types of MSs (50 < Ø < 100 microm) were prepared by crosslinking two RGs, Syn-RG, and the arginine-glycine-aspartate-containing Hu-RG. The MSs were characterized, and their tissue reaction and degradation in rats was examined. Histological analysis of the explants after 14 and 28 days in vivo also showed that Syn-RG was degraded slower than Hu-RG, which correlated with the in vitro degradation assay. Hu-RG explants displayed more cellular ingrowth (60% vs. 15% for Syn-RG at day 14), which was associated with extracellular matrix deposition and vascularization. The infiltrating cells consisted of mainly macrophages, part of which fused to giant cells locally, and fibroblasts. No differences were found in matrix metalloproteinase mRNA levels, whereas gelatinase activity was clearly higher in Hu-RG explants. In conclusion, the in vitro and in vivo results of these novel formulations pave the way for cell- and/or factor-driven TR by these RG MSs. PMID:20102269

  3. Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.

    PubMed Central

    Milne, G T; Jin, S; Shannon, K B; Weaver, D T

    1996-01-01

    DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells. PMID:8754818

  4. 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. PMID:26337087

  5. 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

  6. 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

    2015-08-01

    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. PMID:26253028

  7. A dominant mutation in human RAD51 reveals its function in DNA interstrand crosslink repair independent of homologous recombination

    PubMed Central

    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

    2015-01-01

    Summary 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 wildtype 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. PMID:26253028

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

    PubMed Central

    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

    2016-01-01

    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. PMID:26792895

  9. 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 HRin 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. PMID:26792895

  10. 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

  11. 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. PMID:27236213

  12. Sources of DNA Double-Strand Breaks and Models of Recombinational DNA Repair

    PubMed Central

    Mehta, Anuja; Haber, James E.

    2014-01-01

    DNA is subject to many endogenous and exogenous insults that impair DNA replication and proper chromosome segregation. DNA double-strand breaks (DSBs) are one of the most toxic of these lesions and must be repaired to preserve chromosomal integrity. Eukaryotes are equipped with several different, but related, repair mechanisms involving homologous recombination, including single-strand annealing, gene conversion, and break-induced replication. In this review, we highlight the chief sources of DSBs and crucial requirements for each of these repair processes, as well as the methods to identify and study intermediate steps in DSB repair by homologous recombination. PMID:25104768

  13. Molecular recombination and the repair of DNA double-strand breaks in CHO cells.

    PubMed Central

    Resnick, M A; Moore, P D

    1979-01-01

    Molecular recombination and the repair of DNA double-strand breaks (DSB) have been examined in the G-0 and S phase of the cell cycle using a temperature-sensitive CHO cell line to test i) if there are cell cycle restrictions on the repair of DSB's' ii) the extent to which molecular recombination can be induced between either sister chromatids or homologous chromosomes and iii) whether repair of DSB's involves recombination (3). Mitomycin C (1-2 micrograms/ml) or ionizing radiation (50 krad) followed by incubation resulted in molecular recombination (hybrid DNA) in S phase cells. Approximately 0.03 to 0.10% of the molecules (number average molecular weight: 5.6 x 10(6) Daltons after shearing) had hybrid regions for more than 75% of their length. However, no recombination was detected in G-0 cells. Since the repair of DSB was observed in both stages with more than 50% of the breaks repaired in 5 hours, it appears that DSB repair in G-0 cells does not involve recombination between homologous chromosomes. The possibility is not excluded that repair in G-0 cells involves only small regions (less than 4 x 10(6) Daltons). PMID:493136

  14. DNA double-strand break repair: a tale of pathway choices.

    PubMed

    Li, Jing; Xu, Xingzhi

    2016-07-01

    Deoxyribonucleic acid double-strand breaks (DSBs) are cytotoxic lesions that must be repaired either through homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways. DSB repair is critical for genome integrity, cellular homeostasis and also constitutes the biological foundation for radiotherapy and the majority of chemotherapy. The choice between HR and NHEJ is a complex yet not completely understood process that will entail more future efforts. Herein we review our current understandings about how the choice is made over an antagonizing balance between p53-binding protein 1 and breast cancer 1 in the context of cell cycle stages, downstream effects, and distinct chromosomal histone marks. These exciting areas of research will surely bring more mechanistic insights about DSB repair and be utilized in the clinical settings. PMID:27217474

  15. Mutations in recombinational repair and in checkpoint control genes suppress the lethal combination of srs2Delta with other DNA repair genes in Saccharomyces cerevisiae.

    PubMed Central

    Klein, H L

    2001-01-01

    The SRS2 gene of Saccharomyces cerevisiae encodes a DNA helicase that is active in the postreplication repair pathway and homologous recombination. srs2 mutations are lethal in a rad54Delta background and cause poor growth or lethality in rdh54Delta, rad50Delta, mre11Delta, xrs2Delta, rad27Delta, sgs1Delta, and top3Delta backgrounds. Some of these genotypes are known to be defective in double-strand break repair. Many of these lethalities or poor growth can be suppressed by mutations in other genes in the DSB repair pathway, namely rad51, rad52, rad55, and rad57, suggesting that inhibition of recombination at a prior step prevents formation of a lethal intermediate. Lethality of the srs2Delta rad54Delta and srs2Delta rdh54Delta double mutants can also be rescued by mutations in the DNA damage checkpoint functions RAD9, RAD17, RAD24, and MEC3, indicating that the srs2 rad54 and srs2 rdh54 mutant combinations lead to an intermediate that is sensed by these checkpoint functions. When the checkpoints are intact the cells never reverse from the arrest, but loss of the checkpoints releases the arrest. However, cells do not achieve wild-type growth rates, suggesting that unrepaired damage is still present and may lead to chromosome loss. PMID:11156978

  16. Role of 53BP1 in the Regulation of DNA Double-Strand Break Repair Pathway Choice

    PubMed Central

    Gupta, Arun; Hunt, Clayton R.; Chakraborty, Sharmistha; Pandita, Raj K.; Yordy, John; Ramnarain, Deepti B.; Horikoshi, Nobuo; Pandita, Tej K.

    2014-01-01

    The p53-binding protein 1 (53BP1) is a well-known DNA damage response (DDR) factor, which is recruited to nuclear structures at the site of DNA damage and forms readily visualized ionizing radiation (IR) induced foci. Depletion of 53BP1 results in cell cycle arrest in G2/M phase as well as genomic instability in human as well as mouse cells. Within the DNA damage response mechanism, 53BP1 is classified as an adaptor/mediator, required for processing of the DNA damage response signal and as a platform for recruitment of other repair factors. More recently, specific 53BP1 contributions to DSB repair pathway choice have been recognized and are being characterized. In this review, we have summarized recent advances in understanding the role of 53BP1 in regulating DNA DSBs repair pathway choice, variable diversity joining [V(D)J] recombination and class-switch recombination (CSR). PMID:24320053

  17. Role of 53BP1 in the regulation of DNA double-strand break repair pathway choice.

    PubMed

    Gupta, Arun; Hunt, Clayton R; Chakraborty, Sharmistha; Pandita, Raj K; Yordy, John; Ramnarain, Deepti B; Horikoshi, Nobuo; Pandita, Tej K

    2014-01-01

    The p53-binding protein 1 (53BP1) is a well-known DNA damage response (DDR) factor, which is recruited to nuclear structures at the site of DNA damage and forms readily visualized ionizing radiation (IR) induced foci. Depletion of 53BP1 results in cell cycle arrest in G2/M phase as well as genomic instability in human as well as mouse cells. Within the DNA damage response mechanism, 53BP1 is classified as an adaptor/mediator, required for processing of the DNA damage response signal and as a platform for recruitment of other repair factors. More recently, specific 53BP1 contributions to DSB repair pathway choice have been recognized and are being characterized. In this review, we have summarized recent advances in understanding the role of 53BP1 in regulating DNA DSBs repair pathway choice, variable diversity joining [V(D)J] recombination and class-switch recombination (CSR). PMID:24320053

  18. DNA replication meets genetic exchange: Chromosomal damage and its repair by homologous recombination

    PubMed Central

    Kuzminov, Andrei

    2001-01-01

    Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair. PMID:11459990

  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. 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. PMID:24461734

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

    PubMed Central

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

    2014-01-01

    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. PMID:24461734

  2. Regulation of homologous recombinational repair by lamin B1 in radiation-induced DNA damage.

    PubMed

    Liu, Ning-Ang; Sun, Jiying; Kono, Kazuteru; Horikoshi, Yasunori; Ikura, Tsuyoshi; Tong, Xing; Haraguchi, Tokuko; Tashiro, Satoshi

    2015-06-01

    DNA double-strand breaks (DSBs) are the major lethal lesion induced by ionizing radiation (IR). RAD51-dependent homologous recombination (HR) is one of the most important pathways in DSB repair and genome integrity maintenance. However, the mechanism of HR regulation by RAD51 remains unclear. To understand the mechanism of RAD51-dependent HR, we searched for interacting partners of RAD51 by a proteomics analysis and identified lamin B1 in human cells. Lamins are nuclear lamina proteins that play important roles in the structural organization of the nucleus and the regulation of chromosome functions. Immunoblotting analyses revealed that siRNA-mediated lamin B1 depletion repressed the DNA damage-dependent increase of RAD51 after IR. The repression was abolished by the proteasome inhibitor MG132, suggesting that lamin B1 stabilizes RAD51 by preventing proteasome-mediated degradation in cells with IR-induced DNA damage. We also showed that lamin B1 depletion repressed RAD51 focus formation and decreased the survival rates after IR. On the basis of these results, we propose that lamin B1 promotes DSB repair and cell survival by maintaining the RAD51 protein levels for HR upon DSB induction after IR. PMID:25733566

  3. An exonuclease I-sensitive DNA repair pathway in Deinococcus radiodurans: a major determinant of radiation resistance.

    PubMed

    Misra, Hari S; Khairnar, Nivedita P; Kota, Swathi; Shrivastava, Smriti; Joshi, Vasudha P; Apte, Shree K

    2006-02-01

    Deinococcus radiodurans R1 recovering from acute dose of gamma radiation shows a biphasic mechanism of DNA double-strand break repair. The possible involvement of microsequence homology-dependent, or non-homologous end joining type mechanisms during initial period followed by RecA-dependent homologous recombination pathways has been suggested for the reconstruction of complete genomes in this microbe. We have exploited the known roles of exonuclease I in DNA recombination to elucidate the nature of recombination involved in DNA double-strand break repair during post-irradiation recovery of D. radiodurans. Transgenic Deinococcus cells expressing exonuclease I functions of Escherichia coli showed significant reduction in gamma radiation radioresistance, while the resistance to far-UV and hydrogen peroxide remained unaffected. The overexpression of E. coli exonuclease I in Deinococcus inhibited DNA double-strand break repair. Such cells exhibited normal post-irradiation expression kinetics of RecA, PprA and single-stranded DNA-binding proteins but lacked the divalent cation manganese [(Mn(II)]-dependent protection from gamma radiation. The results strongly suggest that 3' (rho) 5' single-stranded DNA ends constitute an important component in recombination pathway involved in DNA double-strand break repair and that absence of sbcB from deinococcal genome may significantly aid its extreme radioresistance phenotype. PMID:16430702

  4. Involvement of homologous recombination repair after proton-induced DNA damage.

    PubMed

    Rostek, C; Turner, E L; Robbins, M; Rightnar, S; Xiao, W; Obenaus, A; Harkness, T A A

    2008-03-01

    Protection from chronic exposure to cosmic radiation, which is primarily composed of protons, in future manned missions to Mars and beyond is considered to be a key unresolved issue. To model the effects of cosmic radiation on a living cell, we used Saccharomyces cerevisiae cells harboring various deletions of DNA repair genes to investigate the response of cells to DNA strand breaks caused by exposure to 250 MeV proton irradiation (linear energy transfer of 0.41 keV/microm). In our study, DNA strand breaks induced by exposure to protons were predominantly repaired via the homologous recombination and postreplication repair pathways. We simulated chronic exposure to proton irradiation by treating cells from colonies that survived proton treatment, after several rounds of subculturing, to a second proton dose, as well as additional cell stressors. In general, cells cultured from proton surviving colonies were not more sensitive to secondary cell stressors. However, cells from rad52delta colonies that survived proton treatment showed increased resistance to secondary stressors, such as gamma-rays (1.17 and 1.33 MeV; 0.267 keV/microm), ultraviolet (UV) and proton irradiation and elevated temperatures. Resistance to secondary stressors was also observed in rad52delta cells that survived exposure to gamma-rays, rather than protons, but this was not observed to occur in rad52delta cells after UV irradiation. rad52delta cells that survived exposure to protons, followed by gamma-rays (proton surviving colonies were cultured prior to gamma-ray exposure), exhibited an additive effect, whereby these cells had a further increase in stress resistance. A genetic analysis indicated that increased stress resistance is most likely due to a second-site mutation that suppresses the rad52delta phenotype. We will discuss possible origins of these second-site mutations. PMID:18267950

  5. 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. PMID:27075725

  6. 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

  7. 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

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

    PubMed

    Grin, Inga; Ishchenko, Alexander A

    2016-05-01

    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

  9. 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

  10. 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.

  11. Repair and recombination induced by triple helix DNA.

    PubMed

    Chin, Joanna Y; Schleifman, Erica B; Glazer, Peter M

    2007-01-01

    Triple-helix DNA structures can form endogenously at mirror repeat polypurine/polypyrimidine sequences or by introduction of triplex-forming oligonucleotides (TFOs). Recent evidence suggests that triple helices are sources of genetic instability, and are subject to increased rates of mutagenesis and recruitment of repair factors. Indeed, observations using TFOs suggest that triple helices provoke a variety of biological processes which can be harnessed to modulate gene expression and induce heritable changes in targeted genes. This review surveys the biological applications of TFOs, with particular attention to their recombinogenic and mutagenic potential, and summarizes available evidence for the mechanism of triplex and triplex-associated repair. PMID:17485375

  12. 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

  13. 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. PMID:25017126

  14. 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

  15. 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

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

    PubMed

    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

    1996-11-01

    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

  17. 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.

  18. 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. PMID:22105360

  19. 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

  20. 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

    2015-03-01

    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. PMID:25661491

  1. Chemotherapeutic Compounds Targeting the DNA Double-Strand Break Repair Pathways: The Good, the Bad, and the Promising

    PubMed Central

    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. PMID:24795863

  2. 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

  3. 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

  4. The λ Integrase Site-specific Recombination Pathway.

    PubMed

    Landy, Arthur

    2015-04-01

    The site-specific recombinase encoded by bacteriophage λ (Int) is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. Int carries out a reaction that is highly directional, tightly regulated, and depends upon an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein and each of the four Int protomers, within a multiprotein 400 kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. In the 12 years since the publication of the last review focused solely on the λ site-specific recombination pathway in Mobile DNA II, there has been a great deal of progress in elucidating the molecular details of this pathway. The most dramatic advances in our understanding of the reaction have been in the area of X-ray crystallography where protein-DNA structures have now been determined for of all of the DNA-protein interfaces driving the Int pathway. Building on this foundation of structures, it has been possible to derive models for the assembly of components that determine the regulatory apparatus in the P-arm, and for the overall architectures that define excisive and integrative recombinogenic complexes. The most fundamental additional mechanistic insights derive from the application of hexapeptide inhibitors and single molecule kinetics. PMID:26104711

  5. (CA/TG) microsatellite sequences escape the inhibition of recombination by mismatch repair in Saccharomyces cerevisiae.

