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Sample records for break repair proteins

  1. Double strand break (DSB) repair in heterochromatin and heterochromatin proteins in DSB repair.

    PubMed

    Lemaître, Charlène; Soutoglou, Evi

    2014-07-01

    Chromosomal translocations are a hallmark of cancer cells and they represent a major cause of tumorigenesis. To avoid chromosomal translocations, faithful repair of DNA double strand breaks (DSBs) has to be ensured in the context of high ordered chromatin structure. However, chromatin compaction is proposed to represent a barrier for DSB repair. Here we review the different mechanisms cells use to alleviate the heterochromatic barrier for DNA repair. At the same time, we discuss the activating role of heterochromatin-associated proteins in this process, therefore proposing that chromatin structure, more than being a simple barrier, is a key modulator of DNA repair.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Jette, Nicholas; Lees-Miller, Susan P

    2015-03-01

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

  4. Cockayne syndrome group B protein regulates DNA double-strand break repair and checkpoint activation.

    PubMed

    Batenburg, Nicole L; Thompson, Elizabeth L; Hendrickson, Eric A; Zhu, Xu-Dong

    2015-05-12

    Mutations of CSB account for the majority of Cockayne syndrome (CS), a devastating hereditary disorder characterized by physical impairment, neurological degeneration and segmental premature aging. Here we report the generation of a human CSB-knockout cell line. We find that CSB facilitates HR and represses NHEJ. Loss of CSB or a CS-associated CSB mutation abrogating its ATPase activity impairs the recruitment of BRCA1, RPA and Rad51 proteins to damaged chromatin but promotes the formation of 53BP1-Rif1 damage foci in S and G2 cells. Depletion of 53BP1 rescues the formation of BRCA1 damage foci in CSB-knockout cells. In addition, knockout of CSB impairs the ATM- and Chk2-mediated DNA damage responses, promoting a premature entry into mitosis. Furthermore, we show that CSB accumulates at sites of DNA double-strand breaks (DSBs) in a transcription-dependent manner. The kinetics of DSB-induced chromatin association of CSB is distinct from that of its UV-induced chromatin association. These results reveal novel, important functions of CSB in regulating the DNA DSB repair pathway choice as well as G2/M checkpoint activation.

  5. Cockayne syndrome group B protein regulates DNA double-strand break repair and checkpoint activation

    PubMed Central

    Batenburg, Nicole L; Thompson, Elizabeth L; Hendrickson, Eric A; Zhu, Xu-Dong

    2015-01-01

    Mutations of CSB account for the majority of Cockayne syndrome (CS), a devastating hereditary disorder characterized by physical impairment, neurological degeneration and segmental premature aging. Here we report the generation of a human CSB-knockout cell line. We find that CSB facilitates HR and represses NHEJ. Loss of CSB or a CS-associated CSB mutation abrogating its ATPase activity impairs the recruitment of BRCA1, RPA and Rad51 proteins to damaged chromatin but promotes the formation of 53BP1-Rif1 damage foci in S and G2 cells. Depletion of 53BP1 rescues the formation of BRCA1 damage foci in CSB-knockout cells. In addition, knockout of CSB impairs the ATM- and Chk2-mediated DNA damage responses, promoting a premature entry into mitosis. Furthermore, we show that CSB accumulates at sites of DNA double-strand breaks (DSBs) in a transcription-dependent manner. The kinetics of DSB-induced chromatin association of CSB is distinct from that of its UV-induced chromatin association. These results reveal novel, important functions of CSB in regulating the DNA DSB repair pathway choice as well as G2/M checkpoint activation. PMID:25820262

  6. 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.

  7. Rapid repair of DNA double strand breaks in Arabidopsis thaliana is dependent on proteins involved in chromosome structure maintenance.

    PubMed

    Kozak, Jaroslav; West, Christopher E; White, Charles; da Costa-Nunes, José A; Angelis, Karel J

    2009-03-01

    DNA double strand breaks (DSBs) are one of the most cytotoxic forms of DNA damage and must be repaired by recombination, predominantly via non-homologous joining of DNA ends (NHEJ) in higher eukaryotes. However, analysis of DSB repair kinetics of plant NHEJ mutants atlig4-4 and atku80 with the neutral comet assay shows that alternative DSB repair pathways are active. Surprisingly, these kinetic measurements show that DSB repair was faster in the NHEJ mutant lines than in wild-type Arabidopsis. Here we provide the first characterization of this KU-independent, rapid DSB repair pathway operating in Arabidopsis. The alternate pathway that rapidly removes the majority of DSBs present in nuclear DNA depends upon structural maintenance of chromosomes (SMC) complex proteins, namely MIM/AtRAD18 and AtRAD21.1. An absolute requirement for SMC proteins and kleisin for rapid repair of DSBs in Arabidopsis opens new insight into the mechanism of DSB removal in plants.

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

    PubMed

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

    2014-12-01

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

  9. The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

    PubMed Central

    Henriksson, Sofia; Rassoolzadeh, Hanif; Hedström, Elisabeth; Coucoravas, Christos; Julner, Alexander; Goldstein, Michael; Imreh, Gabriela; Zhivotovsky, Boris; Kastan, Michael B.; Helleday, Thomas

    2014-01-01

    The WD40 domain-containing protein WRAP53β (WD40 encoding RNA antisense to p53; also referred to as WDR79/TCAB1) controls trafficking of splicing factors and the telomerase enzyme to Cajal bodies, and its functional loss has been linked to carcinogenesis, premature aging, and neurodegeneration. Here, we identify WRAP53β as an essential regulator of DNA double-strand break (DSB) repair. WRAP53β rapidly localizes to DSBs in an ATM-, H2AX-, and MDC1-dependent manner. We show that WRAP53β targets the E3 ligase RNF8 to DNA lesions by facilitating the interaction between RNF8 and its upstream partner, MDC1, in response to DNA damage. Simultaneous binding of MDC1 and RNF8 to the highly conserved WD40 scaffold domain of WRAP53β facilitates their interaction and accumulation of RNF8 at DSBs. In this manner, WRAP53β controls proper ubiquitylation at DNA damage sites and the downstream assembly of 53BP1, BRCA1, and RAD51. Furthermore, we reveal that knockdown of WRAP53β impairs DSB repair by both homologous recombination (HR) and nonhomologous end-joining (NHEJ), causes accumulation of spontaneous DNA breaks, and delays recovery from radiation-induced cell cycle arrest. Our findings establish WRAP53β as a novel regulator of DSB repair by providing a scaffold for DNA repair factors. PMID:25512560

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

    PubMed Central

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

    2009-01-01

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

  11. Melatonin sensitizes human breast cancer cells to ionizing radiation by downregulating proteins involved in double-strand DNA break repair.

    PubMed

    Alonso-González, Carolina; González, Alicia; Martínez-Campa, Carlos; Gómez-Arozamena, José; Cos, Samuel

    2015-03-01

    Radiation and adjuvant endocrine therapy are nowadays considered a standard treatment option after surgery in breast cancer. Melatonin exerts oncostatic actions on human breast cancer cells. In the current study, we investigated the effects of a combination of radiotherapy and melatonin on human breast cancer cells. Melatonin (1 mm, 10 μm and 1 nm) significantly inhibited the proliferation of MCF-7 cells. Radiation alone inhibited the MCF-7 cell proliferation in a dose-dependent manner. Pretreatment of breast cancer cells with melatonin 1 wk before radiation led to a significantly greater decrease of MCF-7 cell proliferation compared with radiation alone. Melatonin pretreatment before radiation also decreased G2 -M phase arrest compared with irradiation alone, with a higher percentage of cells in the G0 -G1 phase and a lower percentage of cells in S phase. Radiation alone diminished RAD51 and DNA-protein kinase (PKcs) mRNA expression, two main proteins involved in double-strand DNA break repair. Treatment with melatonin for 7 days before radiation led to a significantly greater decrease in RAD51 and DNA-PKcs mRNA expression compared with radiation alone. Our findings suggest that melatonin pretreatment before radiation sensitizes breast cancer cells to the ionizing effects of radiation by decreasing cell proliferation, inducing cell cycle arrest and downregulating proteins involved in double-strand DNA break repair. These findings may have implications for designing clinical trials using melatonin and radiotherapy.

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

    NASA Astrophysics Data System (ADS)

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

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

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

    DTIC Science & Technology

    2002-07-01

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

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

    DTIC Science & Technology

    2003-07-01

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

  15. Overexpression of the scaffold WD40 protein WRAP53β enhances the repair of and cell survival from DNA double-strand breaks

    PubMed Central

    Rassoolzadeh, H; Böhm, S; Hedström, E; Gad, H; Helleday, T; Henriksson, S; Farnebo, M

    2016-01-01

    Altered expression of the multifunctional protein WRAP53β (WD40 encoding RNA Antisense to p53), which targets repair factors to DNA double-strand breaks and factors involved in telomere elongation to Cajal bodies, is linked to carcinogenesis. While loss of WRAP53β function has been shown to disrupt processes regulated by this protein, the consequences of its overexpression remain unclear. Here we demonstrate that overexpression of WRAP53β disrupts the formation of and impairs the localization of coilin to Cajal bodies. At the same time, the function of this protein in the repair of DNA double-strand breaks is enhanced. Following irradiation, cells overexpressing WRAP53β exhibit more rapid clearance of phospho-histone H2AX (γH2AX), and more efficient homologous recombination and non-homologous end-joining, in association with fewer DNA breaks. Moreover, in these cells the ubiquitylation of damaged chromatin, which is known to facilitate the recruitment of repair factors and subsequent repair, is elevated. Knockdown of the ubiquitin ligase involved, ring-finger protein 8 (RNF8), which is recruited to DNA breaks by WRAP53β, attenuated this effect, suggesting that overexpression of WRAP53β leads to more rapid repair, as well as improved cell survival, by enhancing RNF8-mediated ubiquitylation at DNA breaks. Our present findings indicate that WRAP53β and RNF8 are rate-limiting factors in the repair of DNA double-strand breaks and raise the possibility that upregulation of WRAP53β may contribute to genomic stability in and survival of cancer cells. PMID:27310875

  16. Protein phosphatases pph3, ptc2, and ptc3 play redundant roles in DNA double-strand break repair by homologous recombination.

    PubMed

    Kim, Jung-Ae; Hicks, Wade M; Li, Jin; Tay, Sue Yen; Haber, James E

    2011-02-01

    In response to a DNA double-strand break (DSB), cells undergo a transient cell cycle arrest prior to mitosis until the break is repaired. In budding yeast (Saccharomyces cerevisiae), the DNA damage checkpoint is regulated by a signaling cascade of protein kinases, including Mec1 and Rad53. When DSB repair is complete, cells resume cell cycle progression (a process called "recovery") by turning off the checkpoint. Recovery involves two members of the protein phosphatase 2C (PP2C) family, Ptc2 and Ptc3, as well as the protein phosphatase 4 (PP4) enzyme, Pph3. Here, we demonstrate a new function of these three phosphatases in DSB repair. Cells lacking all three phosphatases Pph3, Ptc2, and Ptc3 exhibit synergistic sensitivities to the DNA-damaging agents camptothecin and methyl methanesulfonate, as well as hydroxyurea but not to UV light. Moreover, the simultaneous absence of Pph3, Ptc2, and Ptc3 results in defects in completing DSB repair, whereas neither single nor double deletion of the phosphatases causes a repair defect. Specifically, cells lacking all three phosphatases are defective in the repair-mediated DNA synthesis. Interestingly, the repair defect caused by the triple deletion of Pph3, Ptc2, and Ptc3 is most prominent when a DSB is slowly repaired and the DNA damage checkpoint is fully activated.

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

    DTIC Science & Technology

    2001-07-01

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

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

    PubMed

    Johnson, Nicholas M; Lemmens, Bennie B L G; Tijsterman, Marcel

    2013-01-01

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

  19. Non-homologous end-joining protein expression screen from radiosensitive cancer patients yields a novel DNA double strand break repair phenotype

    PubMed Central

    Goh, Su Kak; McKay, Jeremy N.; Chao, Michael; McKay, Timothy M.

    2017-01-01

    Background Clinical radiosensitivity is a significant impediment to tumour control and cure, in that it restricts the total doses which can safely be delivered to the whole radiotherapy population, within the tissue tolerance of potentially radiosensitive (RS) individuals. Understanding its causes could lead to personalization of radiotherapy. Methods We screened tissues from a unique bank of RS cancer patients for expression defects in major DNA double-strand break repair proteins, using Western blot analysis and subsequently reverse-transcriptase polymerase chain reaction and pulsed-field gel electrophoresis. Results We hypothesized that abnormalities in expression of these proteins may explain the radiosensitivity of some of our cancer patients. The cells from one patient showed a reproducibly consistent expression reduction in two complex-forming DNA double-strand break repair protein components (DNA Ligase IV and XRCC4). We also showed a corresponding reduction in both gene products at the mRNA level. Additionally, the mRNA inducibility by ionizing radiation was increased for one of the proteins in the patient’s cells. We confirmed the likely functional significance of the non-homologous end-joining (NHEJ) expression abnormalities with a DNA double strand break (DNA DSB) repair assay. Conclusions We have identified a novel biological phenotype linked to clinical radiosensitivity. This is important in that very few molecular defects are known in human radiotherapy subjects. Such knowledge may contribute to the understanding of radiation response mechanisms in cancer patients and to personalization of radiotherapy. PMID:28361061

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

    PubMed

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

    2015-06-01

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

  1. Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120.

    PubMed

    Hu, Sheng; Wang, Jinglan; Wang, Li; Zhang, Cheng-Cai; Chen, Wen-Li

    2015-01-01

    DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N2 fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells.

  2. Poly(ADP-Ribose) Polymerase-1 and DNA-Dependent Protein Kinase Have Equivalent Roles in Double Strand Break Repair Following Ionizing Radiation

    SciTech Connect

    Mitchell, Jody; Smith, Graeme; Curtin, Nicola J.

    2009-12-01

    Purpose: Radiation-induced DNA double strand breaks (DSBs) are predominantly repaired by nonhomologous end joining (NHEJ), involving DNA-dependent protein kinase (DNA-PK). Poly(ADP-ribose) polymerase-1 (PARP-1), well characterized for its role in single strand break repair, may also facilitate DSB repair. We investigated the activation of these enzymes by differing DNA ends and their interaction in the cellular response to ionizing radiation (IR). Methods and Materials: The effect of PARP and DNA-PK inhibitors (KU-0058684 and NU7441) on repair of IR-induced DSBs was investigated in DNA-PK and PARP-1 proficient and deficient cells by measuring gammaH2AX foci and neutral comets. Complementary in vitro enzyme kinetics assays demonstrated the affinities of DNA-PK and PARP-1 for DSBs with varying DNA termini. Results: DNA-PK and PARP-1 both promoted the fast phase of resolution of IR-induced DSBs in cells. Inactivation of both enzymes was not additive, suggesting that PARP-1 and DNA-PK cooperate within the same pathway to promote DSB repair. The affinities of the two enzymes for oligonucleotides with blunt, 3' GGG or 5' GGG overhanging termini were similar and overlapping (K{sub dapp} = 2.6-6.4nM for DNA-PK; 1.7-4.5nM for PARP-1). DNA-PK showed a slightly greater affinity for overhanging DNA and was significantly more efficient when activated by a 5' GGG overhang. PARP-1 had a preference for blunt-ended DNA and required a separate factor for efficient stimulation by a 5' GGG overhang. Conclusion: DNA-PK and PARP-1 are both required in a pathway facilitating the fast phase of DNA DSB repair.

  3. Acetyltransferase p300 collaborates with chromodomain helicase DNA-binding protein 4 (CHD4) to facilitate DNA double-strand break repair.

    PubMed

    Qi, Wenjing; Chen, Hongyu; Xiao, Ting; Wang, Ruoxi; Li, Ting; Han, Liping; Zeng, Xianlu

    2016-03-01

    Chromatin remodelling is critical for repairing DNA damage and maintaining genomic integrity. Previous studies have reported that histone acetyltransferase p300 and ATP-dependent chromatin remodeler chromodomain helicase DNA-binding protein 4 (CHD4) functions, respectively, in DNA double-strand breaks (DSBs) repair. But the physiological significance of their interaction remains elusive. Here, we showed that p300 and CHD4 were both recruited to the sites of DSBs. Their ablation led to impaired DSBs repair and sensitised cells to laser and the anti-cancer drug, etoposide. Using DR-GFP and EJ5-GFP reporter systems, we found that knockdown of p300 or CHD4 impaired the homologous recombination (HR) repair but no the non-homologous end joining (NHEJ) repair. Furthermore, p300 or CHD4 knockdown respectively suppressed the recruitment of replication protein A (RPA), a key protein for HR, to the DSB sites. In addition, immunofluorescence results showed that knockdown of p300 reduced the recruitment of CHD4 at DSB sites. In turn, CHD4 knockdown also decreased p300 assembly. Moreover, immunoprecipitation and purified protein pull down assay revealed that p300 physically interacted with CHD4 at DNA damage sites, and this interaction was dependent on the chromodomain and ATPase/helicase domain of CHD4 and the CH2, Bd and HAT domains of p300. These results indicate that p300 and CHD4 could function cooperatively at DSB sites and provide a new insight into the detailed crosstalk among the chromatin remodelling proteins.

  4. The catalytic subunit of DNA-dependent protein kinase is required for cellular resistance to oxidative stress independent of DNA double-strand break repair.

    PubMed

    Li, Mengxia; Lin, Yu-Fen; Palchik, Guillermo A; Matsunaga, Shinji; Wang, Dong; Chen, Benjamin P C

    2014-11-01

    DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) are the two major kinases involved in DNA double-strand break (DSB) repair, and are required for cellular resistance to ionizing radiation. Whereas ATM is the key upstream kinase for DSB signaling, DNA-PKcs is primarily involved in DSB repair through the nonhomologous end-joining (NHEJ) mechanism. In addition to DSB repair, ATM has been shown to be involved in the oxidative stress response and could be activated directly in vitro on hydrogen peroxide (H2O2) treatment. However, the role of DNA-PKcs in cellular response to oxidative stress is not clear. We hypothesize that DNA-PKcs may participate in the regulation of ATM activation in response to oxidative stress, and that this regulatory role is independent of its role in DNA double-strand break repair. Our findings reveal that H2O2 induces hyperactivation of ATM signaling in DNA-PKcs-deficient, but not Ligase 4-deficient cells, suggesting an NHEJ-independent role for DNA-PKcs. Furthermore, DNA-PKcs deficiency leads to the elevation of reactive oxygen species (ROS) production, and to a decrease in cellular survival against H2O2. For the first time, our results reveal that DNA-PKcs plays a noncanonical role in the cellular response to oxidative stress, which is independent from its role in NHEJ. In addition, DNA-PKcs is a critical regulator of the oxidative stress response and contributes to the maintenance of redox homeostasis. Our findings reveal that DNA-PKcs is required for cellular resistance to oxidative stress and suppression of ROS buildup independently of its function in DSB repair.

  5. Alignment of Homologous Chromosomes and Effective Repair of Programmed DNA Double-Strand Breaks during Mouse Meiosis Require the Minichromosome Maintenance Domain Containing 2 (MCMDC2) Protein

    PubMed Central

    Ravindranathan, Ramya; Dereli, Ihsan; Stanzione, Marcello; Tóth, Attila

    2016-01-01

    Orderly chromosome segregation during the first meiotic division requires meiotic recombination to form crossovers between homologous chromosomes (homologues). Members of the minichromosome maintenance (MCM) helicase family have been implicated in meiotic recombination. In addition, they have roles in initiation of DNA replication, DNA mismatch repair and mitotic DNA double-strand break repair. Here, we addressed the function of MCMDC2, an atypical yet conserved MCM protein, whose function in vertebrates has not been reported. While we did not find an important role for MCMDC2 in mitotically dividing cells, our work revealed that MCMDC2 is essential for fertility in both sexes due to a crucial function in meiotic recombination. Meiotic recombination begins with the introduction of DNA double-strand breaks into the genome. DNA ends at break sites are resected. The resultant 3-prime single-stranded DNA overhangs recruit RAD51 and DMC1 recombinases that promote the invasion of homologous duplex DNAs by the resected DNA ends. Multiple strand invasions on each chromosome promote the alignment of homologous chromosomes, which is a prerequisite for inter-homologue crossover formation during meiosis. We found that although DNA ends at break sites were evidently resected, and they recruited RAD51 and DMC1 recombinases, these recombinases were ineffective in promoting alignment of homologous chromosomes in the absence of MCMDC2. Consequently, RAD51 and DMC1 foci, which are thought to mark early recombination intermediates, were abnormally persistent in Mcmdc2-/- meiocytes. Importantly, the strand invasion stabilizing MSH4 protein, which marks more advanced recombination intermediates, did not efficiently form foci in Mcmdc2-/- meiocytes. Thus, our work suggests that MCMDC2 plays an important role in either the formation, or the stabilization, of DNA strand invasion events that promote homologue alignment and provide the basis for inter-homologue crossover formation during

  6. The RSF1 histone-remodelling factor facilitates DNA double-strand break repair by recruiting centromeric and Fanconi Anaemia proteins.

    PubMed

    Pessina, Fabio; Lowndes, Noel F

    2014-05-01

    ATM is a central regulator of the cellular responses to DNA double-strand breaks (DSBs). Here we identify a biochemical interaction between ATM and RSF1 and we characterise the role of RSF1 in this response. The ATM-RSF1 interaction is dependent upon both DSBs and ATM kinase activity. Together with SNF2H/SMARCA5, RSF1 forms the RSF chromatin-remodelling complex. Although RSF1 is specific to the RSF complex, SNF2H/SMARCA5 is a catalytic subunit of several other chromatin-remodelling complexes. Although not required for checkpoint signalling, RSF1 is required for efficient repair of DSBs via both end-joining and homology-directed repair. Specifically, the ATM-dependent recruitment to sites of DSBs of the histone fold proteins CENPS/MHF1 and CENPX/MHF2, previously identified at centromeres, is RSF1-dependent. In turn these proteins recruit and regulate the mono-ubiquitination of the Fanconi Anaemia proteins FANCD2 and FANCI. We propose that by depositing CENPS/MHF1 and CENPX/MHF2, the RSF complex either directly or indirectly contributes to the reorganisation of chromatin around DSBs that is required for efficient DNA repair.

  7. Regulation of DNA-end resection by hnRNPU-like proteins promotes DNA double-strand break signaling and repair.

    PubMed

    Polo, Sophie E; Blackford, Andrew N; Chapman, J Ross; Baskcomb, Linda; Gravel, Serge; Rusch, Andre; Thomas, Anoushka; Blundred, Rachel; Smith, Philippa; Kzhyshkowska, Julia; Dobner, Thomas; Taylor, A Malcolm R; Turnell, Andrew S; Stewart, Grant S; Grand, Roger J; Jackson, Stephen P

    2012-02-24

    DNA double-strand break (DSB) signaling and repair are critical for cell viability, and rely on highly coordinated pathways whose molecular organization is still incompletely understood. Here, we show that heterogeneous nuclear ribonucleoprotein U-like (hnRNPUL) proteins 1 and 2 play key roles in cellular responses to DSBs. We identify human hnRNPUL1 and -2 as binding partners for the DSB sensor complex MRE11-RAD50-NBS1 (MRN) and demonstrate that hnRNPUL1 and -2 are recruited to DNA damage in an interdependent manner that requires MRN. Moreover, we show that hnRNPUL1 and -2 stimulate DNA-end resection and promote ATR-dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents. Finally, we establish that hnRNPUL1 and -2 function downstream of MRN and CtBP-interacting protein (CtIP) to promote recruitment of the BLM helicase to DNA breaks. Collectively, these results provide insights into how mammalian cells respond to DSBs.

  8. Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins.

    PubMed Central

    Hays, S L; Firmenich, A A; Berg, P

    1995-01-01

    The repair of DNA double-strand breaks in Saccharomyces cerevisiae requires genes of the RAD52 epistasis group, of which RAD55 and RAD57 are members. Here, we show that the x-ray sensitivity of rad55 and rad57 mutant strains is suppressible by overexpression of RAD51 or RAD52. Virtually complete suppression is provided by the simultaneous overexpression of RAD51 and RAD52. This suppression occurs at 23 degrees C, where these mutants are more sensitive to x-rays, as well as at 30 degrees C and 36 degrees C. In addition, a recombination defect of rad55 and rad57 mutants is similarly suppressed. Direct in vivo interactions between the Rad51 and Rad55 proteins, and between Rad55 and Rad57, have also been identified by using the two-hybrid system. These results indicate that these four proteins constitute part of a complex, a "recombinosome," to effect the recombinational repair of double-strand breaks. PMID:7624345

  9. Repair of DNA Double-Strand Breaks

    NASA Astrophysics Data System (ADS)

    Falk, Martin; Lukasova, Emilie; Kozubek, Stanislav

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  12. Regulation of 53BP1 Protein Stability by RNF8 and RNF168 Is Important for Efficient DNA Double-Strand Break Repair

    PubMed Central

    Hu, Yiheng; Wang, Chao; Huang, Kun; Xia, Fen; Parvin, Jeffrey D.; Mondal, Neelima

    2014-01-01

    53BP1 regulates DNA double-strand break (DSB) repair. In functional assays for specific DSB repair pathways, we found that 53BP1 was important in the conservative non-homologous end-joining (C-NHEJ) pathway, and this activity was dependent upon RNF8 and RNF168. We observed that 53BP1 protein was diffusely abundant in nuclei, and upon ionizing radiation, 53BP1 was everywhere degraded except at DNA damage sites. Depletion of RNF8 or RNF168 blocked the degradation of the diffusely localized nuclear 53BP1, and ionizing radiation induced foci (IRIF) did not form. Furthermore, when 53BP1 degradation was inhibited, a subset of 53BP1 was bound to DNA damage sites but bulk, unbound 53BP1 remained in the nucleoplasm, and localization of its downstream effector RIF1 at DSBs was abolished. Our data suggest a novel mechanism for responding to DSB that upon ionizing radiation, 53BP1 was divided into two populations, ensuring functional DSB repair: damage site-bound 53BP1 whose binding signal is known to be generated by RNF8 and RNF168; and unbound bulk 53BP1 whose ensuing degradation is regulated by RNF8 and RNF168. PMID:25337968

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

    PubMed

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

    2011-05-15

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

  14. Genetics of x-ray induced double strand break repair in saccharomyces cerevisiae

    SciTech Connect

    Budd, M.E.

    1982-07-01

    The possible fates of x-ray-induced double-strand breaks in Saccharomyces cerevisiae were examined. One possible pathway which breaks can follow, the repair pathway, was studied by assaying strains with mutations in the RAD51, RAD54, and RAD57 loci for double-strand break repair. In order of increasing radiation sensitivity one finds: rad57-1(23/sup 0/)> rad51-1(30/sup 0/)> rad54-3(36/sup 0/). At 36/sup 0/, rad54-3 cells cannot repair double-strand breaks, while 23/sup 0/, they can. Strains with the rad57-1 mutation can rejoin broken chromosomes at both temperatures. However, the low survival at 36/sup 0/ shows that the assay is not distinguishing large DNA fragments which allow cell survival from those which cause cell death. A rad51-1 strain could also rejoin broken chromosomes, and was thus capable of incomplete repair. The data can be explained with the hypothesis that rad54-3 cells are blocked in an early step of repair, while rad51-1 and rad57-1 strains are blocked in a later step of repair. The fate of double-strand breaks when they are left unrepaired was investigated with the rad54-3 mutation. If breaks are prevented from entering the RAD54 repair pathway they become uncommitted lesions. These lesions are repaired slower than the original breaks. One possible fate for an uncommitted lesion is conversion into a fixed lesion, which is likely to be an unrepairable or misrepaired double-strand break. The presence of protein synthesis after irradiation increases the probability that a break will enter the repair pathway. Evidence shows that increased probability of repair results from enhanced synthesis of repair proteins shortly after radiation. (ERB)

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-03-20

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

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

    PubMed

    Miné-Hattab, Judith; Rothstein, Rodney

    2013-11-01

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

  18. MTE1 Functions with MPH1 in Double-Strand Break Repair.

    PubMed

    Yimit, Askar; Kim, TaeHyung; Anand, Ranjith P; Meister, Sarah; Ou, Jiongwen; Haber, James E; Zhang, Zhaolei; Brown, Grant W

    2016-05-01

    Double-strand DNA breaks occur upon exposure of cells to ionizing radiation and certain chemical agents or indirectly through replication fork collapse at DNA damage sites. If left unrepaired, double-strand breaks can cause genome instability and cell death, and their repair can result in loss of heterozygosity. In response to DNA damage, proteins involved in double-strand break repair by homologous recombination relocalize into discrete nuclear foci. We identified 29 proteins that colocalize with recombination repair protein Rad52 in response to DNA damage. Of particular interest, Ygr042w/Mte1, a protein of unknown function, showed robust colocalization with Rad52. Mte1 foci fail to form when the DNA helicase gene MPH1 is absent. Mte1 and Mph1 form a complex and are recruited to double-strand breaks in vivo in a mutually dependent manner. MTE1 is important for resolution of Rad52 foci during double-strand break repair and for suppressing break-induced replication. Together our data indicate that Mte1 functions with Mph1 in double-strand break repair.

  19. Hepatitis B virus pre-S2 mutant large surface protein inhibits DNA double-strand break repair and leads to genome instability in hepatocarcinogenesis.

    PubMed

    Hsieh, Yi-Hsuan; Chang, Yu-Ying; Su, Ih-Jen; Yen, Chia-Jui; Liu, Yi-Ru; Liu, Ren-Jei; Hsieh, Wen-Chuan; Tsai, Hung-Wen; Wang, Lily Hui-Ching; Huang, Wenya

    2015-07-01

    Although hepatitis B virus (HBV) has been established to cause hepatocellular carcinoma (HCC), the exact mechanism remains to be clarified. Type II ground glass hepatocytes (GGHs) harbouring the HBV pre-S2 mutant large surface protein (LHBS) have been recognized as a morphologically distinct hallmark of HCC in the advanced stages of chronic HBV infection. Considering its preneoplastic nature, we hypothesized that type II GGH may exhibit high genomic instability, which is important for the carcinogenic process in chronic HBV carriers. In this study we found that pre-S2 mutant LHBS directly interacted with importin α1, the key factor that recognizes cargos undergoing nuclear transportation mediated by the importin α/β-associated nuclear pore complex (NPC). By interacting with importin α1, which inhibits its function as an NPC factor, pre-S2 mutant LHBS blocked nuclear transport of an essential DNA repair and recombination factor, Nijmegen breakage syndrome 1 (NBS1), upon DNA damage, thereby delaying the formation of nuclear foci at the sites of DNA double-strand breaks (DSBs). Pre-S2 mutant LHBS was also found to block NBS1-mediated homologous recombination repair and induce multi-nucleation of cells. In addition, pre-S2 mutant LHBS transgenic mice showed genomic instability, indicated by increased global gene copy number variations (CNVs), which were significantly higher than those in hepatitis B virus X mice, indicating that pre-S2 mutant LHBS is the major viral oncoprotein inducing genomic instability in HBV-infected hepatocytes. Consistently, the human type II GGHs in HCC patients exhibited increased DNA DSBs representing significant genomic instability. In conclusion, type II GGHs harbouring HBV pre-S2 mutant oncoprotein represent a high-risk marker for the loss of genome integrity in chronic HBV carriers and explain the complex chromosome changes in HCCs. Mouse array CGH raw data: GEO Accession No. GSE61378 (http://www.ncbi

  20. Single-stranded DNA oligomers stimulate error-prone alternative repair of DNA double-strand breaks through hijacking Ku protein

    PubMed Central

    Yuan, Ying; Britton, Sébastien; Delteil, Christine; Coates, Julia; Jackson, Stephen P.; Barboule, Nadia; Frit, Philippe; Calsou, Patrick

    2015-01-01

    In humans, DNA double-strand breaks (DSBs) are repaired by two mutually-exclusive mechanisms, homologous recombination or end-joining. Among end-joining mechanisms, the main process is classical non-homologous end-joining (C-NHEJ) which relies on Ku binding to DNA ends and DNA Ligase IV (Lig4)-mediated ligation. Mostly under Ku- or Lig4-defective conditions, an alternative end-joining process (A-EJ) can operate and exhibits a trend toward microhomology usage at the break junction. Homologous recombination relies on an initial MRN-dependent nucleolytic degradation of one strand at DNA ends. This process, named DNA resection generates 3′ single-stranded tails necessary for homologous pairing with the sister chromatid. While it is believed from the current literature that the balance between joining and recombination processes at DSBs ends is mainly dependent on the initiation of resection, it has also been shown that MRN activity can generate short single-stranded DNA oligonucleotides (ssO) that may also be implicated in repair regulation. Here, we evaluate the effect of ssO on end-joining at DSB sites both in vitro and in cells. We report that under both conditions, ssO inhibit C-NHEJ through binding to Ku and favor repair by the Lig4-independent microhomology-mediated A-EJ process. PMID:26350212

  1. Repair of DNA Double-Strand Breaks in Heterochromatin

    PubMed Central

    Watts, Felicity Z.

    2016-01-01

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

  2. A requirement for polymerized actin in DNA double-strand break repair.

    PubMed

    Andrin, Christi; McDonald, Darin; Attwood, Kathleen M; Rodrigue, Amélie; Ghosh, Sunita; Mirzayans, Razmik; Masson, Jean-Yves; Dellaire, Graham; Hendzel, Michael J

    2012-07-01

    Nuclear actin is involved in several nuclear processes from chromatin remodeling to transcription. Here we examined the requirement for actin polymerization in DNA double-strand break repair. Double-strand breaks are considered the most dangerous type of DNA lesion. Double-strand break repair consists of a complex set of events that are tightly regulated. Failure at any step can have catastrophic consequences such as genomic instability, oncogenesis or cell death. Many proteins involved in this repair process have been identified and their roles characterized. We discovered that some DNA double-strand break repair factors are capable of associating with polymeric actin in vitro and specifically, that purified Ku70/80 interacts with polymerized actin under these conditions. We find that the disruption of polymeric actin inhibits DNA double strand break repair both in vitro and in vivo. Introduction of nuclear targeted mutant actin that cannot polymerize, or the depolymerization of endogenous actin filaments by the addition of cytochalasin D, alters the retention of Ku80 at sites of DNA damage in live cells. Our results suggest that polymeric actin is required for proper DNA double-strand break repair and may function through the stabilization of the Ku heterodimer at the DNA damage site.

  3. Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control double-strand break repair by homologous recombination.

    PubMed

    Limbo, Oliver; Chahwan, Charly; Yamada, Yoshiki; de Bruin, Robertus A M; Wittenberg, Curt; Russell, Paul

    2007-10-12

    The Mre11-Rad50-Nbs1 (MRN) complex is a primary sensor of DNA double-strand breaks (DSBs). Upon recruitment to DSBs, it plays a critical role in catalyzing 5' --> 3' single-strand resection that is required for repair by homologous recombination (HR). Unknown mechanisms repress HR in G1 phase of the cell cycle during which nonhomologous end-joining (NHEJ) is the favored mode of DSB repair. Here we describe fission yeast Ctp1, so-named because it shares conserved domains with the mammalian tumor suppressor CtIP. Ctp1 is recruited to DSBs where it is essential for repair by HR. Ctp1 is required for efficient formation of RPA-coated single-strand DNA adjacent to DSBs, indicating that it functions with the MRN complex in 5' --> 3' resection. Transcription of ctp1(+) is periodic during the cell cycle, with the onset of its expression coinciding with the start of DNA replication. These data suggest that regulation of Ctp1 underlies cell-cycle control of HR.

  4. Aberrant DNA Double-strand Break Repair Threads in Breast Carcinoma: Orchestrating Genomic Insult Survival.

    PubMed

    Kumar, Azad; Purohit, Shruti; Sharma, Nilesh Kumar

    2016-12-01

    Breast carcinoma is a heterogeneous disease that has exhibited rapid resistance to treatment in the last decade. Depending genotype and phenotype of breast cancer, there are discernible differences in DNA repair protein responses including DNA double strand break repair. It is a fact that different molecular sub-types of breast carcinoma activate these dedicated protein pathways in a distinct manner. The DNA double-strand damage repair machinery is manipulated by breast carcinoma to selectively repair the damage or insults inflicted by the genotoxic effects of chemotherapy or radiation therapy. The two DNA double-strand break repair pathways employed by breast carcinoma are homologous recombination and non-homologous end joining. In recent decades, therapeutic interventions targeting one or more factors involved in repairing DNA double-strand breaks inflicted by chemo/radiation therapy have been widely studied. Herein, this review paper summarizes the recent evidence and ongoing clinical trials citing potential therapeutic combinatorial interventions targeting DNA double-strand break repair pathways in breast carcinoma.

  5. Aberrant DNA Double-strand Break Repair Threads in Breast Carcinoma: Orchestrating Genomic Insult Survival

    PubMed Central

    Kumar, Azad; Purohit, Shruti; Sharma, Nilesh Kumar

    2016-01-01

    Breast carcinoma is a heterogeneous disease that has exhibited rapid resistance to treatment in the last decade. Depending genotype and phenotype of breast cancer, there are discernible differences in DNA repair protein responses including DNA double strand break repair. It is a fact that different molecular sub-types of breast carcinoma activate these dedicated protein pathways in a distinct manner. The DNA double-strand damage repair machinery is manipulated by breast carcinoma to selectively repair the damage or insults inflicted by the genotoxic effects of chemotherapy or radiation therapy. The two DNA double-strand break repair pathways employed by breast carcinoma are homologous recombination and non-homologous end joining. In recent decades, therapeutic interventions targeting one or more factors involved in repairing DNA double-strand breaks inflicted by chemo/radiation therapy have been widely studied. Herein, this review paper summarizes the recent evidence and ongoing clinical trials citing potential therapeutic combinatorial interventions targeting DNA double-strand break repair pathways in breast carcinoma. PMID:28053956

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Aparicio, Tomas; Baer, Richard; Gautier, Jean

    2014-07-01

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

  9. Differential requirement for SUB1 in chromosomal and plasmid double-strand DNA break repair.

    PubMed

    Yu, Lijian; Volkert, Michael R

    2013-01-01

    Non homologous end joining (NHEJ) is an important process that repairs double strand DNA breaks (DSBs) in eukaryotic cells. Cells defective in NHEJ are unable to join chromosomal breaks. Two different NHEJ assays are typically used to determine the efficiency of NHEJ. One requires NHEJ of linearized plasmid DNA transformed into the test organism; the other requires NHEJ of a single chromosomal break induced either by HO endonuclease or the I-SceI restriction enzyme. These two assays are generally considered equivalent and rely on the same set of NHEJ genes. PC4 is an abundant DNA binding protein that has been suggested to stimulate NHEJ. Here we tested the role of PC4's yeast homolog SUB1 in repair of DNA double strand breaks using different assays. We found SUB1 is required for NHEJ repair of DSBs in plasmid DNA, but not in chromosomal DNA. Our results suggest that these two assays, while similar are not equivalent and that repair of plasmid DNA requires additional factor(s) that are not required for NHEJ repair of chromosomal double-strand DNA breaks. Possible roles for Sub1 proteins in NHEJ of plasmid DNA are discussed.

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

    PubMed

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

    2004-01-01

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

  11. Repair of DNA double strand breaks: in vivo biochemistry.

    PubMed

    Sugawara, Neal; Haber, James E

    2006-01-01

    Double strand breaks (DSBs) can cause damage to the genomic integrity of a cell as well as initiate genetic recombination processes. The HO and I-SceI endonucleases from budding yeast have provided a way to study these events by inducing a unique DSB in vivo under the control of a galactose-inducible promoter. The GAL::HO construct has been used extensively to study processes such as nonhomologous end joining, intra- and interchromosomal gene conversion, single strand annealing and break-induced recombination. Synchronously induced DSBs have also been important in the study of the DNA damage checkpoint, adaptation, and recovery pathways of yeast. This chapter describes methods of using GAL::HO to physically monitor the progression of events following a DSB, specifically the events leading to the switching of mating type by gene conversion of MAT using the silent donors at HML and HMR. Southern blot analysis can be used to follow the overall events in this process such as the formation of the DSB and product. Denaturing alkaline gels and slot blot techniques can be employed to follow the 5' to 3' resection of DNA starting at the DSB. After resection, the 3' tail initiates a homology search and then strand invades its homologous sequence at the donor cassette. Polymerase chain reaction is an important means to assay strand invasion and the priming of new DNA synthesis as well as the completion of gene conversion. Methods such as chromatin immunoprecipitation have provided a means to study many proteins that associate with a DSB, including not only recombination proteins, but also proteins involved in nonhomologous end joining, cell cycle arrest, chromatin remodeling, cohesin function, and mismatch repair.