    PubMed Central

    Gendrel, C G; Dutreix, M

    2001-01-01

    Sequence divergence reduces the frequency of recombination, a process that is dependent on the activity of the mismatch repair system. In the yeast Saccharomyces cerevisiae, repair of mismatches results in gene conversion or restoration, whereas failure to repair mismatches results in postmeiotic segregation (PMS). By examining the conversion and PMS in yeast strains deficient in various MMR genes and heterozygous for large inserts (107 bp) with either a mixed sequence or a 39 (CA/TG) repetitive microsatellite sequence, we demonstrate that: (1) the inhibition of conversion by large inserts depends upon a complex containing both Msh2 and Pms1 proteins; (2) conversion is not inhibited if the single-stranded DNA loop in the heteroduplex is the microsatellite sequence; and (3) large heteroduplex loops with random sequence or repetitive sequence might be repaired by two complexes, containing either Msh2 or Pms1. Our results suggest that inhibition of recombination by heterologous inserts and large loop repair are not processed by the same MMR complexes. We propose that the inhibition of conversion by large inserts is due to recognition by the Msh2/Pms1 complex of mismatches created by intrastrand interactions in the heteroduplex loop. PMID:11779795

  6. RRP6/EXOSC10 is required for the repair of DNA double-strand breaks by homologous recombination.

    PubMed

    Marin-Vicente, Consuelo; Domingo-Prim, Judit; Eberle, Andrea B; Visa, Neus

    2015-03-15

    The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively. Depletion of RRP6/EXOSC10 does not interfere with the phosphorylation of the histone variant H2Av (Drosophila) or H2AX (humans), but impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A-V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway. Taken together, our results suggest that the ribonucleolytic activity of RRP6/EXOSC10 is required for the recruitment of RAD51 to DSBs. PMID:25632158

  7. Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli.

    PubMed Central

    Kolodner, R; Fishel, R A; Howard, M

    1985-01-01

    Tn5 insertion mutations in the recN gene, and in what appears to be a new RecF pathway gene designated recO and mapping at approximately 55.4 min on the standard genetic map, were isolated by screening Tn5 insertion mutations that cotransduced with tyrA. The recO1504::Tn5 mutation decreased the frequency of recombination during Hfr-mediated crosses and increased the susceptibility to killing by UV irradiation and mitomycin C when present in a recB recC sbcB background, but only increased the sensitivity to killing by UV irradiation when present in an otherwise Rec+ background. The effects of these and other RecF pathway mutations on plasmid recombination were tested. Mutations in the recJ, recO, and ssb genes, when present in otherwise Rec+ E. coli strains, decreased the frequency of plasmid recombination, whereas the lexA3, recAo281, recN, and ruv mutations had no effect on plasmid recombination. Tn5 insertion mutations in the lexA gene increased the frequency of plasmid recombination. These data indicate that plasmid recombination events in wild-type Escherichia coli strains are catalyzed by a recombination pathway that is related to the RecF recombination pathway and that some component of this pathway besides the recA gene product is regulated by the lexA gene product. PMID:2993230

  8. Temporal and Spatial Uncoupling of DNA Double Strand Break Repair Pathways within Mammalian Heterochromatin.

    PubMed

    Tsouroula, Katerina; Furst, Audrey; Rogier, Melanie; Heyer, Vincent; Maglott-Roth, Anne; Ferrand, Alexia; Reina-San-Martin, Bernardo; Soutoglou, Evi

    2016-07-21

    Repetitive DNA is packaged into heterochromatin to maintain its integrity. We use CRISPR/Cas9 to induce DSBs in different mammalian heterochromatin structures. We demonstrate that in pericentric heterochromatin, DSBs are positionally stable in G1 and recruit NHEJ factors. In S/G2, DSBs are resected and relocate to the periphery of heterochromatin, where they are retained by RAD51. This is independent of chromatin relaxation but requires end resection and RAD51 exclusion from the core. DSBs that fail to relocate are engaged by NHEJ or SSA proteins. We propose that the spatial disconnection between end resection and RAD51 binding prevents the activation of mutagenic pathways and illegitimate recombination. Interestingly, in centromeric heterochromatin, DSBs recruit both NHEJ and HR proteins throughout the cell cycle. Our results highlight striking differences in the recruitment of DNA repair factors between pericentric and centromeric heterochromatin and suggest a model in which the commitment to specific DNA repair pathways regulates DSB position. PMID:27397684

  9. 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

  10. Genetic polymorphisms in homologous recombination repair genes in healthy Slovenian population and their influence on DNA damage

    PubMed Central

    Goricar, Katja; Erculj, Nina; Zadel, Maja; Dolzan, Vita

    2012-01-01

    Background Homologous recombination (HR) repair is an important mechanism involved in repairing double-strand breaks in DNA and for maintaining genomic stability. Polymorphisms in genes coding for enzymes involved in this pathway may influence the capacity for DNA repair. The aim of this study was to select tag single nucleotide polymorphisms (SNPs) in specific genes involved in HR repair, to determine their allele frequencies in a healthy Slovenian population and their influence on DNA damage detected with comet assay. Materials and methods In total 373 individuals were genotyped for nine tag SNPs in three genes: XRCC3 722C>T, XRCC3 -316A>G, RAD51 -98G>C, RAD51 -61G>T, RAD51 1522T>G, NBS1 553G>C, NBS1 1197A>G, NBS1 37117C>T and NBS1 3474A>C using competitive allele-specific amplification (KASPar assay). Comet assay was performed in a subgroup of 26 individuals to determine the influence of selected SNPs on DNA damage. Results We observed that age significantly affected genotype frequencies distribution of XRCC3 -316A>G (P = 0.039) in healthy male blood donors. XRCC3 722C>T (P = 0.005), RAD51 -61G>T (P = 0.023) and NBS1 553G>C (P = 0.008) had a statistically significant influence on DNA damage. Conclusions XRCC3 722C>T, RAD51 -61G>T and NBS1 553G>C polymorphisms significantly affect the repair of damaged DNA and may be of clinical importance as they are common in Slovenian population. PMID:22933979

  11. 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. PMID:24732344

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

    PubMed Central

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

    2014-01-01

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

  13. Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways.

    PubMed

    Qi, Yiping; Zhang, Yong; Zhang, Feng; Baller, Joshua A; Cleland, Spencer C; Ryu, Yungil; Starker, Colby G; Voytas, Daniel F

    2013-03-01

    Improved methods for engineering sequence-specific nucleases, including zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs), have made it possible to precisely modify plant genomes. However, the success of genome modification is largely dependent on the intrinsic activity of the engineered nucleases. In this study, we sought to enhance ZFN-mediated targeted mutagenesis and gene targeting (GT) in Arabidopsis by manipulating DNA repair pathways. Using a ZFN that creates a double-strand break (DSB) at the endogenous ADH1 locus, we analyzed repair outcomes in the absence of DNA repair proteins such as KU70 and LIG4 (both involved in classic nonhomologous end-joining, NHEJ) and SMC6B (involved in sister-chromatid-based homologous recombination, HR). We achieved a fivefold to 16-fold enhancement in HR-based GT in a ku70 mutant and a threefold to fourfold enhancement in GT in the lig4 mutant. Although the NHEJ mutagenesis frequency was not significantly changed in ku70 or lig4, DNA repair was shifted to microhomology-dependent alternative NHEJ. As a result, mutations in both ku70 and lig4 were predominantly large deletions, which facilitates easy screening for mutations by PCR. Interestingly, NHEJ mutagenesis and GT at the ADH1 locus were enhanced by sixfold to eightfold and threefold to fourfold, respectively, in a smc6b mutant. The increase in NHEJ-mediated mutagenesis by loss of SMC6B was further confirmed using ZFNs that target two other Arabidopsis genes, namely, TT4 and MPK8. Considering that components of DNA repair pathways are highly conserved across species, mutations in DNA repair genes likely provide a universal strategy for harnessing repair pathways to achieve desired targeted genome modifications. PMID:23282329

  14. Fanconi Anemia Repair Pathway Dysfunction, a Potential Therapeutic Target in Lung Cancer

    PubMed Central

    Duan, Wenrui; Gao, Li; Aguila, Brittany; Kalvala, Arjun; Otterson, Gregory A.; Villalona-Calero, Miguel A.

    2014-01-01

    The Fanconi anemia (FA) pathway is a major mechanism of homologous recombination DNA repair. The functional readout of the pathway is activation through mono-ubiquitination of FANCD2 leading to nuclear foci of repair. We have recently developed an FA triple-staining immunofluorescence based method (FATSI) to evaluate FANCD2 foci formation in formalin fixed paraffin-embedded (FFPE) tumor samples. DNA-repair deficiencies have been considered of interest in lung cancer prevention, given the persistence of damage produced by cigarette smoke in this setting, as well as in treatment, given potential increased efficacy of DNA-damaging drugs. We screened 139 non-small cell lung cancer (NSCLC) FFPE tumors for FANCD2 foci formation by FATSI analysis. Among 104 evaluable tumors, 23 (22%) were FANCD2 foci negative, thus repair deficient. To evaluate and compare novel-targeted agents in the background of FA deficiency, we utilized RNAi technology to render several lung cancer cell lines FANCD2 deficient. Successful FANCD2 knockdown was confirmed by reduction in the FANCD2 protein. Subsequently, we treated the FA defective H1299D2-down and A549D2-down NSCLC cells and their FA competent counterparts (empty vector controls) with the PARP inhibitors veliparib (ABT-888) (5 μM) and BMN673 (0.5 μM), as well as the CHK1 inhibitor Arry-575 at a dose of 0.5 μM. We also treated the FA defective small cell lung cancer cell lines H719D2-down and H792D2-down and their controls with the BCL-2/XL inhibitor ABT-263 at a dose of 2 μM. The treated cells were harvested at 24, 48, and 72 h post treatment. MTT cell viability analysis showed that each agent was more cytotoxic to the FANCD2 knock-down cells. In all tests, the FA defective lung cancer cells had less viable cells as comparing to controls 72 h post treatment. Both MTT and clonogenic analyses comparing the two PARP inhibitors, showed that BMN673 was more potent compared to veliparib. Given that FA pathway plays essential

  15. 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

  16. Oxidative stress-related DNA damage and homologous recombination repairing induced by N,N-dimethylformamide.

    PubMed

    Wang, Cui; Yang, Jinhuan; Lu, Dezhao; Fan, Yongsheng; Zhao, Meirong; Li, Zhuoyu

    2016-07-01

    The intensified anthropogenic release of N,N-dimethylformamide (DMF) has been proven to have hepatotoxic effects. However, the potential mechanism for DMF-induced toxicity has rarely been investigated. Our research implicated that DMF induced a significantly dose-dependent increase in reactive oxygen species (ROS) in HL-7702 human liver cells. Moreover, oxidative stress-related DNA damage, marked as 8-hydroxy-2'-deoxyguanosine, was increased 1.5-fold at 100 mmol l(-1) . The most severe DNA lesion (double-strand break, DSB), measured as the formation of γH2AX foci, was increased at/above 6.4 mmol l(-1) , and approximately 50% of cells underwent DSB at the peak induction. Subsequently, the DNA repair system triggered by molecules of RAD50 and MRE11A induced the homologous recombination (HR) pathway by upregulation of both gene and protein levels of RAD50, RAD51, XRCC2 and XRCC3 at 16 mmol l(-1) and was attenuated at 40 mmol l(-1) . Consequently, cellular death observed at 40 mmol l(-1) was exaggerated compared with exposure at 16 mmol l(-1) . Although the exact mechanism relying on the DMF-induced hepatotoxicity needs further clarification, oxidative stress and DNA damage involved in DSBs partially explain the reason for DMF-induced liver injury. Oxidative stress-induced DNA damage should be first considered during risk assessment on liver-targeted chemicals. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26387567

  17. Genetic evidence that the elevated levels of Escherichia coli helicase II antagonize recombinational DNA repair.

    PubMed

    Petranović, M; Zahradka, K; Zahradka, D; Petranović, D; Nagy, B; Salaj-Smic, E; Petranović, D

    2001-01-01

    Some phages survive irradiation much better upon multiple than upon single infection, a phenomenon known as multiplicity reactivation (MR). Long ago MR of UV-irradiated lambda red phage in E. coli cells was shown to be a manifestation of recA-dependent recombinational DNA repair. We used this experimental model to assess the influence of helicase II on the type of recombinational repair responsible for MR. Since helicase II is encoded by the SOS-inducible uvrD gene, SOS-inducing treatments such as irradiating recA(+) or heating recA441 cells were used. We found: i) that MR was abolished by the SOS-inducing treatments; ii) that in uvrD background these treatments did not affect MR; and iii) that the presence of a high-copy plasmid vector carrying the uvrD(+) allele together with its natural promoter region was sufficient to block MR. From these results we infer that helicase II is able to antagonize the type of recA-dependent recombinational repair acting on multiple copies of UV-damaged lambda DNA and that its anti-recombinogenic activity is operative at elevated levels only. PMID:11879732

  18. Mismatch repair ensures fidelity of replication and recombination in the radioresistant organism Deinococcus radiodurans.

    PubMed

    Mennecier, S; Coste, G; Servant, P; Bailone, A; Sommer, S

    2004-11-01

    We have characterized the mismatch repair system (MMR) of the highly radiation-resistant type strain of Deinococcus radiodurans, ATCC 13939. We show that the MMR system is functional in this organism, where it participates in ensuring the fidelity of DNA replication and recombination. The system relies on the activity of two key proteins, MutS1 and MutL, which constitute a conserved core involved in mismatch recognition. Inactivation of MutS1 or MutL resulted in a seven-fold increase in the frequency of spontaneous RifR mutagenesis and a ten-fold increase in the efficiency of integration of a donor point-mutation marker during bacterial transformation. Inactivation of the mismatch repair-associated UvrD helicase increased the level of spontaneous mutagenesis, but had no effect on marker integration--suggesting that binding of MutS1 and MutL proteins to a mismatched heteroduplex suffices to inhibit recombination between non identical (homeologous) DNAs. In contrast, inactivation of MutS2, encoded by the second mutS -related gene present in D. radiodurans, had no effect on mutagenesis or recombination. Cells devoid of MutS1 or MutL proteins were as resistant to gamma-rays, mitomycin C and UV-irradiation as wild-type bacteria, suggesting that the mismatch repair system is not essential for the reconstitution of a functional genome after DNA damage. PMID:15503140

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

    PubMed

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

    2016-03-18

    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

  20. 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

  1. 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. PMID:24507154

  2. 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.

  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. Mismatch repair inhibits homeologous recombination via coordinated directional unwinding of trapped DNA structures.

    PubMed

    Tham, Khek-Chian; Hermans, Nicolaas; Winterwerp, Herrie H K; Cox, Michael M; Wyman, Claire; Kanaar, Roland; Lebbink, Joyce H G

    2013-08-01

    Homeologous recombination between divergent DNA sequences is inhibited by DNA mismatch repair. In Escherichia coli, MutS and MutL respond to DNA mismatches within recombination intermediates and prevent strand exchange via an unknown mechanism. Here, using purified proteins and DNA substrates, we find that in addition to mismatches within the heteroduplex region, secondary structures within the displaced single-stranded DNA formed during branch migration within the recombination intermediate are involved in the inhibition. We present a model that explains how higher-order complex formation of MutS, MutL, and DNA blocks branch migration by preventing rotation of the DNA strands within the recombination intermediate. Furthermore, we find that the helicase UvrD is recruited to directionally resolve these trapped intermediates toward DNA substrates. Thus, our results explain on a mechanistic level how the coordinated action between MutS, MutL, and UvrD prevents homeologous recombination and maintains genome stability. PMID:23932715

  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. Three R's of bacterial evolution: how replication, repair, and recombination frame the origin of species.

    PubMed

    Brown, E W; LeClerc, J E; Kotewicz, M L; Cebula, T A

    2001-01-01

    The genetic diversity of bacteria results not only from errors in DNA replication and repair but from horizontal exchange and recombination of DNA sequences from similar and disparate species as well. New individuals carrying adaptive changes are thus being spawned constantly among the population at large. When new selection pressures appear, these are the individuals that survive, at the expense of the general population, to forge new populations. Depending on the severity and uniqueness of the selection pressure, this could lead to new speciation. It is becoming more and more evident that, as nucleotide sequences of numerous loci from many bacterial strains continue to amass, horizontal transfer has played a key role in configuring the Escherichia coli chromosome. Here, we examine views, both old and new, for the role of recombination in the evolution of bacterial chromosomes. We present novel phylogenetic evidence for horizontal transfer of three genes involved in DNA replication and repair (mutS, uvrD, and polA). These data reveal a prominent role for horizontal transfer in the evolution of genes known to play a key role in the fidelity of DNA replication and, thus, ultimate survival of the organism. Our data underscore that recombination plays both a diversifying and a homogenizing role in defining the structure of the E. coli genome. PMID:11746762

  7. Cdk1-dependent regulation of the Mre11 complex couples DNA repair pathways to cell cycle progression

    PubMed Central

    Simoneau, Antoine; Robellet, Xavier; Ladouceur, Anne-Marie; D’Amours, Damien

    2014-01-01

    Homologous recombination (HR) and non-homologous end joining (NHEJ) are the main pathways ensuring the repair of DNA double-stranded breaks (DSBs) in eukaryotes. It has long been known that cell cycle stage is a major determinant of the type of pathway used to repair DSBs in vivo. However, the mechanistic basis for the cell cycle regulation of the DNA damage response is still unclear. Here we show that a major DSB sensor, the Mre11–Rad50–Xrs2 (MRX) complex, is regulated by cell cycle-dependent phosphorylation specifically in mitosis. This modification depends on the cyclin-dependent kinase Cdc28/Cdk1, and abrogation of Xrs2 and Mre11 phosphorylation results in a marked preference for DSB repair through NHEJ. Importantly, we show that phosphorylation of the MRX complex after DNA damage and during mitosis are regulated independently of each other by Tel1/ATM and Cdc28/Cdk1 kinases. Collectively, our results unravel an intricate network of phosphoregulatory mechanisms that act through the MRX complex to modulate DSB repair efficiency during mitosis. PMID:24553123

  8. 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

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

    PubMed

    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

  10. 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. PMID:26503252

  11. Effects of hyperthermia on DNA repair pathways: one treatment to inhibit them all.

    PubMed

    Oei, Arlene L; Vriend, Lianne E M; Crezee, Johannes; Franken, Nicolaas A P; Krawczyk, Przemek M

    2015-01-01

    The currently available arsenal of anticancer modalities includes many DNA damaging agents that can kill malignant cells. However, efficient DNA repair mechanisms protect both healthy and cancer cells against the effects of treatment and contribute to the development of drug resistance. Therefore, anti-cancer treatments based on inflicting DNA damage can benefit from inhibition of DNA repair. Hyperthermia - treatment at elevated temperature - considerably affects DNA repair, among other cellular processes, and can thus sensitize (cancer) cells to DNA damaging agents. This effect has been known and clinically applied for many decades, but how heat inhibits DNA repair and which pathways are targeted has not been fully elucidated. In this review we attempt to summarize the known effects of hyperthermia on DNA repair pathways relevant in clinical treatment of cancer. Furthermore, we outline the relationships between the effects of heat on DNA repair and sensitization of cells to various DNA damaging agents. PMID:26245485

  12. 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

  13. Cdk1 targets Srs2 to complete synthesis-dependent strand annealing and to promote recombinational repair.

    PubMed

    Saponaro, Marco; Callahan, Devon; Zheng, Xiuzhong; Krejci, Lumir; Haber, James E; Klein, Hannah L; Liberi, Giordano

    2010-02-01

    Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and is also required for double-strand break (DSB) repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2, using mutants that constitutively express the phosphorylated or unphosphorylated protein isoforms. We found that Cdk1 targets Srs2 to repair DSB and, in particular, to complete synthesis-dependent strand annealing, likely controlling the disassembly of a D-loop intermediate. Cdk1-dependent phosphorylation controls turnover of Srs2 at the invading strand; and, in absence of this modification, the turnover of Rad51 is not affected. Further analysis of the recombination phenotypes of the srs2 phospho-mutants showed that Srs2 phosphorylation is not required for the removal of toxic Rad51 nucleofilaments, although it is essential for cell survival, when DNA breaks are channeled into homologous recombinational repair. Cdk1-targeted Srs2 displays a PCNA-independent role and appears to have an attenuated ability to inhibit recombination. Finally, the recombination defects of unphosphorylatable Srs2 are primarily due to unscheduled accumulation of the Srs2 protein in a sumoylated form. Thus, the Srs2 anti-recombination function in removing toxic Rad51 filaments is genetically separable from its role in promoting recombinational repair, which depends exclusively on Cdk1-dependent phosphorylation. We suggest that Cdk1 kinase counteracts unscheduled sumoylation of Srs2 and targets Srs2 to dismantle specific DNA structures, such as the D-loops, in a helicase-dependent manner during homologous recombinational repair. PMID:20195513

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

    PubMed Central

    Su, Xiaofeng A.; Dion, Vincent; Gasser, Susan M.

    2015-01-01

    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. PMID:25940904

  15. Assessing the function of homologous recombination DNA repair in malignant pleural effusion (MPE) samples

    PubMed Central

    Patterson, M J; Sutton, R E; Forrest, I; Sharrock, R; Lane, M; Kaufmann, A; O'Donnell, R; Edmondson, R J; Wilson, B T; Curtin, N J

    2014-01-01

    Background: Patients with malignant pleural effusions (MPEs) generally have advanced disease with poor survival and few therapeutic options. Cells within MPEs may be used to stratify patients for targeted therapy. Targeted therapy with poly(ADP ribose) polymerase inhibitors (PARPi) depends on identifying homologous recombination DNA repair (HRR)-defective cancer cells. We aimed to determine the feasibility of assaying HRR status in MPE cells. Methods: A total of 15 MPE samples were collected from consenting patients with non-small-cell lung cancer (NSCLC), mesothelioma and ovarian and breast cancer. Primary cultures were confirmed as epithelial by pancytokeratin, and HRR status was determined by the detection of γH2AX and RAD51 foci following a 24-h exposure to rucaparib, by immunofluorescence microscopy. Massively parallel next-generation sequencing of DNA repair genes was performed on cultured MPE cells. Results: From 15 MPE samples, 13 cultures were successfully established, with HRR function successfully determined in 12 cultures. Four samples – three NSCLC and one mesothelioma – were HRR defective and eight samples – one NSCLC, one mesothelioma, one sarcomatoid, one breast and four ovarian cancers – were HRR functional. No mutations in DNA repair genes were associated with HRR status, but there was probable loss of heterozygosity of FANCG, RPA1 and PARP1. Conclusions: HRR function can be successfully detected in MPE cells demonstrating the potential to stratify patients for targeted therapy with PARPi. PMID:24867690

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

    PubMed

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

    2006-01-01

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

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

    PubMed Central

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

    2006-01-01

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

  19. Divergent genes in potential inoculant Sinorhizobium strains are related to DNA replication, recombination, and repair.

    PubMed

    Penttinen, Petri; Greco, Dario; Muntyan, Victoria; Terefework, Zewdu; De Lajudie, Philippe; Roumiantseva, Marina; Becker, Anke; Auvinen, Petri; Lindström, Kristina

    2016-06-01

    To serve as inoculants of legumes, nitrogen-fixing rhizobium strains should be competitive and tolerant of diverse environments. We hybridized the genomes of symbiotically efficient and salt tolerant Sinorhizobium inoculant strains onto the Sinorhizobium meliloti Rm1021 microarray. The number of variable genes, that is, divergent or putatively multiplied genes, ranged from 503 to 1556 for S. meliloti AK23, S. meliloti STM 1064 and S. arboris HAMBI 1552. The numbers of divergent genes affiliated with the symbiosis plasmid pSymA and related to DNA replication, recombination and repair were significantly higher than expected. The variation was mainly in the accessory genome, implying that it was important in shaping the adaptability of the strains. PMID:26879331

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

    PubMed

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

    2012-03-14

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

  1. Molecular structures of crossover and noncrossover intermediates during gap repair in yeast: implications for recombination mechanisms and the regulation of recombination fidelity

    PubMed Central

    Mitchel, Katrina; Zhang, Hengshan; Welz-Voegele, Caroline; Jinks-Robertson, Sue

    2010-01-01

    SUMMARY The molecular structures of crossover (CO) and noncrossover (NCO) intermediates were determined by sequencing the products formed when a gapped plasmid was repaired using a diverged chromosomal template. Analyses were done in the absence of mismatch repair (MMR) to allow efficient detection of strand-transfer intermediates, and the results reveal striking differences in the extents and locations of heteroduplex DNA (hDNA) in NCO versus CO products. These data indicate that most NCOs are produced by synthesis-dependent strand annealing rather than by a canonical double-strand break repair pathway, and that resolution of Holliday junctions formed as part of the latter pathway is highly constrained to generate CO products. We suggest a model in which the length of hDNA formed by the initiating strand invasion event determines susceptibility of the resulting intermediate to antirecombination and ultimately whether a CO- or a NCO-producing pathway is followed. PMID:20417600

  2. 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. PMID:22342069

  3. 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

  4. 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

    2016-01-01

    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

  5. The yeast recombinational repair protein Rad59 interacts with Rad52 and stimulates single-strand annealing.

    PubMed Central

    Davis, A P; Symington, L S

    2001-01-01

    The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing. PMID:11606529

  6. 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

    2014-01-01

    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. PMID:25275598

  7. Assembling an orchestra: Fanconi anemia pathway of DNA repair.

    PubMed

    Yuan, Fenghua; Song, Limin; Qian, Liangyue; Hu, Jennifer J; Zhang, Yanbin

    2010-01-01

    Fanconi anemia (FA) is a recessive genetic disorder characterized by developmental defects, bone marrow failure, and cancer susceptibility. The complete set of FA genes has only been identified recently and seems to be uniquely conserved among vertebrates. Fanconi anemia proteins have been implicated in the repair of interstrand DNA crosslinks that block DNA replication and transcription. Although all thirteen FA complementation groups show similar clinical and cellular phenotypes, approximately 85% of patients presented defective FANCA, FANCC, or FANCG. The established DNA interacting components (FANCM, FANCI, FANCD2, and FANCJ) account only for approximately 5% of all FA patients, an observation that raises doubt concerning the roles of FA proteins in DNA repair. In recent years, rapid progress in the area of FA research has provided great insights into the critical roles of FA proteins in DNA repair. However, many FA proteins do not have identifiable domains to indicate how they contribute to biological processes, particularly DNA repair. Therefore, future biochemical studies are warranted to understand the biological functions of FA proteins and their implications in human diseases. PMID:20515746

  8. Effects of Tumor Suppressor Lysyl Oxidase Propeptide on Prostate Cancer Xenograft Growth and Its Direct Interactions with DNA Repair Pathways

    PubMed Central

    Bais, Manish V.; Ozdener, Gokhan Baris; Sonenshein, Gail E.; Trackman, Philip C.

    2014-01-01

    Lysyl oxidase (LOX) is a multifunctional protein required for normal collagen and elastin biosynthesis and maturation. In addition, LOX has complex roles in cancer in which the lysyl oxidase propeptide (LOX-PP) domain of secreted pro-LOX has tumor suppressor activity, while the active enzyme promotes metastasis. In prostate cancer cell lines, recombinant LOX-PP (rLOX-PP) inhibits the growth of PC3 cells in vitro by mechanisms which were not characterized, while in DU145 cells rLOX-PP targeted FGF signaling. Because rLOX-PP can enhance effects of a genotoxic chemotherapeutic on breast cancer cell apoptosis, we reasoned that rLOX-PP could target DNA repair pathways typically elevated in cancer. Here we demonstrate for the first time that rLOX-PP inhibits prostate xenograft growth in vivo and that activating phosphorylations of the key DNA repair molecules ATM and CHK2 are inhibited by rLOX-PP expression in vivo. In addition, in vitro studies showed that rLOX-PP inhibits radiation induced activating phosphorylations of ATM and CHK2, and that exogenously added rLOX-PP protein can localize to the nucleus in both DU145 and PC3 cells. rLOX-PP pull-down studies resulted in detection of a protein complex with the nuclear DNA repair regulator MRE11 in both cell lines, and rLOX-PP localized to radiation-induced nuclear DNA repair foci. Finally, rLOX-PP was shown to sensitize both DU145 and PC3 cells to radiation-induced cell death determined in colony formation assays. These data provide evidence that rLOX-PP has a nuclear mechanism of action in which it directly interacts with DNA repair proteins to sensitize prostate cancer cells to the effects of ionizing radiation. PMID:24882580

  9. Topoisomerase II-Mediated DNA Damage Is Differently Repaired during the Cell Cycle by Non-Homologous End Joining and Homologous Recombination

    PubMed Central

    de Campos-Nebel, Marcelo; Larripa, Irene; González-Cid, Marcela

    2010-01-01

    Topoisomerase II (Top2) is a nuclear enzyme involved in several metabolic processes of DNA. Chemotherapy agents that poison Top2 are known to induce persistent protein-mediated DNA double strand breaks (DSB). In this report, by using knock down experiments, we demonstrated that Top2α was largely responsible for the induction of γH2AX and cytotoxicity by the Top2 poisons idarubicin and etoposide in normal human cells. As DSB resulting from Top2 poisons-mediated damage may be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR), we aimed to analyze both DNA repair pathways. We found that DNA-PKcs was rapidly activated in human cells, as evidenced by autophosphorylation at serine 2056, following Top2-mediated DNA damage. The chemical inhibition of DNA-PKcs by wortmannin and vanillin resulted in an increased accumulation of DNA DSB, as evaluated by the comet assay. This was supported by a hypersensitive phenotype to Top2 poisons of Ku80- and DNA-PKcs- defective Chinese hamster cell lines. We also showed that Rad51 protein levels, Rad51 foci formation and sister chromatid exchanges were increased in human cells following Top2-mediated DNA damage. In support, BRCA2- and Rad51C- defective Chinese hamster cells displayed hypersensitivity to Top2 poisons. The analysis by immunofluorescence of the DNA DSB repair response in synchronized human cell cultures revealed activation of DNA-PKcs throughout the cell cycle and Rad51 foci formation in S and late S/G2 cells. Additionally, we found an increase of DNA-PKcs-mediated residual repair events, but not Rad51 residual foci, into micronucleated and apoptotic cells. Therefore, we conclude that in human cells both NHEJ and HR are required, with cell cycle stage specificity, for the repair of Top2-mediated reversible DNA damage. Moreover, NHEJ-mediated residual repair events are more frequently associated to irreversibly damaged cells. PMID:20824055