  12. Nonhomologous Mechanisms of Repair of Chromosomal Breaks

    SciTech Connect

    Haber, J. E.

    2001-12-19

    Discovered three new proteins involved in DNA damage assessment. Interestingly they are all proteins involved in recombination, but they have very different roles in that process and other proteins that might be expected to be equivalently involved are not. This is developing into a very significant area of research.

  13. In vitro binding kinetics of DNA double strand break repair proteins Ku70/80 and DNA-PKcs quantified by fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Abdisalaam, Salim; Chen, David J.; Alexandrakis, George

    2012-02-01

    DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage that occurs in eukaryotic cells. There are two distinct pathways of repairing DSBs, homologous recombination (HR) and non-homologous end joining (NHEJ). In the NHEJ repairing pathway, DSB recognition and repair initiation is directed by the interaction of DNAbinding subunit Ku70/80 heterodimer with the DNA-PK protein catalytic subunit (DNA-PKcs). Mutations in these proteins result in repair stalling and eventual DNA misrepair that may lead to genomic instability. Studying the binding kinetics of these repair proteins is therefore important for understanding the conditions under which DSB repair stalls. Currently open questions are, what is the minimum DNA length that this complex needs to get a foothold onto a DSB and how tightly does DNA-PKcs bind onto the DNA-Ku70/80 complex. Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross-Correlation Spectroscopy (FCCS) techniques have the potential to give information about the binding kinetics of DNA-protein and protein-protein interactions at the single-molecule level. In this work, FCS/FCCS measurements were performed to explore the minimum DNA base-pair (bp) length that Ku70/80 needed as a foothold to bind effectively onto the tips of different lengths of double-stranded DNA (dsDNA) fragments that mimic DSBs. 25 bp, 33 bp and 50 bp of dsDNA were used for these experiments and binding was studied as a function of salt concentration in solution. It was found that the 25 bp binding was weak even at physiological salt concentrations while the dissociation constant (Kd) remained constant for 33 and 50 bp dsDNA strand lengths. These studies indicated that the minimum binding length for the Ku70/8 is in the vicinity of 25 bp. The specificity of binding of Ku70/80 was proven by competitive binding FCCS experiments between Cy5-labeled DNA, GFP-Ku70/80 and titrations of unlabeled Ku70/80. Finally, using FCCS it was possible to estimate

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

    PubMed

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

    2016-01-01

    Etoposide is a widely used anticancer drug and a DNA topoisomerase II (Top2) inhibitor. Etoposide produces Top2-attached single-strand breaks (Top2-SSB complex) and double-strand breaks (Top2-DSB complex) that are thought to induce cell death in tumor cells. The Top2-SSB complex is more abundant than the Top2-DSB complex. Human tyrosyl-DNA phosphodiesterase 2 (TDP2) is required for efficient repair of Top2-DSB complexes. However, the identities of the proteins involved in the repair of Top2-SSB complexes are unknown, although yeast genetic data indicate that 5' to 3' structure-specific DNA endonuclease activity is required for alternative repair of Top2 DNA damage. In this study, we purified a flap endonuclease 1 (FEN1) and xeroderma pigmentosum group G protein (XPG) in the 5' to 3' structure-specific DNA endonuclease family and synthesized single-strand break DNA substrates containing a 5'-phoshotyrosyl bond, mimicking the Top2-SSB complex. We found that FEN1 and XPG did not remove the 5'-phoshotyrosyl bond-containing DSB substrates but removed the 5'-phoshotyrosyl bond-containing SSB substrates. Under DNA repair conditions, FEN1 efficiently repaired the 5'-phoshotyrosyl bond-containing SSB substrates in the presence of DNA ligase and DNA polymerase. Therefore, FEN1 may play an important role in the repair of Top2-SSB complexes in etoposide-treated cells.

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

    PubMed

    Bao, Yunhe; Shen, Xuetong

    2007-04-01

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

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

    PubMed

    Jeggo, Penny A; Downs, Jessica A

    2014-11-15

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

  17. Chromatin organization and dynamics in double-strand break repair.

    PubMed

    Seeber, Andrew; Gasser, Susan M

    2016-10-31

    Chromatin is organized and segmented into a landscape of domains that serve multiple purposes. In contrast to transcription, which is controlled by defined sequences at distinct sites, DNA damage can occur anywhere. Repair accordingly must occur everywhere, yet it is inevitably affected by its chromatin environment. In this review, we summarize recent work investigating how changes in chromatin organization facilitate and/or guide DNA double-strand break repair. In addition, we examine new live cell studies on the dynamics of chromatin and the mechanisms that regulate its movement.

  18. Nucleosome remodelers in double-strand break repair.

    PubMed

    Seeber, Andrew; Hauer, Michael; Gasser, Susan M

    2013-04-01

    ATP-dependent nucleosome remodelers use ATP hydrolysis to shift, evict and exchange histone dimers or octamers and have well-established roles in transcription. Earlier work has suggested a role for nucleosome remodelers such as INO80 in double-strand break (DSB) repair. This review will begin with an update on recent studies that explore how remodelers are recruited to DSBs. We then examine their impact on various steps of repair, focusing on resection and the formation of the Rad51-ssDNA nucleofilament. Finally, we will explore new studies that implicate remodelers in the physical movement of chromatin in response to damage.

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

    PubMed

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

    2016-05-08

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

  20. Correct End Use during End Joining of Multiple Chromosomal Double Strand Breaks Is Influenced by Repair Protein RAD50, DNA-dependent Protein Kinase DNA-PKcs, and Transcription Context*

    PubMed Central

    Gunn, Amanda; Bennardo, Nicole; Cheng, Anita; Stark, Jeremy M.

    2011-01-01

    During repair of multiple chromosomal double strand breaks (DSBs), matching the correct DSB ends is essential to limit rearrangements. To investigate the maintenance of correct end use, we examined repair of two tandem noncohesive DSBs generated by endonuclease I-SceI and the 3′ nonprocessive exonuclease Trex2, which can be expressed as an I-SceI-Trex2 fusion. We examined end joining (EJ) repair that maintains correct ends (proximal-EJ) versus using incorrect ends (distal-EJ), which provides a relative measure of incorrect end use (distal end use). Previous studies showed that ATM is important to limit distal end use. Here we show that DNA-PKcs kinase activity and RAD50 are also important to limit distal end use, but that H2AX is dispensable. In contrast, we find that ATM, DNA-PKcs, and RAD50 have distinct effects on repair events requiring end processing. Furthermore, we developed reporters to examine the effects of the transcription context on DSB repair, using an inducible promoter. We find that a DSB downstream from an active promoter shows a higher frequency of distal end use, and a greater reliance on ATM for limiting incorrect end use. Conversely, DSB transcription context does not affect end processing during EJ, the frequency of homology-directed repair, or the role of RAD50 and DNA-PKcs in limiting distal end use. We suggest that RAD50, DNA-PKcs kinase activity, and transcription context are each important to limit incorrect end use during EJ repair of multiple DSBs, but that these factors and conditions have distinct roles during repair events requiring end processing. PMID:22027841

  1. Correct end use during end joining of multiple chromosomal double strand breaks is influenced by repair protein RAD50, DNA-dependent protein kinase DNA-PKcs, and transcription context.

    PubMed

    Gunn, Amanda; Bennardo, Nicole; Cheng, Anita; Stark, Jeremy M

    2011-12-09

    During repair of multiple chromosomal double strand breaks (DSBs), matching the correct DSB ends is essential to limit rearrangements. To investigate the maintenance of correct end use, we examined repair of two tandem noncohesive DSBs generated by endonuclease I-SceI and the 3' nonprocessive exonuclease Trex2, which can be expressed as an I-SceI-Trex2 fusion. We examined end joining (EJ) repair that maintains correct ends (proximal-EJ) versus using incorrect ends (distal-EJ), which provides a relative measure of incorrect end use (distal end use). Previous studies showed that ATM is important to limit distal end use. Here we show that DNA-PKcs kinase activity and RAD50 are also important to limit distal end use, but that H2AX is dispensable. In contrast, we find that ATM, DNA-PKcs, and RAD50 have distinct effects on repair events requiring end processing. Furthermore, we developed reporters to examine the effects of the transcription context on DSB repair, using an inducible promoter. We find that a DSB downstream from an active promoter shows a higher frequency of distal end use, and a greater reliance on ATM for limiting incorrect end use. Conversely, DSB transcription context does not affect end processing during EJ, the frequency of homology-directed repair, or the role of RAD50 and DNA-PKcs in limiting distal end use. We suggest that RAD50, DNA-PKcs kinase activity, and transcription context are each important to limit incorrect end use during EJ repair of multiple DSBs, but that these factors and conditions have distinct roles during repair events requiring end processing.

  2. Evidence that the Nijmegen breakage syndrome protein, an early sensor of double-strand DNA breaks (DSB), is involved in HIV-1 post-integration repair by recruiting the ataxia telangiectasia-mutated kinase in a process similar to, but distinct from, cellular DSB repair.

    PubMed

    Smith, Johanna A; Wang, Feng-Xiang; Zhang, Hui; Wu, Kou-Juey; Williams, Kevin Jon; Daniel, René

    2008-01-22

    Retroviral transduction involves integrase-dependent linkage of viral and host DNA that leaves an intermediate that requires post-integration repair (PIR). We and others proposed that PIR hijacks the host cell double-strand DNA break (DSB) repair pathways. Nevertheless, the geometry of retroviral DNA integration differs considerably from that of DSB repair and so the precise role of host-cell mechanisms in PIR remains unclear. In the current study, we found that the Nijmegen breakage syndrome 1 protein (NBS1), an early sensor of DSBs, associates with HIV-1 DNA, recruits the ataxia telangiectasia-mutated (ATM) kinase, promotes stable retroviral transduction, mediates efficient integration of viral DNA and blocks integrase-dependent apoptosis that can arise from unrepaired viral-host DNA linkages. Moreover, we demonstrate that the ATM kinase, recruited by NBS1, is itself required for efficient retroviral transduction. Surprisingly, recruitment of the ATR kinase, which in the context of DSB requires both NBS1 and ATM, proceeds independently of these two proteins. A model is proposed emphasizing similarities and differences between PIR and DSB repair. Differences between the pathways may eventually allow strategies to block PIR while still allowing DSB repair.

  3. Splicing controls the ubiquitin response during DNA double-strand break repair

    PubMed Central

    Pederiva, C; Böhm, S; Julner, A; Farnebo, M

    2016-01-01

    Although evidence that splicing regulates DNA repair is accumulating, the underlying mechanism(s) remain unclear. Here, we report that short-term inhibition of pre-mRNA splicing by spliceosomal inhibitors impairs cellular repair of DNA double-strand breaks. Indeed, interference with splicing as little as 1 h prior to irradiation reduced ubiquitylation of damaged chromatin and impaired recruitment of the repair factors WRAP53β, RNF168, 53BP1, BRCA1 and RAD51 to sites of DNA damage. Consequently, splicing-deficient cells exhibited significant numbers of residual γH2AX foci, as would be expected if DNA repair is defective. Furthermore, we show that this is due to downregulation of the E3 ubiquitin ligase RNF8 and that re-introduction of this protein into splicing-deficient cells restores ubiquitylation at sites of DNA damage, accumulation of downstream factors and subsequent repair. Moreover, downregulation of RNF8 explains the defective repair associated with knockdown of various splicing factors in recent genome-wide siRNA screens and, significantly, overexpression of RNF8 counteracts this defect. These discoveries reveal a mechanism that may not only explain how splicing regulates repair of double-strand breaks, but also may underlie various diseases caused by deregulation of splicing factors, including cancer. PMID:27315300

  4. Writers, Readers, and Erasers of Histone Ubiquitylation in DNA Double-Strand Break Repair.

    PubMed

    Smeenk, Godelieve; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose faulty repair may alter the content and organization of cellular genomes. To counteract this threat, numerous signaling and repair proteins are recruited hierarchically to the chromatin areas surrounding DSBs to facilitate accurate lesion repair and restoration of genome integrity. In vertebrate cells, ubiquitin-dependent modifications of histones adjacent to DSBs by RNF8, RNF168, and other ubiquitin ligases have a key role in promoting the assembly of repair protein complexes, serving as direct recruitment platforms for a range of genome caretaker proteins and their associated factors. These DNA damage-induced chromatin ubiquitylation marks provide an essential component of a histone code for DSB repair that is controlled by multifaceted regulatory circuits, underscoring its importance for genome stability maintenance. In this review, we provide a comprehensive account of how DSB-induced histone ubiquitylation is sensed, decoded and modulated by an elaborate array of repair factors and regulators. We discuss how these mechanisms impact DSB repair pathway choice and functionality for optimal protection of genome integrity, as well as cell and organismal fitness.

  5. Writers, Readers, and Erasers of Histone Ubiquitylation in DNA Double-Strand Break Repair

    PubMed Central

    Smeenk, Godelieve; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose faulty repair may alter the content and organization of cellular genomes. To counteract this threat, numerous signaling and repair proteins are recruited hierarchically to the chromatin areas surrounding DSBs to facilitate accurate lesion repair and restoration of genome integrity. In vertebrate cells, ubiquitin-dependent modifications of histones adjacent to DSBs by RNF8, RNF168, and other ubiquitin ligases have a key role in promoting the assembly of repair protein complexes, serving as direct recruitment platforms for a range of genome caretaker proteins and their associated factors. These DNA damage-induced chromatin ubiquitylation marks provide an essential component of a histone code for DSB repair that is controlled by multifaceted regulatory circuits, underscoring its importance for genome stability maintenance. In this review, we provide a comprehensive account of how DSB-induced histone ubiquitylation is sensed, decoded and modulated by an elaborate array of repair factors and regulators. We discuss how these mechanisms impact DSB repair pathway choice and functionality for optimal protection of genome integrity, as well as cell and organismal fitness. PMID:27446204

  6. Double-strand break repair-adox: Restoration of suppressed double-strand break repair during mitosis induces genomic instability.

    PubMed

    Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

    2014-12-01

    Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G1 phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability.

  7. 53BP1-mediated DNA double strand break repair: insert bad pun here.

    PubMed

    Noon, Angela T; Goodarzi, Aaron A

    2011-10-10

    53BP1 is an established player in the cellular response to DNA damage and is a canonical component of ionizing-radiation induced foci--that cadre of proteins which assemble at DNA double strand breaks following radiation exposure and which are readily visualized by immunofluorescence microscopy. While its roles in p53 regulation and cell cycle checkpoint activation have been studied for some time, the impact of 53BP1 on DNA double strand break rejoining has only come to light in the past few years. Convincing evidence now exists for 53BP1 significantly affecting the outcome of DNA double strand break repair in several contexts, many of which hint to an important role in modulating chromatin structure surrounding the break site. Here, we highlight the known and emerging roles of 53BP1 in DNA double strand break repair, including the repair of lesions induced within heterochromatin, following telomere uncapping, in long-range V(D)J recombination, during immunoglobulin class switch recombination and its much debated role in regulating resection during homologous recombination.

  8. Double-strand break repair on sex chromosomes: challenges during male meiotic prophase

    PubMed Central

    Lu, Lin-Yu; Yu, Xiaochun

    2015-01-01

    During meiotic prophase, DNA double-strand break (DSB) repair-mediated homologous recombination (HR) occurs for exchange of genetic information between homologous chromosomes. Unlike autosomes or female sex chromosomes, human male sex chromosomes X and Y share little homology. Although DSBs are generated throughout male sex chromosomes, homologous recombination does not occur for most regions and DSB repair process is significantly prolonged. As a result, male sex chromosomes are coated with many DNA damage response proteins and form a unique chromatin structure known as the XY body. Interestingly, associated with the prolonged DSB repair, transcription is repressed in the XY body but not in autosomes, a phenomenon known as meiotic sex chromosome inactivation (MSCI), which is critical for male meiosis. Here using mice as model organisms, we briefly summarize recent progress on DSB repair in meiotic prophase and focus on the mechanism and function of DNA damage response in the XY body. PMID:25565522

  9. Double-strand break repair on sex chromosomes: challenges during male meiotic prophase.

    PubMed

    Lu, Lin-Yu; Yu, Xiaochun

    2015-01-01

    During meiotic prophase, DNA double-strand break (DSB) repair-mediated homologous recombination (HR) occurs for exchange of genetic information between homologous chromosomes. Unlike autosomes or female sex chromosomes, human male sex chromosomes X and Y share little homology. Although DSBs are generated throughout male sex chromosomes, homologous recombination does not occur for most regions and DSB repair process is significantly prolonged. As a result, male sex chromosomes are coated with many DNA damage response proteins and form a unique chromatin structure known as the XY body. Interestingly, associated with the prolonged DSB repair, transcription is repressed in the XY body but not in autosomes, a phenomenon known as meiotic sex chromosome inactivation (MSCI), which is critical for male meiosis. Here using mice as model organisms, we briefly summarize recent progress on DSB repair in meiotic prophase and focus on the mechanism and function of DNA damage response in the XY body.

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

    PubMed

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

    2013-03-01

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

  11. Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, C.; Heilmann, J.; Rink, H.

    The lens epithelium is the initiation site for the development of radiation induced cataracts. While in the cortex and nucleus radiation interacts with proteins, experimental results from cultured lenses and lens epithelial cells demonstrate mutagenic and cytotoxic effects in the epithelium. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the radiation's relative biological effectiveness (RBE), because cosmic rays differ significantly from X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiations. Irradiations were performed either with 300 kV X-rays or at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. For strand break measurements hydroxyapatite chromatography of alka-line unwound DNA (overall strand breaks) and non-denaturing filter elution technique (double strand breaks) were applied. Experiments showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV/μm more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of

  12. Identification and Characterization of a Human DNA Double-Strand Break Repair Complex

    SciTech Connect

    Chen, D.J.; Cary, R.B.

    1999-07-12

    The authors have used atomic force microscopy (AFM) to characterize the assembly and structure of the macromolecular assemblies involved in DNA repair. They have demonstrated using AFM that the DNA-dependent protein kinase can play a structural role in the repair of DNA double-strand breaks (DSBs) by physically holding DNA ends together. They have extended these studies to include other DNA damage response proteins, these efforts have resulted in important and novel findings regarding the ATM protein. Specifically, the work has demonstrated, for the first time, that the ATM protein binds with specificity to a DNA end. This finding is the first to implicate the ATM protein in the detection of DNA damage by direct physical interaction with DSBs.

  13. Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, C.; Heilmann, J.; Rink, H.

    The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV μ -1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non

  14. Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation.