  10. Kidney injury molecule-1 (KIM-1) mediates renal epithelial cell repair via ERK MAPK signaling pathway

    PubMed Central

    Zhang, Zhiwei; Cai, Cindy X

    2016-01-01

    The expression of kidney injury molecule-1 (KIM-1), a very promising sensitive and specific urinary biomarker for acute renal injury, is markedly upregulated in injured and regenerating renal proximal tubular epithelial cells following ischemic or toxic insults, suggesting a possible role for this molecule in renal repair process. In the present study we report that expression of KIM-1 facilitates renal tubular epithelial cell repair by promoting cell migration and proliferation. KIM-1 expression also enhances ERK MAPK activation, and the modulatory effect of KIM-1 on cellular repair process is likely mediated via ERK MAPK signaling pathway. PMID:27084535

  11. Kidney injury molecule-1 (KIM-1) mediates renal epithelial cell repair via ERK MAPK signaling pathway.

    PubMed

    Zhang, Zhiwei; Cai, Cindy X

    2016-05-01

    The expression of kidney injury molecule-1 (KIM-1), a very promising sensitive and specific urinary biomarker for acute renal injury, is markedly upregulated in injured and regenerating renal proximal tubular epithelial cells following ischemic or toxic insults, suggesting a possible role for this molecule in renal repair process. In the present study, we report that expression of KIM-1 facilitates renal tubular epithelial cell repair by promoting cell migration and proliferation. KIM-1 expression also enhances ERK MAPK activation, and the modulatory effect of KIM-1 on cellular repair process is likely mediated via ERK MAPK signaling pathway. PMID:27084535

  12. 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. PMID:26922091

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

    PubMed Central

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

    2016-01-01

    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. PMID:26922091

  14. 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. PMID:17630970

  15. 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

  16. Analysis of Heme Biosynthetic Pathways in a Recombinant Escherichia coli.

    PubMed

    Pranawidjaja, Stephanie; Choi, Su-In; Lay, Bibiana W; Kim, Pil

    2015-06-01

    Bacterial heme was produced from a genetic-engineered Escherichia coli via the porphyrin pathway and it was useful as an iron resource for animal feed. The amount of the E. colisynthesized heme, however, was only few milligrams in a culture broth and it was not enough for industrial applications. To analyze heme biosynthetic pathways, an engineered E. coli artificially overexpressing ALA synthase (hemA from Rhodobacter sphaeroides) and pantothenate kinase (coaA gene from self geneome) was constructed as a bacterial heme-producing strain, and both the transcription levels of pathway genes and the intermediates concentrations were determined from batch and continuous cultures. Transcription levels of the pathway genes were not significantly changed among the tested conditions. Intracellular intermediate concentrations indicated that aminolevulinic acid (ALA) and coenzyme A (CoA) were enhanced by the hemA-coaA co-expression. Intracellular coproporphyrinogen I and protoporphyrin IX accumulation suggested that the bottleneck steps in the heme biosynthetic pathway could be the spontaneous conversion of HMB to coproporphyrinogen I and the limited conversion of protoporphyrin IX to heme, respectively. A strategy to increase the conversion of ALA to heme is discussed based on the results. PMID:25537720

  17. 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.

  18. Impact of DNA repair pathways on the cytotoxicity of piperlongumine in chicken DT40 cell-lines

    PubMed Central

    Okamoto, Saki; Narita, Takeo; Sasanuma, Hiroyuki; Takeda, Shunichi; Masunaga, Shin-ichiro; Bessho, Tadayoshi; Tano, Keizo

    2014-01-01

    Piperlongumine is a naturally-occurring small molecule with various biological activities. Recent studies demonstrate that piperlongumine selectively kills various types of transformed cells with minimal toxicity to non-transformed cells by inducing a high level of reactive oxygen species (ROS). ROS generates various types of DNA lesions, including base modifications and single strand breaks. In order to examine the contribution of ROS-induced DNA damage to the cytotoxicity by piperlongumine, various DNA repair-deficient chicken DT40 cell-lines with a single DNA repair gene deletion were tested for cellular sensitivity to piperlongumine. The results showed that cell lines defective in homologous recombination (HR) display hyper-sensitivity to piperlongumine, while other cell lines with a deficiency in non-homologous end joining (NHEJ), base excision repair (BER), nucleotide excision repair (NER), Fanconi anemia (FA) pathway, or translesion DNA synthesis (TLS) polymerases, show no sensitivity to piperlongumine. The results strongly implicate that double strand breaks (DSBs) generated by piperlongumine are major cytotoxic DNA lesions. Furthermore, a deletion of 53BP1 or Ku70 in the BRCA1-deficient cell line restored cellular resistance to piperlongumine. This strongly supports the idea that piperlongumine induces DSB- mediated cell death. Interestingly, piperlongumine makes the wild type DT40 cell line hypersensitive to a PARP-inhibitor, Olaparib. The results implicate that piperlongumine inhibits HR. Further analysis with cell-based HR assay and the kinetic study of Rad51 foci formation confirmed that piperlongumine suppresses HR activity. Altogether, we revealed novel mechanisms of piperlongumine-induced cytotoxicity. PMID:25221646

  19. Impact of DNA repair pathways on the cytotoxicity of piperlongumine in chicken DT40 cell-lines.

    PubMed

    Okamoto, Saki; Narita, Takeo; Sasanuma, Hiroyuki; Takeda, Shunichi; Masunaga, Shin-Ichiro; Bessho, Tadayoshi; Tano, Keizo

    2014-07-01

    Piperlongumine is a naturally-occurring small molecule with various biological activities. Recent studies demonstrate that piperlongumine selectively kills various types of transformed cells with minimal toxicity to non-transformed cells by inducing a high level of reactive oxygen species (ROS). ROS generates various types of DNA lesions, including base modifications and single strand breaks. In order to examine the contribution of ROS-induced DNA damage to the cytotoxicity by piperlongumine, various DNA repair-deficient chicken DT40 cell-lines with a single DNA repair gene deletion were tested for cellular sensitivity to piperlongumine. The results showed that cell lines defective in homologous recombination (HR) display hyper-sensitivity to piperlongumine, while other cell lines with a deficiency in non-homologous end joining (NHEJ), base excision repair (BER), nucleotide excision repair (NER), Fanconi anemia (FA) pathway, or translesion DNA synthesis (TLS) polymerases, show no sensitivity to piperlongumine. The results strongly implicate that double strand breaks (DSBs) generated by piperlongumine are major cytotoxic DNA lesions. Furthermore, a deletion of 53BP1 or Ku70 in the BRCA1-deficient cell line restored cellular resistance to piperlongumine. This strongly supports the idea that piperlongumine induces DSB- mediated cell death. Interestingly, piperlongumine makes the wild type DT40 cell line hypersensitive to a PARP-inhibitor, Olaparib. The results implicate that piperlongumine inhibits HR. Further analysis with cell-based HR assay and the kinetic study of Rad51 foci formation confirmed that piperlongumine suppresses HR activity. Altogether, we revealed novel mechanisms of piperlongumine-induced cytotoxicity. PMID:25221646

  20. Hodgkin Lymphoma Risk: Role of Genetic Polymorphisms and Gene-Gene Interactions in DNA repair pathways

    PubMed Central

    Monroy, Claudia M.; Cortes, Andrea C.; Lopez, Mirtha; Rourke, Elizabeth; Etzel, Carol J.; Younes, Anas; Strom, Sara S.; El-Zein, Randa

    2011-01-01

    DNA repair variants may play a potentially important role in an individual’s susceptibility to developing cancer. Numerous studies have reported the association between genetic single nucleotide polymorphisms (SNPs) in DNA repair genes and different types of hematologic cancers. However, to date, the effects of such SNPs on modulating Hodgkin Lymphoma (HL) risk have not yet been investigated. We hypothesized that gene-gene interaction between candidate genes in Direct Reversal, Nucleotide excision repair (NER), Base excision repair (BER) and Double strand break (DSB) pathways may contribute to susceptibility to HL. To test this hypothesis, we conducted a study on 200 HL cases and 220 controls to assess associations between HL risk and 21 functional SNPs in DNA repair genes. We evaluated potential gene-gene interactions and the association of multiple polymorphisms in a chromosome region using a multi-analytic strategy combining logistic regression, multi-factor dimensionality reduction and classification and regression tree approaches. We observed that, in combination, allelic variants in the XPC Ala499Val, NBN Glu185Gln, XRCC3 Thr241Me, XRCC1 Arg194Trp and XRCC1 399Gln polymorphisms modify the risk for developing HL. Moreover, the cumulative genetic risk score revealed a significant trend where the risk for developing HL increases as the number of adverse alleles in BER and DSB genes increase. These findings suggest that DNA repair variants in BER and DSB pathways may play an important role in the development of HL. PMID:21374732

  1. The SAGA Deubiquitination Module Promotes DNA Repair and Class Switch Recombination through ATM and DNAPK-Mediated γH2AX Formation.

    PubMed

    Ramachandran, Shaliny; Haddad, Dania; Li, Conglei; Le, Michael X; Ling, Alexanda K; So, Clare C; Nepal, Rajeev M; Gommerman, Jennifer L; Yu, Kefei; Ketela, Troy; Moffat, Jason; Martin, Alberto

    2016-05-17

    Class switch recombination (CSR) requires activation-induced deaminase (AID) to instigate double-stranded DNA breaks at the immunoglobulin locus. DNA breaks activate the DNA damage response (DDR) by inducing phosphorylation of histone H2AX followed by non-homologous end joining (NHEJ) repair. We carried out a genome-wide screen to identify CSR factors. We found that Usp22, Eny2, and Atxn7, members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitination module, are required for deubiquitination of H2BK120ub following DNA damage, are critical for CSR, and function downstream of AID. The SAGA deubiquitinase activity was required for optimal irradiation-induced γH2AX formation, and failure to remove H2BK120ub inhibits ATM- and DNAPK-induced γH2AX formation. Consistent with this effect, these proteins were found to function upstream of various double-stranded DNA repair pathways. This report demonstrates that deubiquitination of histone H2B impacts the early stages of the DDR and is required for the DNA repair phase of CSR. PMID:27160905

  2. The histone-like protein HU binds specifically to DNA recombination and repair intermediates

    PubMed Central

    Kamashev, D.; Rouviere-Yaniv, J.

    2000-01-01

    The heterodimeric HU protein associated with the Escherichia coli nucleoid shares some properties with histones and HMG proteins. HU binds DNA junctions and DNA containing a nick much more avidly than double-stranded (ds-) DNA. Cells lacking HU are extremely sensitive to γ irradiation and we wondered how HU could play a role in maintaining the integrity of the bacterial chromosome. We show that HU binds with high affinity to DNA repair and recombination intermediates, including DNA invasions, DNA overhangs and DNA forks. The DNA structural motif that HU specifically recognizes in all these structures consists of a ds-DNA module joined to a second module containing either ds- or single-stranded (ss-) DNA. The two modules rotate freely relative to one another. Binding specificity results from the simultaneous interaction of HU with these two modules: HU arms bind the ds-DNA module whereas the HU body contacts the ‘variable’ module containing either ds- or ss-DNA. Both structural motifs are recognized by HU at least 1000-fold more avidly than duplex DNA. PMID:11101525

  3. Role of LrpC from Bacillus subtilis in DNA transactions during DNA repair and recombination

    PubMed Central

    López-Torrejón, Gema; Martínez-Jiménez, María I.; Ayora, Silvia

    2006-01-01

    Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein, which binds both single-stranded (ss) and double-stranded (ds) DNA and facilitates the formation of higher order protein–DNA complexes in vitro. LrpC binds at different sites within the same DNA molecule promoting intramolecular ligation. When bound to separate molecules, it promotes intermolecular ligation, and joint molecule formation between a circular ssDNA and a homologous ssDNA-tailed linear dsDNA. LrpC binding showed a higher affinity for 4-way (Holliday) junctions in their open conformation, when compared with curved dsDNA. Consistent with these biochemical activities, an lrpC null mutant strain rendered cells sensitive to DNA damaging agents such as methyl methanesulfonate and 4-nitroquinoline-1-oxide, and showed a segregation defect. These findings collectively suggest that LrpC may be involved in DNA transactions during DNA repair and recombination. PMID:16407330

  4. 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. PMID:24675793

  5. 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.

  6. DNA-PK: a dynamic enzyme in a versatile DSB repair pathway

    PubMed Central

    Davis, Anthony J.; Chen, Benjamin P.C.; Chen, David J.

    2014-01-01

    DNA double stranded breaks (DSBs) are the most cytoxic DNA lesion as the inability to properly repair them can lead to genomic instability and tumorigenesis. The prominent DSB repair pathway in humans is non-homologous end-joining (NHEJ). In the simplest sense, NHEJ mediates the direct re-ligation of the broken DNA molecule. However, NHEJ is a complex and versatile process that can repair DSBs with a variety of damages and ends via the utilization of a significant number of proteins. In this review we will describe the important factors and mechanisms modulating NHEJ with emphasis given to the versatility of this repair process and the DNA-PK complex. PMID:24680878

  7. Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks.

    PubMed

    Kuhar, Ryan; Gwiazda, Kamila S; Humbert, Olivier; Mandt, Tyler; Pangallo, Joey; Brault, Michelle; Khan, Iram; Maizels, Nancy; Rawlings, David J; Scharenberg, Andrew M; Certo, Michael T

    2014-01-01

    The creation of a DNA break at a specific locus by a designer endonuclease can be harnessed to edit a genome. However, DNA breaks may engage one of several competing repair pathways that lead to distinct types of genomic alterations. Therefore, understanding the contribution of different repair pathways following the introduction of a targeted DNA break is essential to further advance the safety and efficiency of nuclease-induced genome modification. To gain insight into the role of different DNA repair pathways in resolving nuclease-induced DNA breaks into genome editing outcomes, we previously developed a fluorescent-based reporter system, designated the Traffic Light Reporter, which provides a readout of gene targeting and gene disruption downstream of a targeted DNA double-strand break. Here we describe two related but novel reporters that extend this technology: one that allows monitoring of the transcriptional activity at the reporter locus, and thus can be applied to interrogate break resolution at active and repressed loci; and a second that reads out single-strand annealing in addition to gene targeting and gene disruption. Application of these reporters to assess repair pathway usage in several common gene editing contexts confirms the importance that chromatin status and initiation of end resection have on the resolution of nuclease-induced breaks. PMID:24121685

  8. Involvement of Caveolin-1 in Repair of DNA Damage through Both Homologous Recombination and Non-Homologous End Joining

    PubMed Central

    Zhu, Hua; Yue, Jingyin; Pan, Zui; Wu, Hao; Cheng, Yan; Lu, Huimei; Ren, Xingcong; Yao, Ming; Shen, Zhiyuan; Yang, Jin-Ming

    2010-01-01

    Background Caveolin-1 (Cav-1), the major component of caveolae, is a 21–24 kDa integral membrane protein that interacts with a number of signaling molecules. By acting as a scaffolding protein, Cav-1 plays crucial roles in the regulation of various physiologic and patho-physiologic processes including oncogenic transformation and tumorigenesis, and tumor invasion and metastasis. Methodology/Principal Findings In the present study we sought to explore the role of Cav-1 in response to DNA damage and the mechanism involved. We found that the level of Cav-1 was up-regulated rapidly in cells treated with ionizing radiation. The up-regulation of Cav-1 following DNA damage occurred only in cells expressing endogenous Cav-1, and was associated with the activation of DNA damage response pathways. Furthermore, we demonstrated that the expression of Cav-1 protected cells against DNA damage through modulating the activities of both the homologous recombination (HR) and non-homologous end joining (NHEJ) repair systems, as evidenced by the inhibitory effects of the Cav-1-targeted siRNA on cell survival, HR frequency, phosphorylation of DNA-dependent protein kinase (DNA-PK), and nuclear translocation of epidermal growth factor receptor (EGFR) following DNA damage, and by the stimulatory effect of the forced expression of Cav-1 on NHEJ frequency. Conclusion/Significance Our results indicate that Cav-1 may play a critical role in sensing genotoxic stress and in orchestrating the response of cells to DNA damage through regulating the important molecules involved in maintaining genomic integrity. PMID:20700465

  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. 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. PMID:26573959

  11. Targeting the DNA repair pathway in Ewing sarcoma.

    PubMed

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

    2014-11-01

    Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice. PMID:25437539

  12. 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

  13. Loss of CtIP disturbs homologous recombination repair and sensitizes breast cancer cells to PARP inhibitors

    PubMed Central

    Fujimori, Hiroaki; Motegi, Akira; Miki, Yoshio; Masutani, Mitsuko

    2016-01-01

    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. PMID:26713604

  14. 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. PMID:26713604

  15. A Delicate Balance Between Repair and Replication Factors Regulates Recombination Between Divergent DNA Sequences in Saccharomyces cerevisiae.

    PubMed

    Chakraborty, Ujani; George, Carolyn M; Lyndaker, Amy M; Alani, Eric

    2016-02-01

    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

  16. RNF20-SNF2H Pathway of Chromatin Relaxation in DNA Double-Strand Break Repair

    PubMed Central

    Kato, Akihiro; Komatsu, Kenshi

    2015-01-01

    Rapid progress in the study on the association of histone modifications with chromatin remodeling factors has broadened our understanding of chromatin dynamics in DNA transactions. In DNA double-strand break (DSB) repair, the well-known mark of histones is the phosphorylation of the H2A variant, H2AX, which has been used as a surrogate marker of DSBs. The ubiquitylation of histone H2B by RNF20 E3 ligase was recently found to be a DNA damage-induced histone modification. This modification is required for DSB repair and regulated by a distinctive pathway from that of histone H2AX phosphorylation. Moreover, the connection between H2B ubiquitylation and the chromatin remodeling activity of SNF2H has been elucidated. In this review, we summarize the current knowledge of RNF20-mediated processes and the molecular link to H2AX-mediated processes during DSB repair. PMID:26184323

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

    PubMed Central

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

    2014-01-01

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

  18. Modeling damage complexity-dependent non-homologous end-joining repair pathway.

    PubMed

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

    2014-01-01

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

  19. 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

  20. 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. PMID:24561002

  1. Either Non-Homologous Ends Joining or Homologous Recombination Is Required to Repair Double-Strand Breaks in the Genome of Macrophage-Internalized Mycobacterium tuberculosis

    PubMed Central

    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. PMID:24658131

  2. 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

  3. Tousled kinase activator, gallic acid, promotes homologous recombinational repair and suppresses radiation cytotoxicity in salivary gland cells.

    PubMed

    Timiri Shanmugam, Prakash Srinivasan; Nair, Renjith Parameshwaran; De Benedetti, Arrigo; Caldito, Gloria; Abreo, Fleurette; Sunavala-Dossabhoy, Gulshan

    2016-04-01

    Accidental or medical radiation exposure of the salivary glands can gravely impact oral health. Previous studies have shown the importance of Tousled-like kinase 1 (TLK1) and its alternate start variant TLK1B in cell survival against genotoxic stresses. Through a high-throughput library screening of natural compounds, the phenolic phytochemical, gallic acid (GA), was identified as a modulator of TLK1/1B. This small molecule possesses anti-oxidant and free radical scavenging properties, but in this study, we report that in vitro it promotes survival of human salivary acinar cells, NS-SV-AC, through repair of ionizing radiation damage. Irradiated cells treated with GA show improved clonogenic survival compared to untreated controls. And, analyses of DNA repair kinetics by alkaline single-cell gel electrophoresis and γ-H2AX foci immunofluorescence indicate rapid resolution of DNA breaks in drug-treated cells. Study of DR-GFP transgene repair indicates GA facilitates homologous recombinational repair to establish a functional GFP gene. In contrast, inactivation of TLK1 or its shRNA knockdown suppressed resolution of radiation-induced DNA tails in NS-SV-AC, and homology directed repair in DR-GFP cells. Consistent with our results in culture, animals treated with GA after exposure to fractionated radiation showed better preservation of salivary function compared to saline-treated animals. Our results suggest that GA-mediated transient modulation of TLK1 activity promotes DNA repair and suppresses radiation cytoxicity in salivary gland cells. PMID:26855419

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

    PubMed

    Nagel, Zachary D; Chaim, Isaac A; Samson, Leona D

    2014-07-01

    Why does a constant barrage of DNA damage lead to disease in some individuals, while others remain healthy? This article surveys current work addressing the implications of inter-individual variation in DNA repair capacity for human health, and discusses the status of DNA repair assays as potential clinical tools for personalized prevention or treatment of disease. In particular, we highlight research showing that there are significant inter-individual variations in DNA repair capacity (DRC), and that measuring these differences provides important biological insight regarding disease susceptibility and cancer treatment efficacy. We emphasize work showing that it is important to measure repair capacity in multiple pathways, and that functional assays are required to fill a gap left by genome wide association studies, global gene expression and proteomics. Finally, we discuss research that will be needed to overcome barriers that currently limit the use of DNA repair assays in the clinic. PMID:24780560

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

    PubMed Central

    Nagel, Zachary D.; Chaim, Isaac. A.; Samson, Leona D.

    2014-01-01

    Why does a constant barrage of DNA damage lead to disease in some individuals, while others remain healthy? This article surveys current work addressing the implications of inter-individual variation in DNA repair capacity for human health, and discusses the status of DNA repair assays as potential clinical tools for personalized prevention or treatment of disease. In particular, we highlight research showing that there are significant inter-individual variations in DNA Repair Capacity (DRC), and that measuring these differences provides important biological insight regarding disease susceptibility and cancer treatment efficacy. We emphasize work showing that it is important to measure repair capacity in multiple pathways, and that functional assays are required to fill a gap left by genome wide association studies, global gene expression and proteomics. Finally, we discuss research that will be needed to overcome barriers that currently limit the use of DNA repair assays in the clinic. PMID:24780560

  6. The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway

    SciTech Connect

    Schiestl, R.H.; Prakash, S.; Prakash, L. )

    1990-04-01

    rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, the authors have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6{Delta}) mutations and show that they also suppress the {gamma}-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of {gamma}-ray sensitivity. The six suppressor mutations they isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. They show that suppression of rad6{Delta} is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6{Delta} SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.

  7. Nucleotide Excision Repair Pathway Polymorphisms and Pancreatic Cancer Risk: Evidence for role of MMS19L

    PubMed Central

    McWilliams, Robert R.; Bamlet, William R.; de Andrade, Mariza; Rider, David N.; Cunningham, Julie M.; Petersen, Gloria M.

    2009-01-01

    Background Nucleotide excision repair (NER) is a vital response to DNA damage, including damage from tobacco exposure. Single nucleotide polymorphisms (SNPs) in the NER pathway may encode alterations that affect DNA repair function and therefore influence risk for pancreatic cancer development. Methods A clinic based case-control study in non-Hispanic white persons compared 1,143 patients with pancreatic adenocarcinoma with 1,097 healthy controls. Twenty-seven genes directly and indirectly involved in the NER pathway were identified and 236 tag-SNPs were selected from 26 of these (one had no SNPs identified). Association studies were performed at the gene level by principal components analysis, while recursive partitioning analysis was utilized to identify potential gene-gene and gene-environment interactions within the pathway. At the individual SNP level, adjusted additive, dominant, and recessive models were investigated, and gene-environment interactions were also assessed. Results Gene level analyses showed an association of MMS19L genotype (chromosome 10q24.1) with altered pancreatic cancer risk (p=0.023). Haplotype analysis of MMS19L also showed a significant association (p=0.0132). Analyses of 7 individual SNPs in this gene showed both protective and risk associations for minor alleles, broadly distributed across patient subgroups defined by smoking status, sex, and age. Conclusion In a candidate pathway SNP association study analysis, common variation in a NER gene, MMS19L, was associated with risk for pancreatic cancer. PMID:19318433

  8. 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. PMID:27398742

  9. Functional variant analyses (FVAs) predict pathogenicity in the BRCA1 DNA double-strand break repair pathway.

    PubMed

    Loke, Johnny; Pearlman, Alexander; Upadhyay, Kinnari; Tesfa, Lydia; Shao, Yongzhao; Ostrer, Harry

    2015-06-01

    Heritable mutations in the BRCA1 and BRCA2 and other genes in the DNA double-strand break (DSB) repair pathway disrupt binding of the encoded proteins, transport into the nucleus and initiation of homologous recombination, thereby increasing cancer risk [Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T. and Livingston, D.M. (1997) Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell, 88, 265-275, Chen, J., Silver, D.P., Walpita, D., Cantor, S.B., Gazdar, A.F., Tomlinson, G., Couch, F.J., Weber, B.L., Ashley, T., Livingston, D.M. et al. (1998) Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells. Mol. Cell, 2, 317-328]. To meet the challenge of correct classification, flow cytometry-based functional variant analyses (FVAs) were developed to determine whether variants in DSB repair genes disrupted the binding of BRCA1 to BARD1, PALB2, BRCA2 and FANCD2, phosphorylation of p53 or BRCA1 nuclear localization in response to DNA damage caused by diepoxybutane, mitomycin C and bleomycin. Lymphoblastoid cells from individuals with BRCA1 pathogenic mutations, benign variants, and variants of uncertain significance or with known BRCA2, FANCC or NBN mutations were tested. Mutations in BRCA1 decreased nuclear localization of BRCA1 in response to individual or combination drug treatment. Mutations in BRCA1 reduced binding to co-factors, PALB2 and FANCD2 and decreased phosphorylation of p53. Mutations in BRCA2, FANCC and NBN decreased nuclear localization of BRCA1 in response to drug treatment, cofactors binding and p53 phosphorylation. Unsupervised cluster analysis of all and as few as two assays demonstrated two apparent clusters, high-risk BRCA1 mutations and phenocopies and low-risk, fully sequenced controls and variants of uncertain significance (VUS). Thus, two FVA assays distinguish BRCA1 mutations and phenocopies from benign variants and categorize most VUS as benign

  10. Role of the double-strand break repair pathway in the maintenance of genomic stability

    PubMed Central

    Le Guen, Tangui; Ragu, Sandrine; Guirouilh-Barbat, Josée; Lopez, Bernard S

    2015-01-01

    DNA double-strand breaks (DSBs) are highly lethal lesions that jeopardize genome integrity. However, DSBs are also used to generate diversity during the physiological processes of meiosis or establishment of the immune repertoire. Therefore, DSB repair must be tightly controlled. Two main strategies are used to repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). HR is generally considered to be error-free, whereas NHEJ is considered to be error-prone. However, recent data challenge these assertions. Here, we present the molecular mechanisms involved in HR and NHEJ and the recently described alternative end-joining mechanism, which is exclusively mutagenic. Whereas NHEJ is not intrinsically error-prone but adaptable, HR has the intrinsic ability to modify the DNA sequence. Importantly, in both cases the initial structure of the DNA impacts the outcome. Finally, the consequences and applications of these repair mechanisms are discussed. Both HR and NHEJ are double-edged swords, essential for maintenance of genome stability and diversity but also able to generate genome instability. PMID:27308383

  11. Inhibition of Wnt/β-catenin pathway promotes regenerative repair of cutaneous and cartilage injury.

    PubMed

    Bastakoty, Dikshya; Saraswati, Sarika; Cates, Justin; Lee, Ethan; Nanney, Lillian B; Young, Pampee P

    2015-12-01

    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. PMID:26268926

  12. 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.

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

    PubMed

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

    2009-07-10

    Structure-specific endonucleases resolve DNA secondary structures generated during DNA repair and recombination. The yeast 5' flap endonuclease Slx1-Slx4 has received particular attention with the finding that Slx4 has Slx1-independent key functions in genome maintenance. Although Slx1 is a highly conserved protein in eukaryotes, no orthologs of Slx4 were reported other than in fungi. Here we report the identification of Slx4 orthologs in metazoa, including fly MUS312, essential for meiotic recombination, and human BTBD12, an ATM/ATR checkpoint kinase substrate. Human SLX1-SLX4 displays robust Holliday junction resolvase activity in addition to 5' flap endonuclease activity. Depletion of SLX1 and SLX4 results in 53BP1 foci accumulation and H2AX phosphorylation as well as cellular hypersensitivity to MMS. Furthermore, we show that SLX4 binds the XPF(ERCC4) and MUS81 subunits of the XPF-ERCC1 and MUS81-EME1 endonucleases and is required for DNA interstrand crosslink repair. We propose that SLX4 acts as a docking platform for multiple structure-specific endonucleases. PMID:19596236

  14. 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

  15. 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

  16. 5-Fluorouracil sensitizes colorectal tumor cells towards double stranded DNA breaks by interfering with homologous recombination repair

    PubMed Central

    Srinivas, Upadhyayula Sai; Dyczkowski, Jerzy; Beißbarth, Tim; Gaedcke, Jochen; Mansour, Wael Y.; Borgmann, Kerstin; Dobbelstein, Matthias

    2015-01-01

    Malignant tumors of the rectum are treated by neoadjuvant radiochemotherapy. This involves a combination of 5-fluorouracil (5-FU) and double stranded DNA-break (DSB)-inducing radiotherapy. Here we explored how 5-FU cooperates with DSB-induction to achieve sustainable DNA damage in colorectal cancer (CRC) cells. After DSB induction by neocarzinostatin, phosphorylated histone 2AX (γ-H2AX) rapidly accumulated but then largely vanished within a few hours. In contrast, when CRC cells were pre-treated with 5-FU, gammaH2AX remained for at least 24 hours. GFP-reporter assays revealed that 5-FU decreases the efficiency of homologous recombination (HR) repair. However, 5-FU did not prevent the initial steps of HR repair, such as the accumulation of RPA and Rad51 at nuclear foci. Thus, we propose that 5-FU interferes with the continuation of HR repair, e. g. the synthesis of new DNA strands. Two key mediators of HR, Rad51 and BRCA2, were found upregulated in CRC biopsies as compared to normal mucosa. Inhibition of HR by targeting Rad51 enhanced DNA damage upon DSB-inducing treatment, outlining an alternative way of enhancing therapeutic efficacy. Taken together, our results strongly suggest that interfering with HR represents a key mechanism to enhance the efficacy when treating CRC with DNA-damaging therapy. PMID:25909291

  17. 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

  18. 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.

  19. 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

  20. 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

  1. The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway

    PubMed Central

    Gros, Laurent; Ishchenko, Alexander A.; Ide, Hiroshi; Elder, Rhoderick H.; Saparbaev, Murat K.