    PubMed

    Baumstark-Khan, C; Heilmann, J; Rink, H

    2003-01-01

    The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV micrometers-1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of

  15. Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells

    PubMed Central

    Alexander, Jessica L.; Beagan, Kelly; Orr-Weaver, Terry L.; McVey, Mitch

    2016-01-01

    Rereplication generates double-strand breaks (DSBs) at sites of fork collisions and causes genomic damage, including repeat instability and chromosomal aberrations. However, the primary mechanism used to repair rereplication DSBs varies across different experimental systems. In Drosophila follicle cells, developmentally regulated rereplication is used to amplify six genomic regions, two of which contain genes encoding eggshell proteins. We have exploited this system to test the roles of several DSB repair pathways during rereplication, using fork progression as a readout for DSB repair efficiency. Here we show that a null mutation in the microhomology-mediated end-joining (MMEJ) component, polymerase θ/mutagen-sensitive 308 (mus308), exhibits a sporadic thin eggshell phenotype and reduced chorion gene expression. Unlike other thin eggshell mutants, mus308 displays normal origin firing but reduced fork progression at two regions of rereplication. We also find that MMEJ compensates for loss of nonhomologous end joining to repair rereplication DSBs in a site-specific manner. Conversely, we show that fork progression is enhanced in the absence of both Drosophila Rad51 homologs, spindle-A and spindle-B, revealing homologous recombination is active and actually impairs fork movement during follicle cell rereplication. These results demonstrate that several DSB repair pathways are used during rereplication in the follicle cells and their contribution to productive fork progression is influenced by genomic position and repair pathway competition. Furthermore, our findings illustrate that specific rereplication DSB repair pathways can have major effects on cellular physiology, dependent upon genomic context. PMID:27849606

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

    PubMed

    Aparicio, Tomas; Gautier, Jean

    2016-07-01

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

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

    PubMed

    Gorbunova, Vera; Seluanov, Andrei

    2016-06-01

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

  18. Mammalian Ino80 mediates double-strand break repair through its role in DNA end strand resection.

    PubMed

    Gospodinov, Anastas; Vaissiere, Thomas; Krastev, Dragomir B; Legube, Gaëlle; Anachkova, Boyka; Herceg, Zdenko

    2011-12-01

    Chromatin modifications/remodeling are important mechanisms by which cells regulate various functions through providing accessibility to chromatin DNA. Recent studies implicated INO80, a conserved chromatin-remodeling complex, in the process of DNA repair. However, the precise underlying mechanism by which this complex mediates repair in mammalian cells remains enigmatic. Here, we studied the effect of silencing of the Ino80 subunit of the complex on double-strand break repair in mammalian cells. Comet assay and homologous recombination repair reporter system analyses indicated that Ino80 is required for efficient double-strand break repair. Ino80 association with chromatin surrounding double-strand breaks suggested the direct involvement of INO80 in the repair process. Ino80 depletion impaired focal recruitment of 53BP1 but did not impede Rad51 focus formation, suggesting that Ino80 is required for the early steps of repair. Further analysis by using bromodeoxyuridine (BrdU)-labeled single-stranded DNA and replication protein A (RPA) immunofluorescent staining showed that INO80 mediates 5'-3' resection of double-strand break ends.

  19. Dynamic monitoring of oxidative DNA double-strand break and repair in cardiomyocytes.

    PubMed

    Ye, Bo; Hou, Ning; Xiao, Lu; Xu, Yifan; Xu, Haodong; Li, Faqian

    2016-01-01

    DNA double-strand breaks (DSBs) are most dangerous lesions. To determine whether oxidative stress can induce DSBs and how they are repaired in cardiomyocytes (CMs), cultured neonatal rat CMs were treated with different doses of H2O2 and followed for up to 72 h for monitoring the spatiotemporal dynamics of DNA repair protein assembly/disassembly at DSB foci. The protein levels and foci numbers of histone H2AX phosphorylated at serine 139 (γ-H2AX) increased proportionally to 50, 100, and 200 μmol/L H2O2 after 30 min treatment. When H2O2 was at or above 400 μmol/L, γ-H2AX became predominantly pannuclear. After 30 min, 200 μmol/L of H2O2 treatment, γ-H2AX levels were highest within the first hour and then gradually declined during the recovery and returned to basal levels at 48 h. Among DNA damage transducer kinases, ataxia telangiectasia mutated (ATM) was significantly activated by H2O2 in contrast to mild activation of ATR (ATM and Rad3-related). A DSB binding protein, p53 binding protein 1, formed distinct nuclear foci that colocalized with γ-H2AX foci and phosphorylated ATM. Our findings indicate that DSBs can be induced by H2O2 and ATM is the main kinase to mediate DSB repair in CMs. Therefore, monitoring DSB repair can assess oxidative injury and response in CMs.

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

    PubMed Central

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

    2014-01-01

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

  1. DNA polymerase θ (POLQ), double-strand break repair, and cancer.

    PubMed

    Wood, Richard D; Doublié, Sylvie

    2016-08-01

    DNA polymerase theta (pol θ) is encoded in the genomes of many eukaryotes, though not in fungi. Pol θ is encoded by the POLQ gene in mammalian cells. The C-terminal third of the protein is a family A DNA polymerase with additional insertion elements relative to prokaryotic homologs. The N-terminal third is a helicase-like domain with DNA-dependent ATPase activity. Pol θ is important in the repair of genomic double-strand breaks (DSBs) from many sources. These include breaks formed by ionizing radiation and topoisomerase inhibitors, breaks arising at stalled DNA replication forks, breaks introduced during diversification steps of the mammalian immune system, and DSB induced by CRISPR-Cas9. Pol θ participates in a route of DSB repair termed "alternative end-joining" (altEJ). AltEJ is independent of the DNA binding Ku protein complex and requires DNA end resection. Pol θ is able to mediate joining of two resected 3' ends harboring DNA sequence microhomology. "Signatures" of Pol θ action during altEJ are the frequent utilization of longer microhomologies, and the insertion of additional sequences at joining sites. The mechanism of end-joining employs the ability of Pol θ to tightly grasp a 3' terminus through unique contacts in the active site, allowing extension from minimally paired primers. Pol θ is involved in controlling the frequency of chromosome translocations and preserves genome integrity by limiting large deletions. It may also play a backup role in DNA base excision repair. POLQ is a member of a cluster of similarly upregulated genes that are strongly correlated with poor clinical outcome for breast cancer, ovarian cancer and other cancer types. Inhibition of pol θ is a compelling approach for combination therapy of radiosensitization.

  2. Visualization of DNA Double-Strand Break Repair at the Single-Molecule Level

    SciTech Connect

    Dynan, William S.; Li, Shuyi; Mernaugh, Raymond; Wragg, Stephanie; Takeda, Yoshihiko

    2003-03-27

    Exposure to low doses of ionizing radiation is universal. The signature injury from ionizing radiation exposure is induction of DNA double-strand breaks (DSBs). The first line of defense against DSBs is direct ligation of broken DNA ends via the nonhomologous end-joining pathway. Because even a relatively high environmental exposure induces only a few DSBs per cell, our current understanding of the response to this exposure is limited by the ability to measure DSB repair events reliably in situ at a single-molecule level. To address this need, we have taken advantage of biological amplification, measuring relocalization of proteins and detection of protein phosphorylation as a surrogate for detection of broken ends themselves. We describe the use of specific antibodies to investigate the kinetics and mechanism of repair of very small numbers of DSBs in human cells by the nonhomologous end-joining pathway.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2004-12-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-07-10

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

  7. DNA ligase III acts as a DNA strand break sensor in the cellular orchestration of DNA strand break repair

    PubMed Central

    Abdou, Ismail; Poirier, Guy G.; Hendzel, Michael J.; Weinfeld, Michael

    2015-01-01

    In the current model of DNA SSBR, PARP1 is regarded as the sensor of single-strand breaks (SSBs). However, biochemical studies have implicated LIG3 as another possible SSB sensor. Using a laser micro-irradiation protocol that predominantly generates SSBs, we were able to demonstrate that PARP1 is dispensable for the accumulation of different single-strand break repair (SSBR) proteins at sites of DNA damage in live cells. Furthermore, we show in live cells for the first time that LIG3 plays a role in mediating the accumulation of the SSBR proteins XRCC1 and PNKP at sites of DNA damage. Importantly, the accumulation of LIG3 at sites of DNA damage did not require the BRCT domain-mediated interaction with XRCC1. We were able to show that the N-terminal ZnF domain of LIG3 plays a key role in the enzyme's SSB sensing function. Finally, we provide cellular evidence that LIG3 and not PARP1 acts as the sensor for DNA damage caused by the topoisomerase I inhibitor, irinotecan. Our results support the existence of a second damage-sensing mechanism in SSBR involving the detection of nicks in the genome by LIG3. PMID:25539916

  8. SAW1 is required for SDSA double-strand break repair in S. cerevisiae.

    PubMed

    Diamante, Graciel; Phan, Claire; Celis, Angie S; Krueger, Jonas; Kelson, Eric P; Fischhaber, Paula L

    2014-03-14

    SAW1, coding for Saw1, is required for single-strand annealing (SSA) DNA double-strand break (DSB) repair in Saccharomycescerevisiae. Saw1 physically associates with Rad1 and Rad52 and recruits the Rad1-Rad10 endonuclease. Herein we show by fluorescence microscopy that SAW1 is similarly required for recruitment of Rad10 to sites of Synthesis-Dependent Strand Annealing (SDSA) and associates with sites of SDSA repair in a manner temporally overlapped with Rad10. The magnitude of induction of colocalized Saw1-CFP/Rad10-YFP/DSB-RFP foci in SDSA is more dramatic in S and G2 phase cells than in M phase, consistent with the known mechanism of SDSA. We observed a substantial fraction of foci in which Rad10 was localized to the repair site without Saw1, but few DSB sites that contained Saw1 without Rad10. Together these data are consistent with a model in which Saw1 recruits Rad1-Rad10 to SDSA sites, possibly even binding as a protein-protein complex, but departs the repair site in advance of Rad1-Rad10.

  9. Chromatin modification and NBS1: their relationship in DNA double-strand break repair.

    PubMed

    Saito, Yuichiro; Zhou, Hui; Kobayashi, Junya

    2016-01-01

    The importance of chromatin modification, including histone modification and chromatin remodeling, for DNA double-strand break (DSB) repair, as well as transcription and replication, has been elucidated. Phosphorylation of H2AX to γ-H2AX is one of the first responses following DSB detection, and this histone modification is important for the DSB damage response by triggering several events, including the accumulation of DNA damage response-related proteins and subsequent homologous recombination (HR) repair. The roles of other histone modifications such as acetylation, methylation and ubiquitination have also been recently clarified, particularly in the context of HR repair. NBS1 is a multifunctional protein that is involved in various DNA damage responses. Its recently identified binding partner RNF20 is an E3 ubiquitin ligase that facilitates the monoubiquitination of histone H2B, a process that is crucial for recruitment of the chromatin remodeler SNF2h to DSB damage sites. Evidence suggests that SNF2h functions in HR repair, probably through regulation of end-resection. Moreover, several recent reports have indicated that SNF2h can function in HR repair pathways as a histone remodeler and that other known histone remodelers can also participate in DSB damage responses. On the other hand, information about the roles of such chromatin modifications and NBS1 in non-homologous end joining (NHEJ) repair of DSBs and stalled fork-related damage responses is very limited; therefore, these aspects and processes need to be further studied to advance our understanding of the mechanisms and molecular players involved.

  10. Subdiffusion Supports Joining Of Correct Ends During Repair Of DNA Double-Strand Breaks

    NASA Astrophysics Data System (ADS)

    Girst, S.; Hable, V.; Drexler, G. A.; Greubel, C.; Siebenwirth, C.; Haum, M.; Friedl, A. A.; Dollinger, G.

    2013-08-01

    The mobility of damaged chromatin regions in the nucleus may affect the probability of mis-repair. In this work, live-cell observation and distance tracking of GFP-tagged DNA damage response protein MDC1 was used to study the random-walk behaviour of chromatin domains containing radiation-induced DNA double-strand breaks (DSB). Our measurements indicate a subdiffusion-type random walk process with similar time dependence for isolated and clustered DSBs that were induced by 20 MeV proton or 43 MeV carbon ion micro-irradiation. As compared to normal diffusion, subdiffusion enhances the probability that both ends of a DSB meet, thus promoting high efficiency DNA repair. It also limits their probability of long-range movements and thus lowers the probability of mis-rejoining and chromosome aberrations.

  11. DNA Repair-Protein Relocalization After Heavy Ion Exposure

    NASA Technical Reports Server (NTRS)

    Metting, N. F.

    1999-01-01

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

  12. Methylating agents and DNA repair responses: methylated bases and sources of strand breaks

    PubMed Central

    Wyatt, Michael D.; Pittman, Douglas L.

    2008-01-01

    The chemical methylating agents methylmethane sulfonate (MMS) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) have been used for decades as classical DNA damaging agents. These agents have been utilized to uncover and explore pathways of DNA repair, DNA damage response, and mutagenesis. MMS and MNNG modify DNA by adding methyl groups to a number of nucleophilic sites on the DNA bases, although MNNG produces a greater percentage of O-methyl adducts. There has been substantial progress elucidating direct reversal proteins that remove methyl groups and base excision repair (BER), which removes and replaces methylated bases. Direct reversal proteins and BER thus counteract the toxic, mutagenic and clastogenic effects of methylating agents. Despite recent progress, the complexity of DNA damage responses to methylating agents is still being discovered. In particular, there is growing understanding of pathways such as homologous recombination, lesion bypass, and mismatch repair that react when the response of direct reversal proteins and BER is insufficient. Furthermore, the importance of proper balance within the steps in BER has been uncovered with the knowledge that DNA structural intermediates during BER are deleterious. A number of issues complicate elucidating the downstream responses when direct reversal is insufficient or BER is imbalanced. These include inter-species differences, cell-type specific differences within mammals and between cancer cell lines, and the type of methyl damage or BER intermediate encountered. MMS also carries a misleading reputation of being a ‘radiomimetic,’ i.e., capable of directly producing strand breaks. This review focuses on the DNA methyl damage caused by MMS and MNNG for each site of potential methylation to summarize what is known about the repair of such damage and the downstream responses and consequences if not repaired. PMID:17173371

  13. RecQ helicases in DNA double strand break repair and telomere maintenance.

    PubMed

    Singh, Dharmendra Kumar; Ghosh, Avik K; Croteau, Deborah L; Bohr, Vilhelm A

    2012-08-01

    Organisms are constantly exposed to various environmental insults which could adversely affect the stability of their genome. To protect their genomes against the harmful effect of these environmental insults, organisms have evolved highly diverse and efficient repair mechanisms. Defective DNA repair processes can lead to various kinds of chromosomal and developmental abnormalities. RecQ helicases are a family of evolutionarily conserved, DNA unwinding proteins which are actively engaged in various DNA metabolic processes, telomere maintenance and genome stability. Bacteria and lower eukaryotes, like yeast, have only one RecQ homolog, whereas higher eukaryotes including humans possess multiple RecQ helicases. These multiple RecQ helicases have redundant and/or non-redundant functions depending on the types of DNA damage and DNA repair pathways. Humans have five different RecQ helicases and defects in three of them cause autosomal recessive diseases leading to various kinds of cancer predisposition and/or aging phenotypes. Emerging evidence also suggests that the RecQ helicases have important roles in telomere maintenance. This review mainly focuses on recent knowledge about the roles of RecQ helicases in DNA double strand break repair and telomere maintenance which are important in preserving genome integrity.

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

    PubMed

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

    2013-07-01

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

  15. The Small RNA GcvB Promotes Mutagenic Break Repair by Opposing the Membrane Stress Response

    PubMed Central

    Barreto, Brittany; Rogers, Elizabeth; Xia, Jun; Frisch, Ryan L.; Richters, Megan; Fitzgerald, Devon M.

    2016-01-01

    ABSTRACT Microbes and human cells possess mechanisms of mutagenesis activated by stress responses. Stress-inducible mutagenesis mechanisms may provide important models for mutagenesis that drives host-pathogen interactions, antibiotic resistance, and possibly much of evolution generally. In Escherichia coli, repair of DNA double-strand breaks is switched to a mutagenic mode, using error-prone DNA polymerases, via the SOS DNA damage and general (σS) stress responses. We investigated small RNA (sRNA) clients of Hfq, an RNA chaperone that promotes mutagenic break repair (MBR), and found that GcvB promotes MBR by allowing a robust σS response, achieved via opposing the membrane stress (σE) response. Cells that lack gcvB were MBR deficient and displayed reduced σS-dependent transcription but not reduced σS protein levels. The defects in MBR and σS-dependent transcription in ΔgcvB cells were alleviated by artificially increasing σS levels, implying that GcvB promotes mutagenesis by allowing a normal σS response. ΔgcvB cells were highly induced for the σE response, and blocking σE response induction restored both mutagenesis and σS-promoted transcription. We suggest that GcvB may promote the σS response and mutagenesis indirectly, by promoting membrane integrity, which keeps σE levels lower. At high levels, σE might outcompete σS for binding RNA polymerase and so reduce the σS response and mutagenesis. The data show the delicate balance of stress response modulation of mutagenesis. IMPORTANCE Mutagenesis mechanisms upregulated by stress responses promote de novo antibiotic resistance and cross-resistance in bacteria, antifungal drug resistance in yeasts, and genome instability in cancer cells under hypoxic stress. This paper describes the role of a small RNA (sRNA) in promoting a stress-inducible-mutagenesis mechanism, mutagenic DNA break repair in Escherichia coli. The roles of many sRNAs in E. coli remain unknown. This study shows that ΔgcvB cells

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

    PubMed

    Kostyrko, Kaja; Mermod, Nicolas

    2016-04-07

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

  17. Deficiency of double-strand DNA break repair does not impair Mycobacterium tuberculosis virulence in multiple animal models of infection.

    PubMed

    Heaton, Brook E; Barkan, Daniel; Bongiorno, Paola; Karakousis, Petros C; Glickman, Michael S

    2014-08-01

    Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed. M. tuberculosis elaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant to M. tuberculosis latency. However, very little information is available about the phenotype of DSB repair-deficient M. tuberculosis in animal models of infection. Here we tested M. tuberculosis strains lacking NHEJ (a Δku ΔligD strain), HR (a ΔrecA strain), or both (a ΔrecA Δku strain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required for M. tuberculosis pathogenesis.

  18. DNA breaks in hypermutating immunoglobulin genes: evidence for a break-and-repair pathway of somatic hypermutation.

    PubMed Central

    Kong, Q; Maizels, N

    2001-01-01

    To test the hypothesis that immunoglobulin gene hypermutation in vivo employs a pathway in which DNA breaks are introduced and subsequently repaired to produce mutations, we have used a PCR-based assay to detect and identify single-strand DNA breaks in lambda1 genes of actively hypermutating primary murine germinal center B cells. We find that there is a two- to threefold excess of breaks in lambda1 genes of hypermutating B cells, relative to nonhypermutating B cells, and that 1.3% of germinal center B cells contain breaks in the lambda1 gene that are associated with hypermutation. Breaks were found in both top and bottom DNA strands and were localized to the region of lambda1 that actively hypermutates, but duplex breaks accounted for only a subset of breaks identified. Almost half of the breaks in hypermutating B cells occurred at hotspots, sites at which two or more independent breaks were identified. Breaksite hotspots were associated with characteristic sequence motifs: a pyrimidine-rich motif, either RCTYT or CCYC; and RGYW, a sequence motif associated with hypermutation hotspots. The sequence motifs identified at breaksite hotspots should inform the design of substrates for characterization of activities that participate in the hypermutation pathway. PMID:11333245

  19. Contribution of double strand break repair gene XRCC3 genotypes to nasopharyngeal carcinoma risk in Taiwan.

    PubMed

    Liu, Juhn-Cherng; Tsai, Chia-Wen; Hsu, Chin-Mu; Chang, Wen-Shin; Li, Chi-Yuan; Liu, Shih-Ping; Shen, Wu-Chung; Bau, Da-Tian

    2015-02-28

    The DNA double strand break repair protein XRCC3 plays a central role in removing double strand breaks from the genome and defects in cellular repair capacity is closely related to human cancer initiation. Therefore, we aimed to investigate the contribution of XRCC3 genotypes to individual nasopharyngeal carcinoma (NPC) susceptibility. In this hospital-based population research, the genotyping and analyzing of XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539, rs28903081 in a large Taiwanese population was performed. Totally, 176 NPC patients and 880 age- and gender-matched healthy controls were genotyped and analyzed by PCR-RFLP method. The results showed that there was a differential distribution among NPC and control subjects in the genotypic (P = 0.000488) and allelic (P = 0.0002) frequencies of XRCC3 rs861539. As for the gene-environment interaction, we have firstly provided evidence showing that there is an obvious joint effect of XRCC3 rs861539 CT and TT genotypes with individual smoking habits on increased NPC risk. In conclusion, the T allele of XRCC3 rs861539, interacts with smoking habit in increasing NPC risk, may be an early detection marker for NPC.

  20. Non-Repairable Strand Breaks Induced by 125I Incorporated into Mammalian DNA

    PubMed Central

    Painter, R. B.; Young, B. R.; Burki, H. J.