    2004-01-01

    In nucleotide incision repair (NIR), an endonuclease nicks oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to the repair of the remaining 5′-dangling modified nucleotide. This mechanistic feature is distinct from DNA glycosylase-mediated base excision repair. Here we report that Ape1, the major apurinic/apyrimidinic endonuclease in human cells, is the damage- specific endonuclease involved in NIR. We show that Ape1 incises DNA containing 5,6-dihydro-2′-deoxyuridine, 5,6-dihydrothymidine, 5-hydroxy-2′-deoxyuridine, alpha-2′-deoxyadenosine and alpha-thymidine adducts, generating 3′-hydroxyl and 5′-phosphate termini. The kinetic constants indicate that Ape1-catalysed NIR activity is highly efficient. The substrate specificity and protein conformation of Ape1 is modulated by MgCl2 concentrations, thus providing conditions under which NIR becomes a major activity in cell-free extracts. While the N-terminal region of Ape1 is not required for AP endonuclease function, we show that it regulates the NIR activity. The physiological relevance of the mammalian NIR pathway is discussed. PMID:14704345

  2. Proteomic Analysis Reveals a Novel Mutator S (MutS) Partner Involved in Mismatch Repair Pathway.

    PubMed

    Chen, Zhen; Tran, Mykim; Tang, Mengfan; Wang, Wenqi; Gong, Zihua; Chen, Junjie

    2016-04-01

    The mismatch repair (MMR) family is a highly conserved group of proteins that function in correcting base-base and insertion-deletion mismatches generated during DNA replication. Disruption of this process results in characteristic microsatellite instability (MSI), repair defects, and susceptibility to cancer. However, a significant fraction of MSI-positive cancers express MMR genes at normal levels and do not carry detectable mutation in known MMR genes, suggesting that additional factors and/or mechanisms may exist to explain these MSI phenotypes in patients. To systematically investigate the MMR pathway, we conducted a proteomic analysis and identified MMR-associated protein complexes using tandem-affinity purification coupled with mass spectrometry (TAP-MS) method. The mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD003014 and DOI 10.6019/PXD003014. We identified 230 high-confidence candidate interaction proteins (HCIPs). We subsequently focused on MSH2, an essential component of the MMR pathway and uncovered a novel MSH2-binding partner, WDHD1. We further demonstrated that WDHD1 forms a stable complex with MSH2 and MSH3 or MSH6,i.e.the MutS complexes. The specific MSH2/WDHD1 interaction is mediated by the second lever domain of MSH2 and Ala(1123)site of WDHD1. Moreover, we showed that, just like MSH2-deficient cells, depletion of WDHD1 also led to 6-thioguanine (6-TG) resistance, indicating that WDHD1 likely contributes to the MMR pathway. Taken together, our study uncovers new components involved in the MMR pathway, which provides candidate genes that may be responsible for the development of MSI-positive cancers. PMID:27037360

  3. Polymorphisms in DNA Repair Pathway Genes, Body Mass Index, and Risk of Non-Hodgkin Lymphoma

    PubMed Central

    Chen, Yingtai; Zheng, Tongzhang; Lan, Qing; Kim, Christopher; Qin, Qin; Foss, Francine; Chen, Xuezhong; Holford, Theodore; Leaderer, Brian; Boyle, Peter; Wang, Chengfeng; Dai, Min; Liu, Zhenjiang; Ma, Shuangge; Chanock, Stephen J.; Rothman, Nathaniel; Zhang, Yawei

    2013-01-01

    We conducted a population-based case-control study in Connecticut women to test the hypothesis that genetic variations in DNA repair pathway genes may modify the relationship between body mass index (BMI) and risk of non-Hodgkin lymphoma (NHL). Compared to those with BMI < 25, women with BMI ≥ 25 had significantly increased risk of NHL among women who carried BRCA1 (rs799917) CT/TT, ERCC2 (rs13181) AA, XRCC1 (rs1799782) CC, and WRN (rs1801195) GG genotypes, but no increase in NHL risk among women who carried BRCA1 CC, ERCC2 AC/CC, XRCC1 CT/TT, and WRN GT/TT genotypes. A significant interaction with BMI was only observed for WRN (rs1801195, P=0.004) for T-cell lymphoma and ERCC2 (rs13181, P=0.002) for diffuse large B-cell lymphoma. The results suggest that common genetic variation in DNA repair pathway genes may modify the association between BMI and NHL risk. PMID:23619945

  4. Epidermal wound repair is regulated by the planar cell polarity signaling pathway.

    PubMed

    Caddy, Jacinta; Wilanowski, Tomasz; Darido, Charbel; Dworkin, Sebastian; Ting, Stephen B; Zhao, Quan; Rank, Gerhard; Auden, Alana; Srivastava, Seema; Papenfuss, Tony A; Murdoch, Jennifer N; Humbert, Patrick O; Parekh, Vishwas; Boulos, Nidal; Weber, Thomas; Zuo, Jian; Cunningham, John M; Jane, Stephen M

    2010-07-20

    The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. Here, we show that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, Celsr1, PTK7, and Scrb1, and the transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects, and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3(-)(/-) mice, we identified RhoGEF19, a homolog of a RhoA activator involved in PCP signaling in Xenopus, as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerization, cellular polarity, and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling and broadly implicate this pathway in epidermal repair. PMID:20643356

  5. Epidermal wound repair is regulated by the planar cell polarity signaling pathway

    PubMed Central

    Caddy, Jacinta; Wilanowski, Tomasz; Darido, Charbel; Dworkin, Sebastian; Ting, Stephen B.; Zhao, Quan; Rank, Gerhard; Auden, Alana; Srivastava, Seema; Papenfuss, Tony A.; Murdoch, Jennifer N.; Humbert, Patrick O.; Boulos, Nidal; Weber, Thomas; Zuo, Jian; Cunningham, John M.; Jane, Stephen M.

    2010-01-01

    SUMMARY The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. Here, we show that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, Celsr1, PTK7, and Scrb1, and the transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3−/− mice, we identified RhoGEF19, a homologue of a RhoA activator involved in PCP signaling in Xenopus, as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerisation, cellular polarity and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling, and broadly implicate this pathway in epidermal repair. PMID:20643356

  6. Biosynthetic pathway for acrylic acid from glycerol in recombinant Escherichia coli.

    PubMed

    Tong, Wenhua; Xu, Ying; Xian, Mo; Niu, Wei; Guo, Jiantao; Liu, Huizhou; Zhao, Guang

    2016-06-01

    Acrylic acid is an important industrial feedstock. In this study, a de novo acrylate biosynthetic pathway from inexpensive carbon source glycerol was constructed in Escherichia coli. The acrylic acid was produced from glycerol via 3-hydroxypropionaldehyde, 3-hydroxypropionyl-CoA, and acrylyl-CoA. The acrylate production was improved by screening and site-directed mutagenesis of key enzyme enoyl-CoA hydratase and chromosomal integration of some exogenous genes. Finally, our recombinant strain produced 37.7 mg/L acrylic acid under shaking flask conditions. Although the acrylate production is low, our study shows feasibility of engineering an acrylate biosynthetic pathway from inexpensive carbon source. Furthermore, the reasons for limited acrylate production and further strain optimization that should be performed in the future were also discussed. PMID:26782744

  7. DNA-repair pathway inhibitors for the treatment of ovarian cancer

    PubMed Central

    Martinek, Igor; Haldar, Krishnayan; Gaitskell, Kezia; Bryant, Andrew; Nicum, Shibani; Kehoe, Sean; Morrison, Jo

    2014-01-01

    Background Ovarian cancer is the sixth most common cancer and seventh most common cause of cancer death in women world-wide.Three-quarters of women present when the disease has spread through-put the abdomen (stage III or IV) and treatment consists of a combination of debulking surgery and platinum-based chemotherapy, with or without taxanes. Although initial responses to chemotherapy are often good, most women will relapse and require further chemotherapy and will eventually develop resistance to chemotherapy agents. Increased understanding about the molecular basis of ovarian cancer has lead to the development of novel agents, which work in different ways to conventional chemotherapy. These include DNA-repair pathway inhibitors, the commonest of which are the PARP (poly (ADP-ribose) polymerase) inhibitors. It is therefore important to compare their effectiveness and side effects of these novel agents to assess their role in the treatment of advanced ovarian cancer, especially as treatment of advanced disease is aiming to improve length of survival and quality of life (QoL). Objectives To compare the effectiveness and harmful effects of interventions, which inhibit DNA-repair pathways, in the treatment of ovarian cancer. Search methods RCTs were identified by searching The Cochrane Central Register of Controlled Trials (CENTRAL, Issue 2, 2009), The Cochrane Gynaecological Cancer Collaborative Review Group’s Trial Register, MEDLINE (1990 to June 2009), EMBASE (1990 to June 2009), ongoing trials on www.controlled-trials.com/rct, www.clinicaltrials.gov, www.cancer.gov/clinicaltrials and the National Research Register (NRR), the FDA database and pharmaceutical industry biomedical literature. Selection criteria Adult women with histologically proven ovarian cancer who were randomised to treatment groups which either compared DNA-repair pathway inhibitors with no treatment or DNA-repair pathway inhibitors together with conventional chemotherapy compared with

  8. Homologous recombination-dependent repair of telomeric DSBs in proliferating human cells.

    PubMed

    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

  9. 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

  10. Sensitization of Pancreatic Cancers to Gemcitabine Chemoradiation by WEE1 Kinase Inhibition Depends on Homologous Recombination Repair12

    PubMed Central

    Kausar, Tasneem; Schreiber, Jason S.; Karnak, David; Parsels, Leslie A.; Parsels, Joshua D.; Davis, Mary A.; Zhao, Lili; Maybaum, Jonathan; Lawrence, Theodore S.; Morgan, Meredith A.

    2015-01-01

    To improve the efficacy of chemoradiation therapy for locally advanced pancreatic cancer and begin to establish patient selection criteria, we investigated the combination of the WEE1 inhibitor AZD1775 with gemcitabine-radiation in homologous recombination (HR) repair proficient and deficient pancreatic cancers. Sensitization to gemcitabine-radiation by AZD1775 was assessed in pancreatic cancer cells by clonogenic survival and in patient-derived xenografts by tumor growth. The contributions of HR repair inhibition and G2 checkpoint abrogation to sensitization were assessed by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistone H3 flow cytometry, respectively. We found that AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type but not BRCA2 mutant pancreatic cancer cells. In all cells, AZD1775 caused inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. However, sensitization by AZD1775 was associated with persistent γH2AX and inhibition of RAD51 focus formation. In HR-proficient (BRCA2 wild-type) or -deficient (BRAC2 null) isogenic cells, AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type, but not in BRCA2 null cells, despite significant G2 checkpoint abrogation. In patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibited tumor growth and impaired RAD51 focus formation in response to gemcitabine-radiation. In conclusion, WEE1 inhibition by AZD1775 is an effective strategy for sensitizing pancreatic cancers to gemcitabine chemoradiation. Although this sensitization is accompanied by inhibition of CDK1 phosphorylation and G2 checkpoint abrogation, this mechanism is not sufficient for sensitization. Our findings demonstrate that sensitization to chemoradiation by WEE1 inhibition results from inhibition of HR repair and suggest that patient tumors without underlying HR defects would benefit most from this therapy. PMID:26585231

  11. 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

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

    PubMed Central

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

    2015-01-01

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

  13. A Role for the Malignant Brain Tumour (MBT) Domain Protein LIN-61 in DNA Double-Strand Break Repair by Homologous Recombination

    PubMed Central

    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. PMID:23505385

  14. 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. PMID:27417934

  15. 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

  16. Interplay of Mre11 Nuclease with Dna2 plus Sgs1 in Rad51-Dependent Recombinational Repair

    PubMed Central

    Budd, Martin E.; Campbell, Judith L.

    2009-01-01

    The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5′ to 3′ exonuclease degradation creating a single-stranded 3′ overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3′ to 5′, rather than 5′ to 3′ activity. Furthermore, mutations that inactivate only the nuclease activity of Mre11 but not its other repair functions, mre11-D56N and mre11-H125N, are resistant to IR. This suggests that another nuclease can catalyze 5′ to 3′ degradation. One candidate nuclease that has not been tested to date because it is encoded by an essential gene is the Dna2 helicase/nuclease. We recently reported the ability to suppress the lethality of a dna2Δ with a pif1Δ. The dna2Δ pif1Δ mutant is IR-resistant. We have determined that dna2Δ pif1Δ mre11-D56N and dna2Δ pif1Δ mre11-H125N strains are equally as sensitive to IR as mre11Δ strains, suggesting that in the absence of Dna2, Mre11 nuclease carries out repair. The dna2Δ pif1Δ mre11-D56N triple mutant is complemented by plasmids expressing Mre11, Dna2 or dna2K1080E, a mutant with defective helicase and functional nuclease, demonstrating that the nuclease of Dna2 compensates for the absence of Mre11 nuclease in IR repair, presumably in 5′ to 3′ degradation at DSB ends. We further show that sgs1Δ mre11-H125N, but not sgs1Δ, is very sensitive to IR, implicating the Sgs1 helicase in the Dna2-mediated pathway. PMID:19165339

  17. A new gene involved in DNA double-strand break repair and V(D)J recombination is located on human chromosome 10p.

    PubMed

    Moshous, D; Li, L; Chasseval, R; Philippe, N; Jabado, N; Cowan, M J; Fischer, A; de Villartay, J P

    2000-03-01

    V(D)J recombination, accountable for the diversity of T cell receptor- and immunoglobulin-encoding genes, is initiated by a lymphoid-specific DNA double-strand break. The general DNA repair machinery is responsible for the resolution of this break. Any defect in one of the known components of the DNA repair/V(D)J recombination machinery (Ku70, Ku80, DNA-PKcs, XRCC4 and DNA ligase IV) leads to abortion of the V(D)J rearrangement process, early block in both T and B cell maturation, and ultimately to severe combined immune deficiency (SCID) in several animal models. A human SCID condition is also characterized by an absence of mature T and B lymphocytes, and is associated with an increase in sensitivity to DNA-damaging agents (RS-SCID). None of the above-mentioned genes are defective in these patients, arguing for the likelihood of the existence of yet another unknown component of the V(D)J recombination/DNA repair apparatus. Athabascan-speaking (SCIDA) Navajo and Apache Native Americans have a very high incidence of T(-)B(-)SCID. The SCIDA locus is highly linked with markers on chromosome 10p, although the exact molecular defect has not been recognized in these patients. We show here that cells with the SCIDA defect are impaired in the DNA repair phase of V(D)J recombination similarly to RS-SCID, precisely an absence of V(D)J coding joint formation. Moreover, genotyping analysis in several RS-SCID families corroborates a linkage of the RS-SCID locus to the SCIDA region on chromosome 10p. These results demonstrate the presence of a new essential DNA repair/V(D)J recombination gene in this region, the mutation of which causes RS-SCID in humans. PMID:10699181

  18. Heterozygous PALB2 c.1592delT mutation channels DNA double-strand break repair into error-prone pathways in breast cancer patients.