    1974-01-01

    When 125I is incorporated into Chinese hamster DNA (via 125I-labeled iododeoxyuridine) and the cells are stored at 77°K, the resulting decays of the isotope cause 4 to 5 breaks/single-strand per disintegration. On the average, about 50% of these breaks are repaired. In contrast, under the same conditions of storage and in the same range of total strand breaks/cell, 70-100% of the breaks induced by x-radiation are repaired. Thus, the extreme toxicity of 125I when incorporated into DNA is correlated with the unrepaired breaks caused by decay of this isotope. These results suggest that unrepaired DNA strand breaks may be important in cell killing after treatments which damage DNA. PMID:4531021

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

    PubMed

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

    2014-01-01

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

  2. Repair on the go: E. coli maintains a high proliferation rate while repairing a chronic DNA double-strand break.

    PubMed

    Darmon, Elise; Eykelenboom, John K; Lopez-Vernaza, Manuel A; White, Martin A; Leach, David R F

    2014-01-01

    DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.

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

    PubMed

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

    2014-10-17

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

  4. Detection and Repair of Ionizing Radiation-Induced DNA Double Strand Breaks: New Developments in Nonhomologous End Joining

    SciTech Connect

    Wang, Chen; Lees-Miller, Susan P.

    2013-07-01

    DNA damage can occur as a result of endogenous metabolic reactions and replication stress or from exogenous sources such as radiation therapy and chemotherapy. DNA double strand breaks are the most cytotoxic form of DNA damage, and defects in their repair can result in genome instability, a hallmark of cancer. The major pathway for the repair of ionizing radiation-induced DSBs in human cells is nonhomologous end joining. Here we review recent advances on the mechanism of nonhomologous end joining, as well as new findings on its component proteins and regulation.

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

    PubMed

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

    2011-02-22

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

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

    PubMed Central

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

    2009-01-01

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

  7. RecA bundles mediate homology pairing between distant sisters during DNA break repair

    NASA Astrophysics Data System (ADS)

    Lesterlin, Christian; Ball, Graeme; Schermelleh, Lothar; Sherratt, David J.

    2014-02-01

    DNA double-strand break (DSB) repair by homologous recombination has evolved to maintain genetic integrity in all organisms. Although many reactions that occur during homologous recombination are known, it is unclear where, when and how they occur in cells. Here, by using conventional and super-resolution microscopy, we describe the progression of DSB repair in live Escherichia coli. Specifically, we investigate whether homologous recombination can occur efficiently between distant sister loci that have segregated to opposite halves of an E. coli cell. We show that a site-specific DSB in one sister can be repaired efficiently using distant sister homology. After RecBCD processing of the DSB, RecA is recruited to the cut locus, where it nucleates into a bundle that contains many more RecA molecules than can associate with the two single-stranded DNA regions that form at the DSB. Mature bundles extend along the long axis of the cell, in the space between the bulk nucleoid and the inner membrane. Bundle formation is followed by pairing, in which the two ends of the cut locus relocate at the periphery of the nucleoid and together move rapidly towards the homology of the uncut sister. After sister locus pairing, RecA bundles disassemble and proteins that act late in homologous recombination are recruited to give viable recombinants 1-2-generation-time equivalents after formation of the initial DSB. Mutated RecA proteins that do not form bundles are defective in sister pairing and in DSB-induced repair. This work reveals an unanticipated role of RecA bundles in channelling the movement of the DNA DSB ends, thereby facilitating the long-range homology search that occurs before the strand invasion and transfer reactions.

  8. DNA double strand break repair, chromosome synapsis and transcriptional silencing in meiosis.

    PubMed

    Inagaki, Akiko; Schoenmakers, Sam; Baarends, Willy M

    2010-05-16

    Chromosome pairing and synapsis during meiotic prophase requires the formation and repair of DNA double-strand breaks (DSBs) by the topoisomerase-like enzyme SPO11. Chromosomes, or chromosomal regions, that lack a pairing partner, such as the largely heterologous X and Y chromosomes, show delayed meiotic DSB repair and are transcriptionally silenced. Herein, we review meiosis-specific aspects of DSB repair in relation to homology recognition and meiotic silencing of heterologous regions. We propose a dynamic interplay between progression of synapsis and persistent meiotic DSBs. Signaling from these persistent breaks could inhibit heterologous synapsis and stimulate meiotic silencing of the X and Y chromosomes.

  9. JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks.

    PubMed

    Van Meter, Michael; Simon, Matthew; Tombline, Gregory; May, Alfred; Morello, Timothy D; Hubbard, Basil P; Bredbenner, Katie; Park, Rosa; Sinclair, David A; Bohr, Vilhelm A; Gorbunova, Vera; Seluanov, Andrei

    2016-09-06

    The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6), promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB) repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

  10. Impact of a stress-inducible switch to mutagenic repair of DNA breaks on mutation in Escherichia coli

    PubMed Central

    Shee, Chandan; Gibson, Janet L.; Darrow, Michele C.; Gonzalez, Caleb; Rosenberg, Susan M.

    2011-01-01

    Basic ideas about the constancy and randomness of mutagenesis that drives evolution were challenged by the discovery of mutation pathways activated by stress responses. These pathways could promote evolution specifically when cells are maladapted to their environment (i.e., are stressed). However, the clearest example—a general stress-response–controlled switch to error-prone DNA break (double-strand break, DSB) repair—was suggested to be peculiar to an Escherichia coli F′ conjugative plasmid, not generally significant, and to occur by an alternative stress-independent mechanism. Moreover, mechanisms of spontaneous mutation in E. coli remain obscure. First, we demonstrate that this same mechanism occurs in chromosomes of starving F− E. coli. I-SceI endonuclease-induced chromosomal DSBs increase mutation 50-fold, dependent upon general/starvation- and DNA-damage-stress responses, DinB error-prone DNA polymerase, and DSB-repair proteins. Second, DSB repair is also mutagenic if the RpoS general-stress-response activator is expressed in unstressed cells, illustrating a stress-response–controlled switch to mutagenic repair. Third, DSB survival is not improved by RpoS or DinB, indicating that mutagenesis is not an inescapable byproduct of repair. Importantly, fourth, fully half of spontaneous frame-shift and base-substitution mutation during starvation also requires the same stress-response, DSB-repair, and DinB proteins. These data indicate that DSB-repair-dependent stress-induced mutation, driven by spontaneous DNA breaks, is a pathway that cells usually use and a major source of spontaneous mutation. These data also rule out major alternative models for the mechanism. Mechanisms that couple mutagenesis to stress responses can allow cells to evolve rapidly and responsively to their environment. PMID:21808005

  11. Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair.

    PubMed

    Chiolo, Irene; Minoda, Aki; Colmenares, Serafin U; Polyzos, Aris; Costes, Sylvain V; Karpen, Gary H

    2011-03-04

    Double-strand breaks (DSBs) in heterochromatic repetitive DNAs pose significant threats to genome integrity, but information about how such lesions are processed and repaired is sparse. We observe dramatic expansion and dynamic protrusions of the heterochromatin domain in response to ionizing radiation (IR) in Drosophila cells. We also find that heterochromatic DSBs are repaired by homologous recombination (HR) but with striking differences from euchromatin. Proteins involved in early HR events (resection) are rapidly recruited to DSBs within heterochromatin. In contrast, Rad51, which mediates strand invasion, only associates with DSBs that relocalize outside of the domain. Heterochromatin expansion and relocalization of foci require checkpoint and resection proteins. Finally, the Smc5/6 complex is enriched in heterochromatin and is required to exclude Rad51 from the domain and prevent abnormal recombination. We propose that the spatial and temporal control of DSB repair in heterochromatin safeguards genome stability by preventing aberrant exchanges between repeats.

  12. Double-Strand Breaks in Heterochromatin Move Outside of a Dynamic HP1a Domain to Complete Recombinational Repair

    PubMed Central

    Chiolo, Irene; Minoda, Aki; Colmenares, Serafin U.; Polyzos, Aris; Costes, Sylvain V.; Karpen, Gary H.

    2012-01-01

    SUMMARY Double-strand breaks (DSBs) in heterochromatic repetitive DNAs pose significant threats to genome integrity, but information about how such lesions are processed and repaired is sparse. We observe dramatic expansion and dynamic protrusions of the heterochromatin domain in response to ionizing radiation (IR) in Drosophila cells. We also find that heterochromatic DSBs are repaired by homologous recombination (HR) but with striking differences from euchromatin. Proteins involved in early HR events (resection) are rapidly recruited to DSBs within heterochromatin. In contrast, Rad51, which mediates strand invasion, only associates with DSBs that relocalize outside of the domain. Hetero-chromatin expansion and relocalization of foci require checkpoint and resection proteins. Finally, the Smc5/6 complex is enriched in heterochromatin and is required to exclude Rad51 from the domain and prevent abnormal recombination. We propose that the spatial and temporal control of DSB repair in heterochromatin safeguards genome stability by preventing aberrant exchanges between repeats. PMID:21353298

  13. PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair**

    PubMed Central

    Coates, Julia; Jhujh, Satpal; Mehmood, Shahid; Tamura, Naoka; Travers, Jon; Wu, Qian; Draviam, Viji M.; Robinson, Carol V.; Blundell, Tom L.; Jackson, Stephen P.

    2014-01-01

    XRCC4 and XLF are two structurally-related proteins that function in DNA double-strand break (DSB) repair. Here, we identify human PAXX (PAralog of XRCC4 and XLF; also called C9orf142) as a new XRCC4-superfamily member, and show that its crystal structure resembles that of XRCC4. PAXX interacts directly with the DSB-repair protein Ku and is recruited to DNA-damage sites in cells. Using RNA interference and CRISPR-Cas9 to generate PAXX−/− cells, we demonstrate that PAXX functions with XRCC4 and XLF to mediate DSB repair and cell survival in response to DSB-inducing agents. Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro, and assembly of core non-homologous end-joining (NHEJ) factors on damaged chromatin in cells. These findings identify PAXX as a new component of the NHEJ machinery. PMID:25574025

  14. Deletion-bias in DNA double-strand break repair differentially contributes to plant genome shrinkage.

    PubMed

    Vu, Giang T H; Cao, Hieu X; Reiss, Bernd; Schubert, Ingo

    2017-02-28

    In order to prevent genome instability, cells need to be protected by a number of repair mechanisms, including DNA double-strand break (DSB) repair. The extent to which DSB repair, biased towards deletions or insertions, contributes to evolutionary diversification of genome size is still under debate. We analyzed mutation spectra in Arabidopsis thaliana and in barley (Hordeum vulgare) by PacBio sequencing of three DSB-targeted loci each, uncovering repair via gene conversion, single strand annealing (SSA) or nonhomologous end-joining (NHEJ). Furthermore, phylogenomic comparisons between A. thaliana and two related species were used to detect naturally occurring deletions during Arabidopsis evolution. Arabidopsis thaliana revealed significantly more and larger deletions after DSB repair than barley, and barley displayed more and larger insertions. Arabidopsis displayed a clear net loss of DNA after DSB repair, mainly via SSA and NHEJ. Barley revealed a very weak net loss of DNA, apparently due to less active break-end resection and easier copying of template sequences into breaks. Comparative phylogenomics revealed several footprints of SSA in the A. thaliana genome. Quantitative assessment of DNA gain and loss through DSB repair processes suggests deletion-biased DSB repair causing ongoing genome shrinking in A. thaliana, whereas genome size in barley remains nearly constant.

  15. DNA-PKcs structure suggests an allosteric mechanism modulating DNA double-strand break repair.

    PubMed

    Sibanda, Bancinyane L; Chirgadze, Dimitri Y; Ascher, David B; Blundell, Tom L

    2017-02-03

    DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a central component of nonhomologous end joining (NHEJ), repairing DNA double-strand breaks that would otherwise lead to apoptosis or cancer. We have solved its structure in complex with the C-terminal peptide of Ku80 at 4.3 angstrom resolution using x-ray crystallography. We show that the 4128-amino acid structure comprises three large structural units: the N-terminal unit, the Circular Cradle, and the Head. Conformational differences between the two molecules in the asymmetric unit are correlated with changes in accessibility of the kinase active site, which are consistent with an allosteric mechanism to bring about kinase activation. The location of KU80ct194 in the vicinity of the breast cancer 1 (BRCA1) binding site suggests competition with BRCA1, leading to pathway selection between NHEJ and homologous recombination.

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

    PubMed Central

    Yun, Maximina H.; Hiom, Kevin

    2009-01-01

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

  17. Branch migration prevents DNA loss during double-strand break repair.

    PubMed

    Mawer, Julia S P; Leach, David R F

    2014-08-01

    The repair of DNA double-strand breaks must be accurate to avoid genomic rearrangements that can lead to cell death and disease. This can be accomplished by promoting homologous recombination between correctly aligned sister chromosomes. Here, using a unique system for generating a site-specific DNA double-strand break in one copy of two replicating Escherichia coli sister chromosomes, we analyse the intermediates of sister-sister double-strand break repair. Using two-dimensional agarose gel electrophoresis, we show that when double-strand breaks are formed in the absence of RuvAB, 4-way DNA (Holliday) junctions are accumulated in a RecG-dependent manner, arguing against the long-standing view that the redundancy of RuvAB and RecG is in the resolution of Holliday junctions. Using pulsed-field gel electrophoresis, we explain the redundancy by showing that branch migration catalysed by RuvAB and RecG is required for stabilising the intermediates of repair as, when branch migration cannot take place, repair is aborted and DNA is lost at the break locus. We demonstrate that in the repair of correctly aligned sister chromosomes, an unstable early intermediate is stabilised by branch migration. This reliance on branch migration may have evolved to help promote recombination between correctly aligned sister chromosomes to prevent genomic rearrangements.

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

    PubMed

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

    2017-01-24

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

  19. Homology Requirements and Competition between Gene Conversion and Break-Induced Replication during Double-Strand Break Repair.

    PubMed

    Mehta, Anuja; Beach, Annette; Haber, James E

    2017-02-02

    Saccharomyces cerevisiae mating-type switching is initiated by a double-strand break (DSB) at MATa, leaving one cut end perfectly homologous to the HMLα donor, while the second end must be processed to remove a non-homologous tail before completing repair by gene conversion (GC). When homology at the matched end is ≤150 bp, efficient repair depends on the recombination enhancer, which tethers HMLα near the DSB. Thus, homology shorter than an apparent minimum efficient processing segment can be rescued by tethering the donor near the break. When homology at the second end is ≤150 bp, second-end capture becomes inefficient and repair shifts from GC to break-induced replication (BIR). But when pol32 or pif1 mutants block BIR, GC increases 3-fold, indicating that the steps blocked by these mutations are reversible. With short second-end homology, absence of the RecQ helicase Sgs1 promotes gene conversion, whereas deletion of the FANCM-related Mph1 helicase promotes BIR.

  20. Mechanistic Modelling and Bayesian Inference Elucidates the Variable Dynamics of Double-Strand Break Repair.

    PubMed

    Woods, Mae L; Barnes, Chris P

    2016-10-01

    DNA double-strand breaks are lesions that form during metabolism, DNA replication and exposure to mutagens. When a double-strand break occurs one of a number of repair mechanisms is recruited, all of which have differing propensities for mutational events. Despite DNA repair being of crucial importance, the relative contribution of these mechanisms and their regulatory interactions remain to be fully elucidated. Understanding these mutational processes will have a profound impact on our knowledge of genomic instability, with implications across health, disease and evolution. Here we present a new method to model the combined activation of non-homologous end joining, single strand annealing and alternative end joining, following exposure to ionising radiation. We use Bayesian statistics to integrate eight biological data sets of double-strand break repair curves under varying genetic knockouts and confirm that our model is predictive by re-simulating and comparing to additional data. Analysis of the model suggests that there are at least three disjoint modes of repair, which we assign as fast, slow and intermediate. Our results show that when multiple data sets are combined, the rate for intermediate repair is variable amongst genetic knockouts. Further analysis suggests that the ratio between slow and intermediate repair depends on the presence or absence of DNA-PKcs and Ku70, which implies that non-homologous end joining and alternative end joining are not independent. Finally, we consider the proportion of double-strand breaks within each mechanism as a time series and predict activity as a function of repair rate. We outline how our insights can be directly tested using imaging and sequencing techniques and conclude that there is evidence of variable dynamics in alternative repair pathways. Our approach is an important step towards providing a unifying theoretical framework for the dynamics of DNA repair processes.

  1. Mechanistic Modelling and Bayesian Inference Elucidates the Variable Dynamics of Double-Strand Break Repair

    PubMed Central

    2016-01-01

    DNA double-strand breaks are lesions that form during metabolism, DNA replication and exposure to mutagens. When a double-strand break occurs one of a number of repair mechanisms is recruited, all of which have differing propensities for mutational events. Despite DNA repair being of crucial importance, the relative contribution of these mechanisms and their regulatory interactions remain to be fully elucidated. Understanding these mutational processes will have a profound impact on our knowledge of genomic instability, with implications across health, disease and evolution. Here we present a new method to model the combined activation of non-homologous end joining, single strand annealing and alternative end joining, following exposure to ionising radiation. We use Bayesian statistics to integrate eight biological data sets of double-strand break repair curves under varying genetic knockouts and confirm that our model is predictive by re-simulating and comparing to additional data. Analysis of the model suggests that there are at least three disjoint modes of repair, which we assign as fast, slow and intermediate. Our results show that when multiple data sets are combined, the rate for intermediate repair is variable amongst genetic knockouts. Further analysis suggests that the ratio between slow and intermediate repair depends on the presence or absence of DNA-PKcs and Ku70, which implies that non-homologous end joining and alternative end joining are not independent. Finally, we consider the proportion of double-strand breaks within each mechanism as a time series and predict activity as a function of repair rate. We outline how our insights can be directly tested using imaging and sequencing techniques and conclude that there is evidence of variable dynamics in alternative repair pathways. Our approach is an important step towards providing a unifying theoretical framework for the dynamics of DNA repair processes. PMID:27741226

  2. DNA double-strand break repair in a cellular context.

    PubMed

    Shibata, A; Jeggo, P A

    2014-05-01

    Substantial insight into the mechanisms responding to DNA double-strand breaks has been gained from molecular, biochemical and structural approaches. Attention is now focusing on understanding the interplay between the pathways, how they interface through the cell cycle and the communication with other DNA transactions, such as replication and transcription. Understanding these aspects will facilitate an assessment of how cancer cells have modified these processes to achieve unlimited proliferative capacity and adaptability, and pave the way to identify targets suitable for therapy. Here, we briefly overview the processes responding to double-strand breaks and discuss our current understanding of their interplay in a cellular context.

  3. Cell transcriptional state alters genomic patterns of DNA double-strand break repair in human astrocytes.

    PubMed

    Yong, Raymund L; Yang, Chunzhang; Lu, Jie; Wang, Huaien; Schlaff, Cody D; Tandle, Anita; Graves, Christian A; Elkahloun, Abdel G; Chen, Xiaoyuan; Zhuang, Zhengping; Lonser, Russell R

    2014-12-17

    The misrepair of DNA double-strand breaks in close spatial proximity within the nucleus can result in chromosomal rearrangements that are important in the pathogenesis of haematopoietic and solid malignancies. It is unknown why certain epigenetic states, such as those found in stem or progenitor cells, appear to facilitate neoplastic transformation. Here we show that altering the transcriptional state of human astrocytes alters patterns of DNA damage repair from ionizing radiation at a gene locus-specific and genome-wide level. Astrocytes induced into a reactive state exhibit increased DNA repair, compared with non-reactive cells, in actively transcribed chromatin after irradiation. In mapping these repair sites, we identify misrepair events and repair hotspots that are unique to each state. The precise characterization of genomic regions susceptible to mutation in specific transcriptional states provides new opportunities for addressing clonal evolution in solid cancers, in particular those where double-strand break induction is a cornerstone of clinical intervention.

  4. RecG Directs DNA Synthesis during Double-Strand Break Repair.

    PubMed

    Azeroglu, Benura; Mawer, Julia S P; Cockram, Charlotte A; White, Martin A; Hasan, A M Mahedi; Filatenkova, Milana; Leach, David R F

    2016-02-01

    Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability.

  5. Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks.

    PubMed

    Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia; Evangelou, Konstantinos; Da-Ré, Caterina; Huber, Florian; Padayachy, Laura; Tardy, Sebastien; Nicati, Noemie L; Barriot, Samia; Ochs, Fena; Lukas, Claudia; Lukas, Jiri; Gorgoulis, Vassilis G; Scapozza, Leonardo; Halazonetis, Thanos D

    2016-12-15

    Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells.

  6. Re-establishment of nucleosome occupancy during double-strand break repair in budding yeast.

    PubMed

    Tsabar, Michael; Hicks, Wade M; Tsaponina, Olga; Haber, James E

    2016-11-01

    Homologous recombination (HR) is an evolutionarily conserved pathway in eukaryotes that repairs a double-strand break (DSB) by copying homologous sequences from a sister chromatid, a homologous chromosome or an ectopic location. Recombination is challenged by the packaging of DNA into nucleosomes, which may impair the process at many steps, from resection of the DSB ends to the re-establishement of nucleosomes after repair. However, nucleosome dynamics during DSB repair have not been well described, primarily because of a lack of well-ordered nucleosomes around a DSB. We designed a system in budding yeast Saccharomyces cerevisiae to monitor nucleosome dynamics during repair of an HO endonuclease-induced DSB. Nucleosome occupancy around the break is lost following DSB formation, by 5'-3' resection of the DSB end. Soon after repair is complete, nucleosome occupancy is partially restored in a repair-dependent but cell cycle-independent manner. Full re-establishment of nucleosome protection back to the level prior to DSB induction is achieved when the cell cycle resumes following repair. These findings may have implications to the mechanisms by which cells sense the completion of repair.

  7. Modeling the repair of DNA strand breaks caused by γ-radiation in a minichromosome

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

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

  8. Breaking bad: The mutagenic effect of DNA repair

    PubMed Central

    2015-01-01

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

  9. Targeting abnormal DNA double strand break repair in tyrosine kinase inhibitor-resistant chronic myeloid leukemias

    PubMed Central

    Tobin, Lisa A.; Robert, Carine; Rapoport, Aaron P.; Gojo, Ivana; Baer, Maria R.; Tomkinson, Alan E.; Rassool, Feyruz V.

    2013-01-01

    Resistance to imatinib (IM) and other BCR-ABL1 tyrosine kinase inhibitors (TKI)s is an increasing problem in leukemias caused by expression of BCR-ABL1. Since chronic myeloid leukemia (CML) cell lines expressing BCR-ABL1 utilize an alternative non-homologous end-joining pathway (ALT NHEJ) to repair DNA double strand breaks (DSB)s, we asked whether this repair pathway is a novel therapeutic target in TKI-resistant disease. Notably, the steady state levels of two ALT NHEJ proteins, poly-(ADP-ribose) polymerase 1 (PARP1) and DNA ligase IIIα were increased in the BCR-ABL1-positive CML cell line K562 and, to a greater extent, in its imatinib resistant (IMR) derivative. Incubation of these cell lines with a combination of DNA ligase and PARP inhibitors inhibited ALT NHEJ and selectively decreased survival with the effect being greater in the IMR derivative. Similar results were obtained with TKI-resistant derivatives of two hematopoietic cell lines that had been engineered to stably express BCR-ABL1. Together our results show that the sensitivity of cell lines expressing BCR-ABL1 to the combination of DNA ligase and PARP inhibitors correlates with the steady state levels of PARP1 and DNA ligase IIIα, and ALT NHEJ activity. Importantly, analysis of clinical samples from CML patients confirmed that the expression levels of PARP1 and DNA ligase IIIα correlated with sensitivity to the DNA repair inhibitor combination. Thus, the expression levels of PARP1 and DNA ligase IIIα serve as biomarkers to identify a subgroup of CML patients who may be candidates for therapies that target the ALT NHEJ pathway when treatment with TKIs has failed. PMID:22641215

  10. Deficiency of XLF and PAXX prevents DNA double-strand break repair by non-homologous end joining in lymphocytes.

    PubMed

    Hung, Putzer J; Chen, Bo-Ruei; George, Rosmy; Liberman, Caleb; Morales, Abigail J; Colon-Ortiz, Pedro; Tyler, Jessica K; Sleckman, Barry P; Bredemeyer, Andrea L

    2017-02-01

    Non-homologous end joining (NHEJ) is a major DNA double-strand break (DSB) repair pathway that functions in all phases of the cell cycle. NHEJ repairs genotoxic and physiological DSBs, such as those generated by ionizing radiation and during V(D)J recombination at antigen receptor loci, respectively. DNA end joining by NHEJ relies on the core factors Ku70, Ku80, XRCC4, and DNA Ligase IV. Additional proteins also play important roles in NHEJ. The XRCC4-like factor (XLF) participates in NHEJ through its interaction with XRCC4, and XLF deficiency in humans leads to immunodeficiency and increased sensitivity to ionizing radiation. However, XLF is dispensable for NHEJ-mediated DSB repair during V(D)J recombination in murine lymphocytes, where it may have redundant functions with other DSB repair factors. Paralog of XRCC4 and XLF (PAXX) is a recently identified NHEJ factor that has structural similarity to XRCC4 and XLF. Here we show that PAXX is also dispensable for NHEJ during V(D)J recombination and during the repair of genotoxic DSBs in lymphocytes. However, a combined deficiency of PAXX and XLF blocks NHEJ with a severity comparable to that observed in DNA Ligase IV-deficient cells. Similar to XLF, PAXX interacts with Ku through its C-terminal region, and mutations that disrupt Ku binding prevent PAXX from promoting NHEJ in XLF-deficient lymphocytes. Our findings suggest that the PAXX and XLF proteins may have redundant functions during NHEJ.

  11. Contribution of DNA double-strand break repair gene XRCC3 genotypes to oral cancer susceptibility in Taiwan.

    PubMed

    Tsai, Chia-Wen; Chang, Wen-Shin; Liu, Juhn-Cherng; Tsai, Ming-Hsui; Lin, Cheng-Chieh; Bau, Da-Tian

    2014-06-01

    The DNA repair gene X-ray repair cross complementing protein 3 (XRCC3) is thought to play a major role in double-strand break repair and in maintaining genomic stability. Very possibly, defective double-strand break repair of cells can lead to carcinogenesis. Therefore, a case-control study was performed to reveal the contribution of XRCC3 genotypes to individual oral cancer susceptibility. In this hospital-based research, the association of XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539, rs28903081 genotypes with oral cancer risk in a Taiwanese population was investigated. In total, 788 patients with oral cancer and 956 age- and gender-matched healthy controls were genotyped. The results showed that there was significant differential distribution among oral cancer and controls in the genotypic (p=0.001428) and allelic (p=0.0013) frequencies of XRCC3 rs861539. As for the other polymorphisms, there was no difference between case and control groups. In gene-lifestyle interaction analysis, we have provided the first evidence showing that there is an obvious joint effect of XRCC3 rs861539 genotype with individual areca chewing habits on oral cancer risk. In conclusion, the T allele of XRCC3 rs861539, which has an interaction with areca chewing habit in oral carcinogenesis, may be an early marker for oral cancer in Taiwanese.

  12. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

    PubMed Central

    Janssen, Aniek; Breuer, Gregory A.; Brinkman, Eva K.; van der Meulen, Annelot I.; Borden, Sean V.; van Steensel, Bas; Bindra, Ranjit S.; LaRocque, Jeannine R.; Karpen, Gary H.

    2016-01-01

    Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio–temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains. PMID:27474442

  13. Cdc14A and Cdc14B Redundantly Regulate DNA Double-Strand Break Repair

    PubMed Central

    Lin, Han; Ha, Kyungsoo; Lu, Guojun; Fang, Xiao; Cheng, Ranran; Zuo, Qiuhong

    2015-01-01

    Cdc14 is a phosphatase that controls mitotic exit and cytokinesis in budding yeast. In mammals, the two Cdc14 homologues, Cdc14A and Cdc14B, have been proposed to regulate DNA damage repair, whereas the mitotic exit and cytokinesis rely on another phosphatase, PP2A-B55α. It is unclear if the two Cdc14s work redundantly in DNA repair and which repair pathways they participate in. More importantly, their target(s) in DNA repair remains elusive. Here we report that Cdc14B knockout (Cdc14B−/−) mouse embryonic fibroblasts (MEFs) showed defects in repairing ionizing radiation (IR)-induced DNA double-strand breaks (DSBs), which occurred only at late passages when Cdc14A levels were low. This repair defect could occur at early passages if Cdc14A levels were also compromised. These results indicate redundancy between Cdc14B and Cdc14A in DSB repair. Further, we found that Cdc14B deficiency impaired both homologous recombination (HR) and nonhomologous end joining (NHEJ), the two major DSB repair pathways. We also provide evidence that Cdh1 is a downstream target of Cdc14B in DSB repair. PMID:26283732

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

    PubMed

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

    2016-08-18

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-01-01

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

  18. 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

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

    PubMed

    Davis, Luther; Maizels, Nancy

    2014-03-11

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

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

    PubMed

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

    2011-01-02

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

  1. Histone H3K56 Acetylation, Rad52, and Non-DNA Repair Factors Control Double-Strand Break Repair Choice with the Sister Chromatid

    PubMed Central

    Rothstein, Rodney; Aguilera, Andrés

    2013-01-01

    DNA double-strand breaks (DSBs) are harmful lesions that arise mainly during replication. The choice of the sister chromatid as the preferential repair template is critical for genome integrity, but the mechanisms that guarantee this choice are unknown. Here we identify new genes with a specific role in assuring the sister chromatid as the preferred repair template. Physical analyses of sister chromatid recombination (SCR) in 28 selected mutants that increase Rad52 foci and inter-homolog recombination uncovered 8 new genes required for SCR. These include the SUMO/Ub-SUMO protease Wss1, the stress-response proteins Bud27 and Pdr10, the ADA histone acetyl-transferase complex proteins Ahc1 and Ada2, as well as the Hst3 and Hst4 histone deacetylase and the Rtt109 histone acetyl-transferase genes, whose target is histone H3 Lysine 56 (H3K56). Importantly, we use mutations in H3K56 residue to A, R, and Q to reveal that H3K56 acetylation/deacetylation is critical to promote SCR as the major repair mechanism for replication-born DSBs. The same phenotype is observed for a particular class of rad52 alleles, represented by rad52-C180A, with a DSB repair defect but a spontaneous hyper-recombination phenotype. We propose that specific Rad52 residues, as well as the histone H3 acetylation/deacetylation state of chromatin and other specific factors, play an important role in identifying the sister as the choice template for the repair of replication-born DSBs. Our work demonstrates the existence of specific functions to guarantee SCR as the main repair event for replication-born DSBs that can occur by two pathways, one Rad51-dependent and the other Pol32-dependent. A dysfunction can lead to genome instability as manifested by high levels of homolog recombination and DSB accumulation. PMID:23357952

  2. Epidermal growth factor receptor and DNA double strand break repair: the cell's self-defence.

    PubMed

    Szumiel, Irena

    2006-10-01

    The purpose of this review is to discuss the relation between the repair of DNA double strand breaks (DSB)--the main lethal lesion inflicted by ionising radiation-and the function of receptors of epidermal growth factor (EGFR) and similar ligands (other members of the ERBB family). The reviewed experimental data support the assumption that in mammalian cells, one consequence of EGFR/ERBB activation by X-rays is its internalisation and nuclear translocation together with DNA-dependent protein kinase (DNA-PK) subunits present in lipid rafts or cytoplasm. The effect of EGFR/ERBB stimulation on DSB rejoining would be due to an increase in the nuclear content of DNA-PK subunits and hence, in activity increase of the DNA-PK dependent non-homologous end-joining (D-NHEJ) system. Such mechanism explains the radiosensitising action of "membrane-active drugs", hypertonic media, and other agents that affect nuclear translocation of proteins. Also, one radiosensitising effect of the recently introduced into clinical practice EGFR/ERBB inhibitors would consist on counteracting the nuclear translocation of DNA-PK subunits. In result, D-NHEJ may be less active in inhibitor-treated cells and this will contribute to an enhanced lethal effect of irradiation. The reviewed observations point to a heretofore not understood mechanism of the cell's self-defence against X-rays which can be exploited in combined radio- and chemotherapy.

  3. Coordination of Double Strand Break Repair and Meiotic Progression in Yeast by a Mek1- Ndt80 Negative Feedback Loop.

    PubMed

    Prugar, Evelyn; Burnett, Cameron; Chen, Xiangyu; Hollingsworth, Nancy M

    2017-03-01

    During meiosis, homologous chromosomes are physically connected by crossovers and sister chromatid cohesion. Interhomolog crossovers are generated by the highly regulated repair of programmed double strand breaks (DSBs). The meiosis-specific kinase, Mek1, is critical for this regulation. Mek1 down-regulates the mitotic recombinase Rad51, indirectly promoting interhomolog strand invasion by the meiosis-specific recombinase, Dmc1. Mek1 also promotes the formation of crossovers that are distributed throughout the genome by interference and is the effector kinase for a meiosis-specific checkpoint that delays entry into Meiosis I until DSBs have been repaired. The target of this checkpoint is a meiosis-specific transcription factor, Ndt80, which is necessary to express the polo-like kinase, CDC5, and the cyclin, CLB1, thereby allowing completion of recombination and meiotic progression. This work shows that Mek1 and Ndt80 negatively feedback on each other such that when DSB levels are high, Ndt80 is inactive due to high levels of Mek1 activity. As DSBs are repaired, chromosomes synapse and Mek1 activity is reduced below a threshold that allows activation of Ndt80. Ndt80 transcription of CDC5 results in degradation of Red1, a meiosis-specific protein required for Mek1 activation, thereby abolishing Mek1 activity completely. Elimination of Mek1 kinase activity allows Rad51-mediated repair of any remaining DSBs. In this way, cells do not enter Meiosis I until recombination is complete and all DSBs are repaired.

  4. DNA double-strand break repair: Genetic determinants of flanking crossing-over

    SciTech Connect

    Kusano, Kohji; Sunohara, Yukari; Kobayashi, Ichizo; Takahashi, Noriko; Yoshikura, Hiroshi )

    1994-02-01

    Whether or not homologous interaction of two DNA molecules results in crossing-over of the flanking sequences is an important decision in view of genome organization. Several homologous recombination models, including the double-strand break repair models, explain this decision as choice between two alternative modes of resolution of Holliday-type intermediates. The authors have demonstrated that a double-strand gap can be repaired through gene conversion copying a homologous duplex, as predicted by the double-strand break repair models, in the RecE pathway of Escherichia coli. This gap repair is often accompanied by crossing-over of the flanking sequences. Mutations in ruvC and recG, whose products interact with Holliday structures in vitro, do not block double-strand gap repair or its association with flanking crossing-over. However, two mutations in the recJ gene, which encodes a single-strand 5[prime][yields]3[prime] exonuclease, severely decrease association of flanking crossing-over. Two mutations in the recQ gene, which encodes a helicase, moderately decrease association of flanking crossing-over by themselves and suppress the severe effect of a recJ mutation. Similar relationships of recJ and recQ mutations are observed in cell survival after ultraviolet light irradiation, [gamma]-ray irradiation, and H[sub 2]O[sub 2] treatment. The authors discuss how cooperation of the recQ gene product and the recJ gene product brings about double-strand break repair accompanied by flanking crossing-over. They also discuss how this reaction is related to repair of chromosome damages.

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

    SciTech Connect

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

    2006-09-25

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

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

    PubMed

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

    2015-01-01

    Ionizing radiation generates DNA double-strand breaks (DSB) which, unless faithfully repaired, can generate chromosomal rearrangements in hematopoietic stem and/or progenitor cells (HSPC), potentially priming the cells towards a leukemic phenotype. Using an enhanced green fluorescent protein (EGFP)-based reporter system, we recently identified differences in the removal of enzyme-mediated DSB in human HSPC versus mature peripheral blood lymphocytes (PBL), particularly regarding homologous DSB repair (HR). Assessment of chromosomal breaks via premature chromosome condensation or γH2AX foci indicated similar efficiency and kinetics of radiation-induced DSB formation and rejoining in PBL and HSPC. Prolonged persistence of chromosomal breaks was observed for higher LET charged particles which are known to induce more complex DNA damage compared to X-rays. Consistent with HR deficiency in HSPC observed in our previous study, we noticed here pronounced focal accumulation of 53BP1 after X-ray and carbon ion exposure (intermediate LET) in HSPC versus PBL. For higher LET, 53BP1 foci kinetics was similarly delayed in PBL and HSPC suggesting similar failure to repair complex DNA damage. Data obtained with plasmid reporter systems revealed a dose- and LET-dependent HR increase after X-ray, carbon ion and higher LET exposure, particularly in HR-proficient immortalized and primary lymphocytes, confirming preferential use of conservative HR in PBL for intermediate LET damage repair. HR measured adjacent to the leukemia-associated MLL breakpoint cluster sequence in reporter lines revealed dose dependency of potentially leukemogenic rearrangements underscoring the risk of leukemia-induction by radiation treatment.

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

    PubMed Central

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

    2015-01-01

    Ionizing radiation generates DNA double-strand breaks (DSB) which, unless faithfully repaired, can generate chromosomal rearrangements in hematopoietic stem and/or progenitor cells (HSPC), potentially priming the cells towards a leukemic phenotype. Using an enhanced green fluorescent protein (EGFP)-based reporter system, we recently identified differences in the removal of enzyme-mediated DSB in human HSPC versus mature peripheral blood lymphocytes (PBL), particularly regarding homologous DSB repair (HR). Assessment of chromosomal breaks via premature chromosome condensation or γH2AX foci indicated similar efficiency and kinetics of radiation-induced DSB formation and rejoining in PBL and HSPC. Prolonged persistence of chromosomal breaks was observed for higher LET charged particles which are known to induce more complex DNA damage compared to X-rays. Consistent with HR deficiency in HSPC observed in our previous study, we noticed here pronounced focal accumulation of 53BP1 after X-ray and carbon ion exposure (intermediate LET) in HSPC versus PBL. For higher LET, 53BP1 foci kinetics was similarly delayed in PBL and HSPC suggesting similar failure to repair complex DNA damage. Data obtained with plasmid reporter systems revealed a dose- and LET-dependent HR increase after X-ray, carbon ion and higher LET exposure, particularly in HR-proficient immortalized and primary lymphocytes, confirming preferential use of conservative HR in PBL for intermediate LET damage repair. HR measured adjacent to the leukemia-associated MLL breakpoint cluster sequence in reporter lines revealed dose dependency of potentially leukemogenic rearrangements underscoring the risk of leukemia-induction by radiation treatment. PMID:26618143

  8. Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae

    PubMed Central

    Stein, Alexis; Kalifa, Lidza; Sia, Elaine A.

    2015-01-01

    Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs. PMID:26540255

  9. RhoB Promotes γH2AX Dephosphorylation and DNA Double-Strand Break Repair

    PubMed Central

    Mamouni, Kenza; Cristini, Agnese; Guirouilh-Barbat, Josée; Monferran, Sylvie; Lemarié, Anthony; Faye, Jean-Charles; Lopez, Bernard S.

    2014-01-01

    Unlike other Rho GTPases, RhoB is rapidly induced by DNA damage, and its expression level decreases during cancer progression. Because inefficient repair of DNA double-strand breaks (DSBs) can lead to cancer, we investigated whether camptothecin, an anticancer drug that produces DSBs, induces RhoB expression and examined its role in the camptothecin-induced DNA damage response. We show that in camptothecin-treated cells, DSBs induce RhoB expression by a mechanism that depends notably on Chk2 and its substrate HuR, which binds to RhoB mRNA and protects it against degradation. RhoB-deficient cells fail to dephosphorylate γH2AX following camptothecin removal and show reduced efficiency of DSB repair by homologous recombination. These cells also show decreased activity of protein phosphatase 2A (PP2A), a phosphatase for γH2AX and other DNA damage and repair proteins. Thus, we propose that DSBs activate a Chk2-HuR-RhoB pathway that promotes PP2A-mediated dephosphorylation of γH2AX and DSB repair. Finally, we show that RhoB-deficient cells accumulate endogenous γH2AX and chromosomal abnormalities, suggesting that RhoB loss increases DSB-mediated genomic instability and tumor progression. PMID:24912678

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

    PubMed

    Brissett, Nigel C; Doherty, Aidan J

    2009-06-01

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

  11. ATP-dependent chromatin remodeling and DNA double-strand break repair.

    PubMed

    van Attikum, Haico; Gasser, Susan M

    2005-08-01

    The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic stability. Two pathways for the repair of DBSs, nonhomologous end-joining (NHEJ) and homologous recombination (HR), have evolved in eukaryotes. These pathways, like processes such as transcription and replication, act on DNA that is embedded in nucleosomes. Recent studies have shown that DNA repair, like transcription, is facilitated both by histone tail modification and by ATP-dependent chromatin remodeling. This review emphasizes recent reports that demonstrate a function for the ATP-dependent chromatin remodeling complexes INO80 and RSC in NHEJ and HR. We also discuss the possible role of SWR1- and TIP60-mediated nucleosomal histone exchange in DNA repair.