    PubMed

    Obermeier, K; Sachsenweger, J; Friedl, T W P; Pospiech, H; Winqvist, R; Wiesmüller, L

    2016-07-21

    Hereditary heterozygous mutations in a variety of DNA double-strand break (DSB) repair genes have been associated with increased breast cancer risk. In the Finnish population, PALB2 (partner and localizer of BRCA2) represents a major susceptibility gene for female breast cancer, and so far, only one mutation has been described, c.1592delT, which leads to a sixfold increased disease risk. PALB2 is thought to participate in homologous recombination (HR). However, the effect of the Finnish founder mutation on DSB repair has not been investigated. In the current study, we used a panel of lymphoblastoid cell lines (LCLs) derived from seven heterozygous female PALB2 c.1592delT mutation carriers with variable health status and six wild-type matched controls. The results of our DSB repair analysis showed that the PALB2 mutation causes specific changes in pathway usage, namely increases in error-prone single-strand annealing (SSA) and microhomology-mediated end-joining (MMEJ) compared with wild-type LCLs. These data indicated haploinsufficiency regarding the suppression of error-prone DSB repair in PALB2 mutation carriers. To the contrary, neither reduced HR activities, nor impaired RAD51 filament assembly, nor sensitization to PARP inhibition were consistently observed. Expression of truncated mutant versus wild-type PALB2 verified a causal role of PALB2 c.1592delT in the shift to error-prone repair. Discrimination between healthy and malignancy-presenting PALB2 mutation carriers revealed a pathway shift particularly in the breast cancer patients, suggesting interaction of PALB2 c.1592delT with additional genomic lesions. Interestingly, the studied PALB2 mutation was associated with 53BP1 accumulation in the healthy mutation carriers but not the patients, and 53BP1 was limiting for error-prone MMEJ in patients but not in healthy carriers. Our study identified a rise in error-prone DSB repair as a potential threat to genomic integrity in heterozygous PALB2 mutation carriers

  19. Heterozygous PALB2 c.1592delT mutation channels DNA double-strand break repair into error-prone pathways in breast cancer patients

    PubMed Central

    Obermeier, K; Sachsenweger, J; Friedl, T W P; Pospiech, H; Winqvist, R; Wiesmüller, L

    2016-01-01

    Hereditary heterozygous mutations in a variety of DNA double-strand break (DSB) repair genes have been associated with increased breast cancer risk. In the Finnish population, PALB2 (partner and localizer of BRCA2) represents a major susceptibility gene for female breast cancer, and so far, only one mutation has been described, c.1592delT, which leads to a sixfold increased disease risk. PALB2 is thought to participate in homologous recombination (HR). However, the effect of the Finnish founder mutation on DSB repair has not been investigated. In the current study, we used a panel of lymphoblastoid cell lines (LCLs) derived from seven heterozygous female PALB2 c.1592delT mutation carriers with variable health status and six wild-type matched controls. The results of our DSB repair analysis showed that the PALB2 mutation causes specific changes in pathway usage, namely increases in error-prone single-strand annealing (SSA) and microhomology-mediated end-joining (MMEJ) compared with wild-type LCLs. These data indicated haploinsufficiency regarding the suppression of error-prone DSB repair in PALB2 mutation carriers. To the contrary, neither reduced HR activities, nor impaired RAD51 filament assembly, nor sensitization to PARP inhibition were consistently observed. Expression of truncated mutant versus wild-type PALB2 verified a causal role of PALB2 c.1592delT in the shift to error-prone repair. Discrimination between healthy and malignancy-presenting PALB2 mutation carriers revealed a pathway shift particularly in the breast cancer patients, suggesting interaction of PALB2 c.1592delT with additional genomic lesions. Interestingly, the studied PALB2 mutation was associated with 53BP1 accumulation in the healthy mutation carriers but not the patients, and 53BP1 was limiting for error-prone MMEJ in patients but not in healthy carriers. Our study identified a rise in error-prone DSB repair as a potential threat to genomic integrity in heterozygous PALB2 mutation carriers

  20. Recombinant 1F9 spidroin microgels for murine full-thickness wound repairing.

    PubMed

    Moisenovich, M M; Malyuchenko, N V; Arkhipova, A Yu; Goncharenko, A V; Kotlyarova, M S; Davydova, L I; Vasil'eva, T V; Bogush, V G; Agapov, I I; Debabov, V G; Kirpichnikov, M P

    2016-01-01

    The study of the stimulating effect of the microgels (MGs) based on recombinant 1F9 spidroin on the regeneration of the deep skin wound in mice was carried out. The use of spidroin MGs was shown to increase significantly the quality of healing compared to the control. The introduction of the MG in the wound edges led to recovery of all the structural elements of the skin: the epidermis, the dermis, including vascular and nervous network, in the periphery of the wound underlying muscles, and skin appendages (sebaceous and sweat glands and hair follicles) was revealed. PMID:27025477

  1. Elevated metals compromise repair of oxidative DNA damage via the base excision repair pathway: implications of pathologic iron overload in the brain on integrity of neuronal DNA.

    PubMed

    Li, Hui; Swiercz, Rafal; Englander, Ella W

    2009-09-01

    Tissue-specific iron content is tightly regulated to simultaneously satisfy specialized metabolic needs and avoid cytotoxicity. In the brain, disruption of iron homeostasis may occur in acute as well as progressive injuries associated with neuronal dysfunction and death. We hypothesized that adverse effects of disrupted metal homeostasis on brain function may involve impairment of DNA repair processes. Because in the brain, the base excision repair (BER) pathway is central for handling oxidatively damaged DNA, we investigated effects of elevated iron and zinc on key BER enzymes. In vitro DNA repair assays revealed inhibitory effects of metals on BER activities, including the incision of abasic sites, 5'-flap cleavage, gap filling DNA synthesis and ligation. Using the comet assay, we showed that while metals at concentrations which inhibit BER activities in in vitro assays, did not induce direct genomic damage in cultured primary neurons, they significantly delayed repair of genomic DNA damage induced by sublethal exposure to H(2)O(2). Thus, in the brain even a mild transient metal overload, may adversely affect the DNA repair capacity and thereby compromise genomic integrity and initiate long-term deleterious sequelae including neuronal dysfunction and death. PMID:19619136

  2. Polymorphisms within base and nucleotide excision repair pathways and risk of differentiated thyroid carcinoma.

    PubMed

    Cipollini, Monica; Figlioli, Gisella; Maccari, Giuseppe; Garritano, Sonia; De Santi, Chiara; Melaiu, Ombretta; Barone, Elisa; Bambi, Franco; Ermini, Stefano; Pellegrini, Giovanni; Cristaudo, Alfonso; Foddis, Rudy; Bonotti, Alessandra; Romei, Cristina; Vivaldi, Agnese; Agate, Laura; Molinari, Eleonora; Barale, Roberto; Forsti, Asta; Hemminki, Kari; Elisei, Rossella; Gemignani, Federica; Landi, Stefano

    2016-05-01

    The thyrocytes are exposed to high levels of oxidative stress which could induce DNA damages. Base excision repair (BER) is one of the principal mechanisms of defense against oxidative DNA damage, however recent evidences suggest that also nucleotide excision repair (NER) could be involved. The aim of present work was to identify novel differentiated thyroid cancer (DTC) risk variants in BER and NER genes. For this purpose, the most strongly associated SNPs within NER and BER genes found in our previous GWAS on DTC were selected and replicated in an independent series of samples for a new case-control study. Although a positive signal was detected at the nominal level of 0.05 for rs7689099 (encoding for an aminoacid change proline to arginine at codon 117 within NEIL3), none of the considered SNPs (i.e. rs7990340 and rs690860 within RFC3, rs3744767 and rs1131636 within RPA1, rs16962916 and rs3136166 in ERCC4, and rs17739370 and rs7689099 in NEIL3) was associated with the risk of DTC when the correction of multiple testing was applied. In conclusion, a role of NER and BER pathways was evoked in the susceptibility to DTC. However, this seemed to be limited to few polymorphic genes and the overall effect size appeared weak. PMID:27062014

  3. Regulation and Disregulation of Mammalian Nucleotide Excision Repair: a Pathway to Non-germline Breast Carcinogenesis†

    PubMed Central

    Latimer, Jean J.; Majekwana, Vongai J.; Pabón-Padín, Yashira R.; Pimpley, Manasi R.; Grant, Stephen G.

    2015-01-01

    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 paper, 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. PMID:25393451

  4. Human DNA repair genes.

    PubMed

    Wood, R D; Mitchell, M; Sgouros, J; Lindahl, T

    2001-02-16

    Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process. PMID:11181991

  5. 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.

  6. 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.

  7. Regulation of axon regeneration by the RNA repair and splicing pathway.

    PubMed

    Song, Yuanquan; Sretavan, David; Salegio, Ernesto A; Berg, Jim; Huang, Xi; Cheng, Tong; Xiong, Xin; Meltzer, Shan; Han, Chun; Nguyen, Trong-Tuong; Bresnahan, Jacqueline C; Beattie, Michael S; Jan, Lily Yeh; Jan, Yuh Nung

    2015-06-01

    Mechanisms governing a neuron's regenerative ability are important but not well understood. We identify Rtca (RNA 3'-terminal phosphate cyclase) as an inhibitor of axon regeneration. Removal of Rtca cell-autonomously enhanced axon regrowth in the Drosophila CNS, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease operate in the RNA repair and splicing pathway important for stress-induced mRNA splicing, including that of Xbp1, a cellular stress sensor. Drosophila Rtca and Archease had opposing effects on Xbp1 splicing, and deficiency of Archease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. Our study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma. PMID:25961792

  8. 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

  9. 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

    2015-01-01

    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

  10. The study of the relation of DNA repair pathway genes SNPs and the sensitivity to radiotherapy and chemotherapy of NSCLC.

    PubMed

    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

  11. 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

  12. Increasing pentose phosphate pathway flux enhances recombinant protein production in Pichia pastoris.

    PubMed

    Nocon, Justyna; Steiger, Matthias; Mairinger, Teresa; Hohlweg, Jonas; Rußmayer, Hannes; Hann, Stephan; Gasser, Brigitte; Mattanovich, Diethard

    2016-07-01

    Production of heterologous proteins in Pichia pastoris (syn. Komagataella sp.) has been shown to exert a metabolic burden on the host metabolism. This burden is associated with metabolite drain, which redirects nucleotides and amino acids from primary metabolism. On the other hand, recombinant protein production affects energy and redox homeostasis of the host cell. In a previous study, we have demonstrated that overexpression of single genes of the oxidative pentose phosphate pathway (PPP) had a positive influence on recombinant production of cytosolic human superoxide dismutase (hSOD). In this study, different combinations of these genes belonging to the oxidative PPP were generated and analyzed. Thereby, a 3.8-fold increase of hSOD production was detected when glucose-6-phosphate dehydrogenase (ZWF1) and 6-gluconolactonase (SOL3) were simultaneously overexpressed, while the combinations of other genes from PPP had no positive effect on protein production. By measuring isotopologue patterns of (13)C-labelled metabolites, we could detect an upshift in the flux ratio of PPP to glycolysis upon ZWF1 and SOL3 co-overexpression, as well as increased levels of 6-phosphogluconate. The substantial improvement of hSOD production by ZWF1 and SOL3 co-overexpression appeared to be connected to an increase in PPP flux. In conclusion, we show that overexpression of SOL3 together with ZWF1 enhanced both the PPP flux ratio and hSOD accumulation, providing evidence that in P. pastoris Sol3 limits the flux through PPP and recombinant protein production. PMID:27020289

  13. SERBP1 affects homologous recombination-mediated DNA repair by regulation of CtIP translation during S phase

    PubMed Central

    Ahn, Jang-Won; Kim, Sunjik; Na, Wooju; Baek, Su-Jin; Kim, Jeong-Hwan; Min, Keehong; Yeom, Jeonghun; Kwak, Hoyun; Jeong, Sunjoo; Lee, Cheolju; Kim, Seon-Young; Choi, Cheol Yong

    2015-01-01

    DNA double-strand breaks (DSBs) are the most severe type of DNA damage and are primarily repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) in the G1 and S/G2 phase, respectively. Although CtBP-interacting protein (CtIP) is crucial in DNA end resection during HR following DSBs, little is known about how CtIP levels increase in an S phase-specific manner. Here, we show that Serpine mRNA binding protein 1 (SERBP1) regulates CtIP expression at the translational level in S phase. In response to camptothecin-mediated DNA DSBs, CHK1 and RPA2 phosphorylation, which are hallmarks of HR activation, was abrogated in SERBP1-depleted cells. We identified CtIP mRNA as a binding target of SERBP1 using RNA immunoprecipitation-coupled RNA sequencing, and confirmed SERBP1 binding to CtIP mRNA in S phase. SERBP1 depletion resulted in reduction of polysome-associated CtIP mRNA and concomitant loss of CtIP expression in S phase. These effects were reversed by reconstituting cells with wild-type SERBP1, but not by SERBP1 ΔRGG, an RNA binding defective mutant, suggesting regulation of CtIP translation by SERBP1 association with CtIP mRNA. These results indicate that SERBP1 affects HR-mediated DNA repair in response to DNA DSBs by regulation of CtIP translation in S phase. PMID:26068472

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

    PubMed Central

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

    2015-01-01

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

  15. 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

  16. Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers.

    PubMed

    Fonseca, A S; Campos, V M A; Magalhães, L A G; Paoli, F

    2015-10-01

    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

  17. 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

  18. Genome-Wide Screen Reveals Replication Pathway for Quasi-Palindrome Fragility Dependent on Homologous Recombination

    PubMed Central

    Zhang, Yu; Saini, Natalie; Sheng, Ziwei; Lobachev, Kirill S.

    2013-01-01

    Inverted repeats capable of forming hairpin and cruciform structures present a threat to chromosomal integrity. They induce double strand breaks, which lead to gross chromosomal rearrangements, the hallmarks of cancers and hereditary diseases. Secondary structure formation at this motif has been proposed to be the driving force for the instability, albeit the mechanisms leading to the fragility are not well-understood. We carried out a genome-wide screen to uncover the genetic players that govern fragility of homologous and homeologous Alu quasi-palindromes in the yeast Saccharomyces cerevisiae. We found that depletion or lack of components of the DNA replication machinery, proteins involved in Fe-S cluster biogenesis, the replication-pausing checkpoint pathway, the telomere maintenance complex or the Sgs1-Top3-Rmi1 dissolvasome augment fragility at Alu-IRs. Rad51, a component of the homologous recombination pathway, was found to be required for replication arrest and breakage at the repeats specifically in replication-deficient strains. These data demonstrate that Rad51 is required for the formation of breakage-prone secondary structures in situations when replication is compromised while another mechanism operates in DSB formation in replication-proficient strains. PMID:24339793

  19. Genome-wide screen reveals replication pathway for quasi-palindrome fragility dependent on homologous recombination.

    PubMed

    Zhang, Yu; Saini, Natalie; Sheng, Ziwei; Lobachev, Kirill S

    2013-01-01

    Inverted repeats capable of forming hairpin and cruciform structures present a threat to chromosomal integrity. They induce double strand breaks, which lead to gross chromosomal rearrangements, the hallmarks of cancers and hereditary diseases. Secondary structure formation at this motif has been proposed to be the driving force for the instability, albeit the mechanisms leading to the fragility are not well-understood. We carried out a genome-wide screen to uncover the genetic players that govern fragility of homologous and homeologous Alu quasi-palindromes in the yeast Saccharomyces cerevisiae. We found that depletion or lack of components of the DNA replication machinery, proteins involved in Fe-S cluster biogenesis, the replication-pausing checkpoint pathway, the telomere maintenance complex or the Sgs1-Top3-Rmi1 dissolvasome augment fragility at Alu-IRs. Rad51, a component of the homologous recombination pathway, was found to be required for replication arrest and breakage at the repeats specifically in replication-deficient strains. These data demonstrate that Rad51 is required for the formation of breakage-prone secondary structures in situations when replication is compromised while another mechanism operates in DSB formation in replication-proficient strains. PMID:24339793