  12. Pulsed-field gel electrophoresis analysis of multicellular DNA double-strand break damage and repair.

    PubMed

    Joshi, Nina; Grant, Stephen G

    2014-01-01

    This assay quantifies the extent of double-strand break (DSB) DNA damage in cell populations embedded in agarose and analyzed for migratory DNA using pulsed-field gel electrophoresis with ethidium bromide staining. The assay can measure preexisting damage as well as induction of DSB by chemical (e.g., bleomycin), physical (e.g., X-irradiation), or biological (e.g., restriction enzymes) agents. By incubating the cells under physiological conditions prior to processing, the cells can be allowed to repair DSB, primarily via the process of nonhomologous end joining. The amount of repair, corresponding to the repair capacity of the treated cells, is then quantified by determining the ratio of the fractions of activity released in the lanes in comparison to the total amount of DNA fragmentation following determination of an optimal exposure for maximum initial fragmentation. Repair kinetics can also be analyzed through a time-course regimen.

  13. Making Ends Meet: Repairing Breaks in Bacterial DNA by Non-Homologous End-Joining

    PubMed Central

    Bowater, Richard; Doherty, Aidan J

    2006-01-01

    DNA double-strand breaks (DSBs) are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ). Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHEJ apparatus has now been identified and characterized in the prokarya. Here we review the recent discoveries concerning bacterial NHEJ and discuss the possible origins of this repair system. We also examine the insights gained from the recent cellular and biochemical studies of this DSB-repair process and discuss the possible cellular roles of an NHEJ pathway in the life-cycle of prokaryotes and phages. PMID:16518468

  14. XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair

    PubMed Central

    Craxton, A; Somers, J; Munnur, D; Jukes-Jones, R; Cain, K; Malewicz, M

    2015-01-01

    Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protein associated with DNA-PKcs—c9orf142. Computer-based modelling of c9orf142 predicted a structure very similar to XRCC4, hence we have named c9orf142—XLS (XRCC4-like small protein). Depletion of c9orf142/XLS in cells impaired DSB repair consistent with a defect in NHEJ. Furthermore, c9orf142/XLS interacted with other core NHEJ factors. These results demonstrate the existence of a new component of the NHEJ DNA repair pathway in mammalian cells. PMID:25941166

  15. Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: the molecular choreography.

    PubMed

    Thompson, Larry H

    2012-01-01

    The faithful maintenance of chromosome continuity in human cells during DNA replication and repair is critical for preventing the conversion of normal diploid cells to an oncogenic state. The evolution of higher eukaryotic cells endowed them with a large genetic investment in the molecular machinery that ensures chromosome stability. In mammalian and other vertebrate cells, the elimination of double-strand breaks with minimal nucleotide sequence change involves the spatiotemporal orchestration of a seemingly endless number of proteins ranging in their action from the nucleotide level to nucleosome organization and chromosome architecture. DNA DSBs trigger a myriad of post-translational modifications that alter catalytic activities and the specificity of protein interactions: phosphorylation, acetylation, methylation, ubiquitylation, and SUMOylation, followed by the reversal of these changes as repair is completed. "Superfluous" protein recruitment to damage sites, functional redundancy, and alternative pathways ensure that DSB repair is extremely efficient, both quantitatively and qualitatively. This review strives to integrate the information about the molecular mechanisms of DSB repair that has emerged over the last two decades with a focus on DSBs produced by the prototype agent ionizing radiation (IR). The exponential growth of molecular studies, heavily driven by RNA knockdown technology, now reveals an outline of how many key protein players in genome stability and cancer biology perform their interwoven tasks, e.g. ATM, ATR, DNA-PK, Chk1, Chk2, PARP1/2/3, 53BP1, BRCA1, BRCA2, BLM, RAD51, and the MRE11-RAD50-NBS1 complex. Thus, the nature of the intricate coordination of repair processes with cell cycle progression is becoming apparent. This review also links molecular abnormalities to cellular pathology as much a possible and provides a framework of temporal relationships.

  16. The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells.

    PubMed

    Elsen, Sylvie; Collin-Faure, Véronique; Gidrol, Xavier; Lemercier, Claudie

    2013-11-01

    Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.

  17. Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair

    PubMed Central

    Tsabar, Michael; Waterman, David P.; Aguilar, Fiona; Katsnelson, Lizabeth; Eapen, Vinay V.; Memisoglu, Gonen; Haber, James E.

    2016-01-01

    To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair. PMID:27222517

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

    PubMed

    Jeggo, Penny A; Löbrich, Markus

    2015-10-01

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

  19. Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair.

    PubMed

    Tsabar, Michael; Waterman, David P; Aguilar, Fiona; Katsnelson, Lizabeth; Eapen, Vinay V; Memisoglu, Gonen; Haber, James E

    2016-05-15

    To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair.

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

    PubMed

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

    2008-12-12

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

  1. 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.

  2. PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL.

    PubMed

    di Masi, A; Cilli, D; Berardinelli, F; Talarico, A; Pallavicini, I; Pennisi, R; Leone, S; Antoccia, A; Noguera, N I; Lo-Coco, F; Ascenzi, P; Minucci, S; Nervi, C

    2016-07-28

    Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis.

  3. PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL

    PubMed Central

    di Masi, A; Cilli, D; Berardinelli, F; Talarico, A; Pallavicini, I; Pennisi, R; Leone, S; Antoccia, A; Noguera, N I; Lo-Coco, F; Ascenzi, P; Minucci, S; Nervi, C

    2016-01-01

    Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis. PMID:27468685

  4. Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

    PubMed Central

    Nakada, Shinichiro

    2016-01-01

    The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway. PMID:26983989

  5. Mechanisms of double-strand break repair in somatic mammalian cells

    PubMed Central

    Hartlerode, Andrea J.; Scully, Ralph

    2010-01-01

    DNA chromosomal DSBs (double-strand breaks) are potentially hazardous DNA lesions, and their accurate repair is essential for the successful maintenance and propagation of genetic information. Two major pathways have evolved to repair DSBs: HR (homologous recombination) and NHEJ (non-homologous end-joining). Depending on the context in which the break is encountered, HR and NHEJ may either compete or co-operate to fix DSBs in eukaryotic cells. Defects in either pathway are strongly associated with human disease, including immunodeficiency and cancer predisposition. Here we review the current knowledge of how NHEJ and HR are controlled in somatic mammalian cells, and discuss the role of the chromatin context in regulating each pathway. We also review evidence for both co-operation and competition between the two pathways. PMID:19772495

  6. Antibody specific for a DNA repair protein

    DOEpatents

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

    2006-07-11

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

  7. DNA encoding a DNA repair protein

    DOEpatents

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

    2006-08-15

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

  8. VCP/p97 Extracts Sterically Trapped Ku70/80 Rings from DNA in Double-Strand Break Repair.

    PubMed

    van den Boom, Johannes; Wolf, Markus; Weimann, Lena; Schulze, Nina; Li, Fanghua; Kaschani, Farnusch; Riemer, Anne; Zierhut, Christian; Kaiser, Markus; Iliakis, George; Funabiki, Hironori; Meyer, Hemmo

    2016-10-06

    During DNA double-strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin-adaptor complex, and specifically targets Ku80 that is modified by K48-linked ubiquitin chains. In U2OS cells, chemical inhibition of p97 or siRNA-mediated depletion of p97 or its adapters impairs Ku80 removal after non-homologous end joining of DSBs. Moreover, this inhibition attenuates early steps in homologous recombination, consistent with p97-driven Ku release also affecting repair pathway choice. Thus, our data answer a central question regarding regulation of Ku in DSB repair and illustrate the ability of p97 to segregate even tightly bound protein complexes for release from DNA.

  9. Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions.

    PubMed

    Tadi, Satish Kumar; Sebastian, Robin; Dahal, Sumedha; Babu, Ravi K; Choudhary, Bibha; Raghavan, Sathees C

    2016-01-15

    Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.

  10. Suberoylanilide Hydroxyamic Acid Modification of Chromatin Architecture Affects DNA Break Formation and Repair

    SciTech Connect

    Singh, Sheetal; Le Hongan; Shih, S.-J.; Ho, Bay; Vaughan, Andrew T.

    2010-02-01

    Purpose: Chromatin-modifying compounds that inhibit the activity of histone deacetylases have shown potency as radiosensitizers, but the action of these drugs at a molecular level is not clear. Here we investigated the effect of suberoylanilide hydroxyamic acid (SAHA) on DNA breaks and their repair and induction of rearrangements. Methods and Materials: The effect of SAHA on both clonogenic survival and repair was assessed using cell lines SCC-25, MCF7, and TK6. In order to study unique DNA double-strand breaks, anti-CD95 antibody was employed to introduce a DNA double-strand break at a known location within the 11q23 region. The effects of SAHA on DNA cleavage and rearrangements were analyzed by ligation-mediated PCR and inverse PCR, respectively. Results: SAHA acts as radiosensitizer at 1 {mu}M, with dose enhancement factors (DEFs) at 10% survival of: SCC-25 - 1.24 +- 0.05; MCF7 - 1.16 +- 0.09 and TK6 - 1.17 +- 0.05, and it reduced the capacity of SCC-25 cells to repair radiation induced lesions. Additionally, SAHA treatment diffused site-specific fragmentation over at least 1 kbp in TK6 cells. Chromosomal rearrangements produced in TK6 cells exposed to SAHA showed a reduction in microhomology at the breakpoint between 11q23 and partner chromosomes. Conclusions: SAHA shows efficacy as a radiosensitizer at clinically obtainable levels. In its presence, targeted DNA strand breaks occur over an expanded region, indicating increased chromatin access. The rejoining of such breaks is degraded by SAHA when measured as rearrangements at the molecular level and rejoining that contributes to cell survival.

  11. Sp1 facilitates DNA double-strand break repair through a nontranscriptional mechanism.

    PubMed

    Beishline, Kate; Kelly, Crystal M; Olofsson, Beatrix A; Koduri, Sravanthi; Emrich, Jacqueline; Greenberg, Roger A; Azizkhan-Clifford, Jane

    2012-09-01

    Sp1 is a ubiquitously expressed transcription factor that is phosphorylated by ataxia telangiectasia mutated kinase (ATM) in response to ionizing radiation and H(2)O(2). Here, we show by indirect immunofluorescence that Sp1 phosphorylated on serine 101 (pSp1) localizes to ionizing radiation-induced foci with phosphorylated histone variant γH2Ax and members of the MRN (Mre11, Rad50, and Nbs1) complex. More precise analysis of occupancy of DNA double-strand breaks (DSBs) by chromatin immunoprecipitation (ChIP) shows that Sp1, like Nbs1, resides within 200 bp of DSBs. Using laser microirradiation of cells, we demonstrate that pSp1 is present at DNA DSBs by 7.5 min after induction of damage and remains at the break site for at least 8 h. Depletion of Sp1 inhibits repair of site-specific DNA breaks, and the N-terminal 182-amino-acid peptide, which contains targets of ATM kinase but lacks the zinc finger DNA binding domain, is phosphorylated, localizes to DSBs, and rescues the repair defect resulting from Sp1 depletion. Together, these data demonstrate that Sp1 is rapidly recruited to the region immediately adjacent to sites of DNA DSBs and is required for DSB repair, through a mechanism independent of its sequence-directed transcriptional effects.

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

    PubMed

    Kato, Akihiro; Komatsu, Kenshi

    2015-07-14

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

  13. ATM specifically mediates repair of double-strand breaks with blocked DNA ends.

    PubMed

    Álvarez-Quilón, Alejandro; Serrano-Benítez, Almudena; Lieberman, Jenna Ariel; Quintero, Cristina; Sánchez-Gutiérrez, Daniel; Escudero, Luis M; Cortés-Ledesma, Felipe

    2014-02-27

    Ataxia telangiectasia is caused by mutations in ATM and represents a paradigm for cancer predisposition and neurodegenerative syndromes linked to deficiencies in the DNA-damage response. The role of ATM as a key regulator of signalling following DNA double-strand breaks (DSBs) has been dissected in extraordinary detail, but the impact of this process on DSB repair still remains controversial. Here we develop novel genetic and molecular tools to modify the structure of DSB ends and demonstrate that ATM is indeed required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are irreversibly blocked. We therefore identify the nature of ATM involvement in DSB repair, presenting blocked DNA ends as a possible pathogenic trigger of ataxia telangiectasia and related disorders.

  14. ATM specifically mediates repair of double-strand breaks with blocked DNA ends

    PubMed Central

    Álvarez-Quilón, Alejandro; Serrano-Benítez, Almudena; Ariel Lieberman, Jenna; Quintero, Cristina; Sánchez-Gutiérrez, Daniel; Escudero, Luis M.; Cortés-Ledesma, Felipe

    2014-01-01

    Ataxia telangiectasia is caused by mutations in ATM and represents a paradigm for cancer predisposition and neurodegenerative syndromes linked to deficiencies in the DNA-damage response. The role of ATM as a key regulator of signalling following DNA double-strand breaks (DSBs) has been dissected in extraordinary detail, but the impact of this process on DSB repair still remains controversial. Here we develop novel genetic and molecular tools to modify the structure of DSB ends and demonstrate that ATM is indeed required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are irreversibly blocked. We therefore identify the nature of ATM involvement in DSB repair, presenting blocked DNA ends as a possible pathogenic trigger of ataxia telangiectasia and related disorders. PMID:24572510

  15. Preferential repair of DNA double-strand break at the active gene in vivo.

    PubMed

    Chaurasia, Priyasri; Sen, Rwik; Pandita, Tej K; Bhaumik, Sukesh R

    2012-10-19

    Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3' end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells.

  16. The transcriptional histone acetyltransferase cofactor TRRAP associates with the MRN repair complex and plays a role in DNA double-strand break repair.

    PubMed

    Robert, Flavie; Hardy, Sara; Nagy, Zita; Baldeyron, Céline; Murr, Rabih; Déry, Ugo; Masson, Jean-Yves; Papadopoulo, Dora; Herceg, Zdenko; Tora, Làszlò

    2006-01-01

    Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

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

    PubMed

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

    2012-05-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. Modulation of Saccharomyces Cerevisiae DNA Double-Strand Break Repair by Srs2 and Rad51

    PubMed Central

    Milne, G. T.; Ho, T.; Weaver, D. T.

    1995-01-01

    RAD52 function is required for virtually all DNA double-strand break repair and recombination events in Saccharomyces cerevisiae. To gain greater insight into the mechanism of RAD52-mediated repair, we screened for genes that suppress partially active alleles of RAD52 when mutant or overexpressed. Described here is the isolation of a phenotypic null allele of SRS2 that suppressed multiple alleles of RAD52 (rad52B, rad52D, rad52-1 and KlRAD52) and RAD51 (KlRAD51) but failed to suppress either a rad52δ or a rad51δ. These results indicate that SRS2 antagonizes RAD51 and RAD52 function in recombinational repair. The mechanism of suppression of RAD52 alleles by srs2 is distinct from that which has been previously described for RAD51 overexpression, as both conditions were shown to act additively with respect to the rad52B allele. Furthermore, overexpression of either RAD52 or RAD51 enhanced the recombination-dependent sensitivity of an srs2δ RAD52 strain, suggesting that RAD52 and RAD51 positively influence recombinational repair mechanisms. Thus, RAD52-dependent recombinational repair is controlled both negatively and positively. PMID:7768432

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed

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

    2004-01-29

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

  2. TDP1 facilitates chromosomal single-strand break repair in neurons and is neuroprotective in vivo.

    PubMed

    Katyal, Sachin; el-Khamisy, Sherif F; Russell, Helen R; Li, Yang; Ju, Limei; Caldecott, Keith W; McKinnon, Peter J

    2007-11-14

    Defective Tyrosyl-DNA phosphodiesterase 1 (TDP1) can cause spinocerebellar ataxia with axonal neuropathy (SCAN1), a neurodegenerative syndrome associated with marked cerebellar atrophy and peripheral neuropathy. Although SCAN1 lymphoblastoid cells show pronounced defects in the repair of chromosomal single-strand breaks (SSBs), it is unknown if this DNA repair activity is important for neurons or for preventing neurodegeneration. Therefore, we generated Tdp1-/- mice to assess the role of Tdp1 in the nervous system. Using both in vitro and in vivo assays, we found that cerebellar neurons or primary astrocytes derived from Tdp1-/- mice display an inability to rapidly repair DNA SSBs associated with Top1-DNA complexes or oxidative damage. Moreover, loss of Tdp1 resulted in age-dependent and progressive cerebellar atrophy. Tdp1-/- mice treated with topotecan, a drug that increases levels of Top1-DNA complexes, also demonstrated significant loss of intestinal and hematopoietic progenitor cells. These data indicate that TDP1 is required for neural homeostasis, and reveal a widespread requisite for TDP1 function in response to acutely elevated levels of Top1-associated DNA strand breaks.

  3. TDP1 facilitates chromosomal single-strand break repair in neurons and is neuroprotective in vivo

    PubMed Central

    Katyal, Sachin; El-Khamisy, Sherif F; Russell, Helen R; Li, Yang; Ju, Limei; Caldecott, Keith W; McKinnon, Peter J

    2007-01-01

    Defective Tyrosyl-DNA phosphodiesterase 1 (TDP1) can cause spinocerebellar ataxia with axonal neuropathy (SCAN1), a neurodegenerative syndrome associated with marked cerebellar atrophy and peripheral neuropathy. Although SCAN1 lymphoblastoid cells show pronounced defects in the repair of chromosomal single-strand breaks (SSBs), it is unknown if this DNA repair activity is important for neurons or for preventing neurodegeneration. Therefore, we generated Tdp1−/− mice to assess the role of Tdp1 in the nervous system. Using both in vitro and in vivo assays, we found that cerebellar neurons or primary astrocytes derived from Tdp1−/− mice display an inability to rapidly repair DNA SSBs associated with Top1–DNA complexes or oxidative damage. Moreover, loss of Tdp1 resulted in age-dependent and progressive cerebellar atrophy. Tdp1−/− mice treated with topotecan, a drug that increases levels of Top1–DNA complexes, also demonstrated significant loss of intestinal and hematopoietic progenitor cells. These data indicate that TDP1 is required for neural homeostasis, and reveal a widespread requisite for TDP1 function in response to acutely elevated levels of Top1-associated DNA strand breaks. PMID:17914460

  4. Chemical repair of base lesions, AP-sites, and strand breaks on plasmid DNA in dilute aqueous solution by ascorbic acid

    SciTech Connect

    Hata, Kuniki; Urushibara, Ayumi; Yamashita, Shinichi; Shikazono, Naoya; Yokoya, Akinari; Katsumura, Yosuke

    2013-05-03

    Highlights: •We report a novel mechanism of radiation protection of DNA by chemical activity of ascorbic acid. •The “chemical repair” of DNA damage was revealed using biochemical assay and chemical kinetics analysis. •We found that ascorbic acid significantly repairs precursors of nucleobase lesions and abasic sites. •However, ascorbic acid seldom repairs precursors of DNA-strand breaks. -- Abstract: We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10–100 μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield of DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50–60% of base lesions and AP-sites were repaired by 10 μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.

  5. c-Myc directly regulates the transcription of the NBS1 gene involved in DNA double-strand break repair.

    PubMed

    Chiang, Yu-Chi; Teng, Shu-Chun; Su, Yi-Ning; Hsieh, Fon-Jou; Wu, Kou-Juey

    2003-05-23

    The c-myc proto-oncogene encodes a ubiquitous transcription factor involved in the control of cell growth and implicated in inducing tumorigenesis. Understanding the function of c-Myc and its role in cancer depends upon the identification of c-Myc target genes. Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, and chromosomal instability. The NBS gene product, NBS1 (p95 or nibrin), is a part of the hMre11 complex, a central player associated with double-strand break (DSB) repair. NBS1 contains domains characteristic for proteins involved in DNA repair, recombination, and replication. Here we show that c-Myc directly activates NBS1. c-Myc-mediated induction of NBS1 gene transcription occurs in different tissues, is independent of cell proliferation, and is mediated by a c-Myc binding site in the intron 1 region of NBS1 gene. Overexpression of NBS1 in Rat1a cells increased cell proliferation. These results indicate that NBS1 is a direct transcriptional target of c-Myc and links the function of c-Myc to the regulation of DNA DSB repair pathway operating during DNA replication.