  20. DNA double-strand break repair inhibitors as cancer therapeutics.

    PubMed

    Srivastava, Mrinal; Raghavan, Sathees C

    2015-01-22

    Among DNA damages, double-strand breaks (DSBs) are one of the most harmful lesions to a cell. Failure in DSB repair could lead to genomic instability and cancer. Homologous recombination (HR) and nonhomologous end joining (NHEJ) are major DSB repair pathways in higher eukaryotes. It is known that expression of DSB repair genes is altered in various cancers. Activation of DSB repair genes is one of the reasons for chemo- and radioresistance. Therefore, targeting DSB repair is an attractive strategy to eliminate cancer. Besides, therapeutic agents introduce breaks in the genome as an intermediate. Therefore, blocking the residual repair using inhibitors can potentiate the efficacy of cancer treatment. In this review, we discuss the importance of targeting DSB repair pathways for the treatment of cancer. Recent advances in the development of DSB repair inhibitors and their clinical relevance are also addressed. PMID:25579208

  1. A physiological significance of the functional interaction between Mus81 and Rad27 in homologous recombination repair

    PubMed Central

    Thu, Huong Phung Thi; Nguyen, Tuan Anh; Munashingha, Palinda Ruvan; Kwon, Buki; Dao Van, Quy; Seo, Yeon-Soo

    2015-01-01

    Fen1 and Mus81–Mms4 are endonucleases involved in the processing of various DNA structural intermediates, and they were shown to have genetic and functional interactions with each other. Here, we show the in vivo significance of the interactions between Mus81 and Rad27 (yeast Fen1). The N-terminal 120 amino-acid (aa) region of Mus81, although entirely dispensable for its catalytic activity, was essential for the abilities of Mus81 to bind to and be stimulated by Rad27. In the absence of SGS1, the mus81Δ120N mutation lacking the N-terminal 120 aa region exhibited synthetic lethality, and the lethality was rescued by deletion of RAD52, a key homologous recombination mediator. These findings, together with the fact that Sgs1 constitutes a redundant pathway with Mus81–Mms4, indicate that the N-terminus-mediated interaction of Mus81 with Rad27 is physiologically important in resolving toxic recombination intermediates. Mutagenic analyses of the N-terminal region identified two distinct motifs, named N21–26 (aa from 21–26) and N108–114 (aa from 108–114) important for the in vitro and in vivo functions of Mus81. Our findings indicate that the N-terminal region of Mus81 acts as a landing pad to interact with Rad27 and that Mus81 and Rad27 work conjointly for efficient removal of various aberrant DNA structures. PMID:25628354

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

    PubMed Central

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

    2015-01-01

    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. PMID:26389960

  3. Survivin contributes to DNA repair by homologous recombination in breast cancer cells.

    PubMed

    Véquaud, Eloïse; Desplanques, Grégoire; Jézéquel, Pascal; Juin, Philippe; Barillé-Nion, Sophie

    2016-01-01

    Survivin overexpression, frequently found in breast cancers and others, is associated with poor prognosis. Its dual regulation of cell division and apoptosis makes it an attractive therapeutic target but its exact functions that are required for tumor maintenance are still elusive. Survivin protects cancer cells from genotoxic agents and this ability is generally assigned to a universal anti-apoptotic function. However, a specific role in cancer cell protection from DNA damage has been overlooked so far. We assessed DNA damage occurrence in Survivin-depleted breast cancer cells using γH2AX staining and comete assay. QPCR data and a gene conversion assay indicated that homologous recombination (HR) was impaired upon Survivin depletion. We conducted the analysis of Survivin and HR genes' expression in breast tumors. We revealed BRCAness phenotype of Survivin-depleted cells using cell death assays combined to PARP targeting. Survivin silencing leads to DNA double-strand breaks in breast cancer cells and functionally reduces HR. Survivin depletion decreases the transcription of a set of genes involved in HR, decreases RAD51 protein expression and impairs the endonuclease complex MUS81/EME1 involved in the resolution of Holliday junctions. Clinically, EME1, RAD51, EXO1, BLM expressions correlate with that of BIRC5 (coding for Survivin) and are of prognostic value. Functionally, Survivin depletion triggers p53 activation and sensitizes cancer cells to of PARP inhibition. We defined Survivin as a constitutive actor of HR in breast cancers, and implies that its inhibition would enhance cell vulnerability upon PARP inhibition. PMID:26679694

  4. International Lung Cancer Consortium: Pooled Analysis of Sequence Variants in DNA Repair and Cell Cycle Pathways

    PubMed Central

    Hung, Rayjean J.; Christiani, David C.; Risch, Angela; Popanda, Odilia; Haugen, Aage; Zienolddiny, Shan; Benhamou, Simone; Bouchardy, Christine; Lan, Qing; Spitz, Margaret R.; Wichmann, H.-Erich; LeMarchand, Loic; Vineis, Paolo; Matullo, Giuseppe; Kiyohara, Chikako; Zhang, Zuo-Feng; Pezeshki, Benhnaz; Harris, Curtis; Mechanic, Leah; Seow, Adeline; Ng, Daniel P.K.; Szeszenia-Dabrowska, Neonila; Zaridze, David; Lissowska, Jolanta; Rudnai, Peter; Fabianova, Eleonora; Mates, Dana; Foretova, Lenka; Janout, Vladimir; Bencko, Vladimir; Caporaso, Neil; Chen, Chu; Duell, Eric J.; Goodman, Gary; Field, John K.; Houlston, Richard S.; Hong, Yun-Chul; Landi, Maria Teresa; Lazarus, Philip; Muscat, Joshua; McLaughlin, John; Schwartz, Ann G.; Shen, Hongbing; Stucker, Isabelle; Tajima, Kazuo; Matsuo, Keitaro; Thun, Michael; Yang, Ping; Wiencke, John; Andrew, Angeline S.; Monnier, Stephanie; Boffetta, Paolo; Brennan, Paul

    2009-01-01

    Background The International Lung Cancer Consortium was established in 2004. To clarify the role of DNA repair genes in lung cancer susceptibility, we conducted a pooled analysis of genetic variants in DNA repair pathways, whose associations have been investigated by at least 3 individual studies. Methods Data from 14 studies were pooled for 18 sequence variants in 12 DNA repair genes, including APEX1, OGG1, XRCC1, XRCC2, XRCC3, ERCC1, XPD, XPF, XPG, XPA, MGMT, and TP53. The total number of subjects included in the analysis for each variant ranged from 2,073 to 13,955 subjects. Results Four of the variants were found to be weakly associated with lung cancer risk with borderline significance: these were XRCC3 T241M [heterozygote odds ratio (OR), 0.89; 95% confidence interval (95% CI), 0.79–0.99 and homozygote OR, 0.84; 95% CI, 0.71–1.00] based on 3,467 cases and 5,021 controls from 8 studies, XPD K751Q (heterozygote OR, 0.99; 95% CI, 0.89–1.10 and homozygote OR, 1.19; 95% CI, 1.02–1.39) based on 6,463 cases and 6,603 controls from 9 studies, and TP53 R72P (heterozygote OR, 1.14; 95% CI, 1.00–1.29 and homozygote OR, 1.20; 95% CI, 1.02–1.42) based on 3,610 cases and 5,293 controls from 6 studies. OGG1 S326C homozygote was suggested to be associated with lung cancer risk in Caucasians (homozygote OR, 1.34; 95% CI, 1.01–1.79) based on 2,569 cases and 4,178 controls from 4 studies but not in Asians. The other 14 variants did not exhibit main effects on lung cancer risk. Discussion In addition to data pooling, future priorities of International Lung Cancer Consortium include coordinated genotyping and multistage validation for ongoing genome-wide association studies. PMID:18990748

  5. The human F-Box DNA helicase FBH1 faces Saccharomyces cerevisiae Srs2 and postreplication repair pathway roles.

    PubMed

    Chiolo, Irene; Saponaro, Marco; Baryshnikova, Anastasia; Kim, Jeong-Hoon; Seo, Yeon-Soo; Liberi, Giordano

    2007-11-01

    The Saccharomyces cerevisiae Srs2 UvrD DNA helicase controls genome integrity by preventing unscheduled recombination events. While Srs2 orthologues have been identified in prokaryotic and lower eukaryotic organisms, human orthologues of Srs2 have not been described so far. We found that the human F-box DNA helicase hFBH1 suppresses specific recombination defects of S. cerevisiae srs2 mutants, consistent with the finding that the helicase domain of hFBH1 is highly conserved with that of Srs2. Surprisingly, hFBH1 in the absence of SRS2 also suppresses the DNA damage sensitivity caused by inactivation of postreplication repair-dependent functions leading to PCNA ubiquitylation. The F-box domain of hFBH1, which is not present in Srs2, is crucial for hFBH1 functions in substituting for Srs2 and postreplication repair factors. Furthermore, our findings indicate that an intact F-box domain, acting as an SCF ubiquitin ligase, is required for the DNA damage-induced degradation of hFBH1 itself. Overall, our findings suggest that the hFBH1 helicase is a functional human orthologue of budding yeast Srs2 that also possesses self-regulation properties necessary to execute its recombination functions. PMID:17724085

  6. Modeling-independent elucidation of inactivation pathways in recombinant and native A-type Kv channels.

    PubMed

    Fineberg, Jeffrey D; Ritter, David M; Covarrubias, Manuel

    2012-11-01

    A-type voltage-gated K(+) (Kv) channels self-regulate their activity by inactivating directly from the open state (open-state inactivation [OSI]) or by inactivating before they open (closed-state inactivation [CSI]). To determine the inactivation pathways, it is often necessary to apply several pulse protocols, pore blockers, single-channel recording, and kinetic modeling. However, intrinsic hurdles may preclude the standardized application of these methods. Here, we implemented a simple method inspired by earlier studies of Na(+) channels to analyze macroscopic inactivation and conclusively deduce the pathways of inactivation of recombinant and native A-type Kv channels. We investigated two distinct A-type Kv channels expressed heterologously (Kv3.4 and Kv4.2 with accessory subunits) and their native counterparts in dorsal root ganglion and cerebellar granule neurons. This approach applies two conventional pulse protocols to examine inactivation induced by (a) a simple step (single-pulse inactivation) and (b) a conditioning step (double-pulse inactivation). Consistent with OSI, the rate of Kv3.4 inactivation (i.e., the negative first derivative of double-pulse inactivation) precisely superimposes on the profile of the Kv3.4 current evoked by a single pulse because the channels must open to inactivate. In contrast, the rate of Kv4.2 inactivation is asynchronous, already changing at earlier times relative to the profile of the Kv4.2 current evoked by a single pulse. Thus, Kv4.2 inactivation occurs uncoupled from channel opening, indicating CSI. Furthermore, the inactivation time constant versus voltage relation of Kv3.4 decreases monotonically with depolarization and levels off, whereas that of Kv4.2 exhibits a J-shape profile. We also manipulated the inactivation phenotype by changing the subunit composition and show how CSI and CSI combined with OSI might affect spiking properties in a full computational model of the hippocampal CA1 neuron. This work unambiguously

  7. Modeling-independent elucidation of inactivation pathways in recombinant and native A-type Kv channels

    PubMed Central

    Fineberg, Jeffrey D.; Ritter, David M.

    2012-01-01

    A-type voltage-gated K+ (Kv) channels self-regulate their activity by inactivating directly from the open state (open-state inactivation [OSI]) or by inactivating before they open (closed-state inactivation [CSI]). To determine the inactivation pathways, it is often necessary to apply several pulse protocols, pore blockers, single-channel recording, and kinetic modeling. However, intrinsic hurdles may preclude the standardized application of these methods. Here, we implemented a simple method inspired by earlier studies of Na+ channels to analyze macroscopic inactivation and conclusively deduce the pathways of inactivation of recombinant and native A-type Kv channels. We investigated two distinct A-type Kv channels expressed heterologously (Kv3.4 and Kv4.2 with accessory subunits) and their native counterparts in dorsal root ganglion and cerebellar granule neurons. This approach applies two conventional pulse protocols to examine inactivation induced by (a) a simple step (single-pulse inactivation) and (b) a conditioning step (double-pulse inactivation). Consistent with OSI, the rate of Kv3.4 inactivation (i.e., the negative first derivative of double-pulse inactivation) precisely superimposes on the profile of the Kv3.4 current evoked by a single pulse because the channels must open to inactivate. In contrast, the rate of Kv4.2 inactivation is asynchronous, already changing at earlier times relative to the profile of the Kv4.2 current evoked by a single pulse. Thus, Kv4.2 inactivation occurs uncoupled from channel opening, indicating CSI. Furthermore, the inactivation time constant versus voltage relation of Kv3.4 decreases monotonically with depolarization and levels off, whereas that of Kv4.2 exhibits a J-shape profile. We also manipulated the inactivation phenotype by changing the subunit composition and show how CSI and CSI combined with OSI might affect spiking properties in a full computational model of the hippocampal CA1 neuron. This work unambiguously

  8. A major role of the RecFOR pathway in DNA double-strand-break repair through ESDSA in Deinococcus radiodurans.

    PubMed

    Bentchikou, Esma; Servant, Pascale; Coste, Geneviève; Sommer, Suzanne

    2010-01-01

    In Deinococcus radiodurans, the extreme resistance to DNA-shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a DeltarecA mutant: DeltarecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to gamma-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, DeltauvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of DeltauvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA. PMID:20090937

  9. Regulation of axon regeneration by the RNA repair/splicing pathway

    PubMed Central

    Song, Yuanquan; Sretavan, David; Salegio, Ernesto A; Berg, Jim; Huang, Xi; Cheng, Tong; Xiong, Xin; Meltzer, Shan; Han, Chun; Nguyen, Trong-Tuong; Bresnahan, Jacqueline C.; Beattie, Michael S.; Jan, Lily Yeh; Jan, Yuh Nung

    2015-01-01

    Mechanisms governing a neuron’s regenerative ability are important but not well understood. We identified Rtca, RNA 3′-terminal phosphate cyclase, as an inhibitor for axon regeneration. Removal of dRtca cell-autonomously enhanced axon regrowth in the Drosophila central nervous system, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease operate in the RNA repair/splicing pathway important for stress induced mRNA splicing, including that of Xbp1, a cellular stress sensor. dRtca and dArchease had opposing effects on Xbp1 splicing, and deficiency of dArchease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. Our study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma. PMID:25961792

  10. Modulation of DNA Damage and Repair Pathways by Human Tumour Viruses

    PubMed Central

    Hollingworth, Robert; Grand, Roger J

    2015-01-01

    With between 10% and 15% of human cancers attributable to viral infection, there is great interest, from both a scientific and clinical viewpoint, as to how these pathogens modulate host cell functions. Seven human tumour viruses have been identified as being involved in the development of specific malignancies. It has long been known that the introduction of chromosomal aberrations is a common feature of viral infections. Intensive research over the past two decades has subsequently revealed that viruses specifically interact with cellular mechanisms responsible for the recognition and repair of DNA lesions, collectively known as the DNA damage response (DDR). These interactions can involve activation and deactivation of individual DDR pathways as well as the recruitment of specific proteins to sites of viral replication. Since the DDR has evolved to protect the genome from the accumulation of deleterious mutations, deregulation is inevitably associated with an increased risk of tumour formation. This review summarises the current literature regarding the complex relationship between known human tumour viruses and the DDR and aims to shed light on how these interactions can contribute to genomic instability and ultimately the development of human cancers. PMID:26008701