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

    PubMed

    Akamatsu, Yufuko; Jasin, Maria

    2010-10-14

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

  7. Transient RNA-DNA Hybrids Are Required for Efficient Double-Strand Break Repair.

    PubMed

    Ohle, Corina; Tesorero, Rafael; Schermann, Géza; Dobrev, Nikolay; Sinning, Irmgard; Fischer, Tamás

    2016-11-03

    RNA-DNA hybrids are a major internal cause of DNA damage within cells, and their degradation by RNase H enzymes is important for maintaining genomic stability. Here, we identified an unexpected role for RNA-DNA hybrids and RNase H enzymes in DNA repair. Using a site-specific DNA double-strand break (DSB) system in Schizosaccharomyces pombe, we showed that RNA-DNA hybrids form as part of the homologous-recombination (HR)-mediated DSB repair process and that RNase H enzymes are essential for their degradation and efficient completion of DNA repair. Deleting RNase H stabilizes RNA-DNA hybrids around DSB sites and strongly impairs recruitment of the ssDNA-binding RPA complex. In contrast, overexpressing RNase H1 destabilizes these hybrids, leading to excessive strand resection and RPA recruitment and to severe loss of repeat regions around DSBs. Our study challenges the existing model of HR-mediated DSB repair and reveals a surprising role for RNA-DNA hybrids in maintaining genomic stability.

  8. Synergistic decrease of DNA single-strand break repair rates in mouse neural cells lacking both Tdp1 and aprataxin

    PubMed Central

    El-Khamisy, Sherif F.; Katyal, Sachin; Patel, Poorvi; Ju, Limei; McKinnon, Peter J.; Caldecott, Keith W.

    2009-01-01

    Ataxia oculomotor apraxia-1 (AOA1) is an autosomal recessive neurodegenerative disease that results from mutations of aprataxin (APTX). APTX associates with the DNA single- and double-strand break repair machinery and is able to remove AMP from 5′-termini at DNA strand breaks in vitro. However, attempts to establish a DNA strand break repair defect in APTX-defective cells have proved conflicting and unclear. We reasoned that this may reflect that DNA strand breaks with 5′-AMP represent only a minor subset of breaks induced in cells, and/or the availability of alternative mechanisms for removing AMP from 5′-termini. Here, we have attempted to increase the dependency of chromosomal single- and double-strand break repair on aprataxin activity by slowing the rate of repair of 3′-termini in aprataxin-defective neural cells, thereby increasing the likelihood that the 5′-termini at such breaks become adenylated and/or block alternative repair mechanisms. To do this, we generated a mouse model in which APTX is deleted together with tyrosyl DNA phosphodiesterase (TDP1), an enzyme that repairs 3′-termini at a subset of single-strand breaks (SSBs), including those with 3′-topoisomerase-1 (Top1) peptide. Notably, the global rate of repair of oxidative and alkylation-induced SSBs was significantly slower in Tdp1−/−/Aptx−/− double knockout quiescent mouse astrocytes compared with Tdp1−/− or Aptx−/− single knockouts. In contrast, camptothecin-induced Top1-SSBs accumulated to similar levels in Tdp1−/− and Tdp1−/−/Aptx−/− double knockout astrocytes. Finally, we failed to identify a measurable defect in double-strand break repair in Tdp1−/−, Aptx−/− or Tdp1−/−/Aptx−/− astrocytes. These data provide direct evidence for a requirement for aprataxin during chromosomal single-strand break repair in primary neural cells lacking Tdp1. PMID:19303373

  9. Breaking the barriers in membrane protein crystallography.

    PubMed

    Kang, Hae Joo; Lee, Chiara; Drew, David

    2013-03-01

    As we appreciate the importance of stabilising membrane proteins, the barriers towards their structure determination are being broken down. This change in mindset comes hand-in-hand with more effort placed on developing methods focused at screening for membrane proteins which are naturally stable in detergent solution or improving those that are not so. In practice, however, it is not easy to decide the best strategy to monitor and improve detergent stability, requiring a decision-making process that can be even more difficult for those new to the field. In this review we outline the importance of membrane protein stability with discussions of the stabilisation strategies applied in context with the use of crystallisation scaffolds and the different types of crystallisation methods themselves. Where possible we also highlight areas that we think could push this field forward with emerging technologies, such as X-ray free electron lasers (X-feL), which could have a big impact on the membrane protein structural biology community. We hope this review will serve as a useful guide for those striving to solve structures of both pro- and eukaryotic membrane proteins.

  10. Essential role for DNA-PK-mediated phosphorylation of NR4A nuclear orphan receptors in DNA double-strand break repair.

    PubMed

    Malewicz, Michal; Kadkhodaei, Banafsheh; Kee, Nigel; Volakakis, Nikolaos; Hellman, Ulf; Viktorsson, Kristina; Leung, Chuen Yan; Chen, Benjamin; Lewensohn, Rolf; van Gent, Dik C; Chen, David J; Perlmann, Thomas

    2011-10-01

    DNA-dependent protein kinase (DNA-PK) is a central regulator of DNA double-strand break (DSB) repair; however, the identity of relevant DNA-PK substrates has remained elusive. NR4A nuclear orphan receptors function as sequence-specific DNA-binding transcription factors that participate in adaptive and stress-related cell responses. We show here that NR4A proteins interact with the DNA-PK catalytic subunit and, upon exposure to DNA damage, translocate to DSB foci by a mechanism requiring the activity of poly(ADP-ribose) polymerase-1 (PARP-1). At DNA repair foci, NR4A is phosphorylated by DNA-PK and promotes DSB repair. Notably, NR4A transcriptional activity is entirely dispensable in this function, and core components of the DNA repair machinery are not transcriptionally regulated by NR4A. Instead, NR4A functions directly at DNA repair sites by a process that requires phosphorylation by DNA-PK. Furthermore, a severe combined immunodeficiency (SCID)-causing mutation in the human gene encoding the DNA-PK catalytic subunit impairs the interaction and phosphorylation of NR4A at DSBs. Thus, NR4As represent an entirely novel component of DNA damage response and are substrates of DNA-PK in the process of DSB repair.

  11. Rad54B Targeting to DNA Double-Strand Break Repair Sites Requires Complex Formation with S100A11

    PubMed Central

    Murzik, Ulrike; Hemmerich, Peter; Weidtkamp-Peters, Stefanie; Ulbricht, Tobias; Bussen, Wendy; Hentschel, Julia; von Eggeling, Ferdinand

    2008-01-01

    S100A11 is involved in a variety of intracellular activities such as growth regulation and differentiation. To gain more insight into the physiological role of endogenously expressed S100A11, we used a proteomic approach to detect and identify interacting proteins in vivo. Hereby, we were able to detect a specific interaction between S100A11 and Rad54B, which could be confirmed under in vivo conditions. Rad54B, a DNA-dependent ATPase, is described to be involved in recombinational repair of DNA damage, including DNA double-strand breaks (DSBs). Treatment with bleomycin, which induces DSBs, revealed an increase in the degree of colocalization between S100A11 and Rad54B. Furthermore, S100A11/Rad54B foci are spatially associated with sites of DNA DSB repair. Furthermore, while the expression of p21WAF1/CIP1 was increased in parallel with DNA damage, its protein level was drastically down-regulated in damaged cells after S100A11 knockdown. Down-regulation of S100A11 by RNA interference also abolished Rad54B targeting to DSBs. Additionally, S100A11 down-regulated HaCaT cells showed a restricted proliferation capacity and an increase of the apoptotic cell fraction. These observations suggest that S100A11 targets Rad54B to sites of DNA DSB repair sites and identify a novel function for S100A11 in p21-based regulation of cell cycle. PMID:18463164

  12. Mutator Phenotype and DNA Double-Strand Break Repair in BLM Helicase-Deficient Human Cells

    PubMed Central

    Suzuki, Tetsuya; Yasui, Manabu

    2016-01-01

    Bloom syndrome (BS), an autosomal recessive disorder of the BLM gene, predisposes sufferers to various cancers. To investigate the mutator phenotype and genetic consequences of DNA double-strand breaks (DSBs) in BS cells, we developed BLM helicase-deficient human cells by disrupting the BLM gene. Cells with a loss of heterozygosity (LOH) due to homologous recombination (HR) or nonhomologous end joining (NHEJ) can be restored with or without site-directed DSB induction. BLM cells exhibited a high frequency of spontaneous interallelic HR with crossover, but noncrossover events with long-tract gene conversions also occurred. Despite the highly interallelic HR events, BLM cells predominantly produced hemizygous LOH by spontaneous deletion. These phenotypes manifested during repair of DSBs. Both NHEJ and HR appropriately repaired DSBs in BLM cells, resulting in hemizygous and homozygous LOHs, respectively. However, the magnitude of the LOH was exacerbated in BLM cells, as evidenced by large deletions and long-tract gene conversions with crossover. BLM helicase suppresses the elongation of branch migration and crossover of double Holliday junctions (HJs) during HR repair, and a deficiency in this enzyme causes collapse, abnormal elongation, and/or preferable resolution to crossover of double HJs, resulting in a large-scale LOH. This mechanism underlies the predisposition for cancer in BS. PMID:27601585

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

    PubMed

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

    2016-02-29

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

  14. Overlapping mechanisms promote postsynaptic RAD-51 filament disassembly during meiotic double-strand break repair.

    PubMed

    Ward, Jordan D; Muzzini, Diego M; Petalcorin, Mark I R; Martinez-Perez, Enrique; Martin, Julie S; Plevani, Paolo; Cassata, Giuseppe; Marini, Federica; Boulton, Simon J

    2010-01-29

    Homologous recombination (HR) is essential for repair of meiotic DNA double-strand breaks (DSBs). Although the mechanisms of RAD-51-DNA filament assembly and strand exchange are well characterized, the subsequent steps of HR are less well defined. Here, we describe a synthetic lethal interaction between the C. elegans helicase helq-1 and RAD-51 paralog rfs-1, which results in a block to meiotic DSB repair after strand invasion. Whereas RAD-51-ssDNA filaments assemble at meiotic DSBs with normal kinetics in helq-1, rfs-1 double mutants, persistence of RAD-51 foci and genetic interactions with rtel-1 suggest a failure to disassemble RAD-51 from strand invasion intermediates. Indeed, purified HELQ-1 and RFS-1 independently bind to and promote the disassembly of RAD-51 from double-stranded, but not single-stranded, DNA filaments via distinct mechanisms in vitro. These results indicate that two compensating activities are required to promote postsynaptic RAD-51 filament disassembly, which are collectively essential for completion of meiotic DSB repair.

  15. 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

  16. Telomere Dysfunction Triggers Palindrome Formation Independently of Double-Strand Break Repair Mechanisms

    PubMed Central

    Raykov, Vasil; Marvin, Marcus E.; Louis, Edward J.; Maringele, Laura

    2016-01-01

    Inverted chromosome duplications or palindromes are linked with genetic disorders and malignant transformation. They are considered by-products of DNA double-strand break (DSB) repair: the homologous recombination (HR) and the nonhomologous end joining (NHEJ). Palindromes near chromosome ends are often triggered by telomere losses. An important question is to what extent their formation depends upon DSB repair mechanisms. Here we addressed this question using yeast genetics and comparative genomic hybridization. We induced palindrome formation by passaging cells lacking any form of telomere maintenance (telomerase and telomere recombination). Surprisingly, we found that DNA ligase 4, essential for NHEJ, did not make a significant contribution to palindrome formation induced by telomere losses. Moreover RAD51, important for certain HR-derived mechanisms, had little effect. Furthermore RAD52, which is essential for HR in yeast, appeared to decrease the number of palindromes in cells proliferating without telomeres. This study also uncovered an important role for Rev3 and Rev7 (but not for Pol32) subunits of polymerase ζ in the survival of cells undergoing telomere losses and forming palindromes. We propose a model called short-inverted repeat-induced synthesis in which DNA synthesis, rather than DSB repair, drives the inverted duplication triggered by telomere dysfunction. PMID:27334270

  17. Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog.

    PubMed

    Li, Shuyi; Li, Zhentian; Shu, Feng-Jue; Xiong, Hairong; Phillips, Andrew C; Dynan, William S

    2014-09-01

    NONO, SFPQ and PSPC1 make up a family of proteins with diverse roles in transcription, RNA processing and DNA double-strand break (DSB) repair. To understand long-term effects of loss of NONO, we characterized murine embryonic fibroblasts (MEFs) from knockout mice. In the absence of genotoxic stress, wild-type and mutant MEFs showed similar growth rates and cell cycle distributions, and the mutants were only mildly radiosensitive. Further investigation showed that NONO deficiency led to upregulation of PSPC1, which replaced NONO in a stable complex with SFPQ. Knockdown of PSPC1 in a NONO-deficient background led to severe radiosensitivity and delayed resolution of DSB repair foci. The DNA-dependent protein kinase (DNA-PK) inhibitor, NU7741, sensitized wild-type and singly deficient MEFs, but had no additional effect on doubly deficient cells, suggesting that NONO/PSPC1 and DNA-PK function in the same pathway. We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes. Of 12 genes tested, none were downregulated, and several were upregulated. Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.

  18. Structural chromosome abnormalities, increased DNA strand breaks and DNA strand break repair deficiency in dermal fibroblasts from old female human donors

    PubMed Central

    Kalfalah, Faiza; Seggewiß, Sabine; Walter, Regina; Tigges, Julia; Moreno-Villanueva, María; Bürkle, Alexander; Ohse, Sebastian; Busch, Hauke; Boerries, Melanie; Hildebrandt, Barbara; Royer-Pokora, Brigitte; Boege, Fritz

    2015-01-01

    Dermal fibroblasts provide a paradigmatic model of cellular adaptation to long-term exogenous stress and ageing processes driven thereby. Here we addressed whether fibroblast ageing analysed ex vivo entails genome instability. Dermal fibroblasts from human female donors aged 20–67 years were studied in primary culture at low population doubling. Under these conditions, the incidence of replicative senescence and rates of age-correlated telomere shortening were insignificant. Genome-wide gene expression analysis revealed age-related impairment of mitosis, telomere and chromosome maintenance and induction of genes associated with DNA repair and non-homologous end-joining, most notably XRCC4 and ligase 4. We observed an age-correlated drop in proliferative capacity and age-correlated increases in heterochromatin marks, structural chromosome abnormalities (deletions, translocations and chromatid breaks), DNA strand breaks and histone H2AX-phosphorylation. In a third of the cells from old and middle-aged donors repair of X-ray induced DNA strand breaks was impaired despite up-regulation of DNA repair genes. The distinct phenotype of genome instability, increased heterochromatinisation and (in 30% of the cases futile) up-regulation of DNA repair genes was stably maintained over several cell passages indicating that it represents a feature of geroconversion that is distinct from cellular senescence, as it does not encompass a block of proliferation. PMID:25678531

  19. Quantitative measurement of DNA strand breaks and repair in. gamma. -irradiated human leukocytes from normal and ataxia telangiectasia donors

    SciTech Connect

    Thierry, D.; Rigaud, O.; Duranton, I.; Moustacchi, E.; Magdelenat, H.

    1985-06-01

    Fluorimetric analysis of DNA unwinding, which allows measurement of DNA strand breaks in human leukocytes, has been optimized by reducing the amount of cells required for the test and by modifying the DNA alkali unwinding conditions. The permitted measurement of DNA strand-break induction in cells irradiated with low (0.5-7 Gy) or high doses (5-20 Gy) of ..gamma.. rays. Linear dose-response curves were obtained for both dose ranges. Presence of cysteamine during irradiation caused a decrease in the extent of DNA strand breaks. The kinetics of the DNA standard-break rejoining process appeared to be biphasic over the dose range of 2-20 Gy when plotted on a linear vs linear axis (percentage of damage as a function of time). No difference in the level of DNA strand breaks and the rate of repair of these breaks was observed between leukocytes from three ataxia telangiectasia patients and those from normal donors.

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

    PubMed

    Skory, C D

    2002-11-01

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

  1. MEIOTIC F-BOX Is Essential for Male Meiotic DNA Double-Strand Break Repair in Rice[OPEN

    PubMed Central

    Wang, Chong; Yu, Junping; Zong, Jie; Lu, Pingli

    2016-01-01

    F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression. PMID:27436711

  2. Lack of dependence on p53 for DNA double strand break repair of episomal vectors in human lymphoblasts

    NASA Technical Reports Server (NTRS)

    Kohli, M.; Jorgensen, T. J.

    1999-01-01

    The p53 tumor suppressor gene has been shown to be involved in a variety of repair processes, and recent findings have suggested that p53 may be involved in DNA double strand break repair in irradiated cells. The role of p53 in DNA double strand break repair, however, has not been fully investigated. In this study, we have constructed a novel Epstein-Barr virus (EBV)-based shuttle vector, designated as pZEBNA, to explore the influence of p53 on DNA strand break repair in human lymphoblasts, since EBV-based vectors do not inactivate the p53 pathway. We have compared plasmid survival of irradiated, restriction enzyme linearized, and calf intestinal alkaline phosphatase (CIP)-treated pZEBNA with a Simian virus 40 (SV40)-based shuttle vector, pZ189, in TK6 (wild-type p53) and WTK1 (mutant p53) lymphoblasts and determined that p53 does not modulate DNA double strand break repair in these cell lines. Copyright 1999 Academic Press.

  3. NF-κB regulates DNA double-strand break repair in conjunction with BRCA1-CtIP complexes.

    PubMed

    Volcic, Meta; Karl, Sabine; Baumann, Bernd; Salles, Daniela; Daniel, Peter; Fulda, Simone; Wiesmüller, Lisa

    2012-01-01

    NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP-BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP-BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation.

  4. Up-regulation of WRN and DNA ligase IIIalpha in chronic myeloid leukemia: consequences for the repair of DNA double-strand breaks.

    PubMed

    Sallmyr, Annahita; Tomkinson, Alan E; Rassool, Feyruz V

    2008-08-15

    Expression of oncogenic BCR-ABL in chronic myeloid leukemia (CML) results in increased reactive oxygen species (ROS) that in turn cause increased DNA damage, including DNA double-strand breaks (DSBs). We have previously shown increased error-prone repair of DSBs by nonhomologous end-joining (NHEJ) in CML cells. Recent reports have identified alternative NHEJ pathways that are highly error prone, prompting us to examine the role of the alternative NHEJ pathways in BCR-ABL-positive CML. Importantly, we show that key proteins in the major NHEJ pathway, Artemis and DNA ligase IV, are down-regulated, whereas DNA ligase IIIalpha, and the protein deleted in Werner syndrome, WRN, are up-regulated. DNA ligase IIIalpha and WRN form a complex that is recruited to DSBs in CML cells. Furthermore, "knockdown" of either DNA ligase IIIalpha or WRN leads to increased accumulation of unrepaired DSBs, demonstrating that they contribute to the repair of DSBs. These results indicate that altered DSB repair in CML cells is caused by the increased activity of an alternative NHEJ repair pathway, involving DNA ligase IIIalpha and WRN. We suggest that, although the repair of ROS-induced DSBs by this pathway contributes to the survival of CML cells, the resultant genomic instability drives disease progression.

  5. Orphan receptor TR3 enhances p53 transactivation and represses DNA double-strand break repair in hepatoma cells under ionizing radiation.

    PubMed

    Zhao, Bi-xing; Chen, Hang-zi; Du, Xiao-dan; Luo, Jie; He, Jian-ping; Wang, Rong-hao; Wang, Yuan; Wu, Rong; Hou, Ru-rong; Hong, Ming; Wu, Qiao

    2011-08-01

    In response to ionizing radiation (IR)-induced DNA double-strand breaks (DSB), cells elicit an evolutionarily conserved checkpoint response that induces cell cycle arrest and either DNA repair or apoptosis, thereby maintaining genomic stability. DNA-dependent protein kinase (DNA-PK) is a central enzyme involved in DSB repair for mammalian cells that comprises a DNA-PK catalytic subunit and the Ku protein, which act as regulatory elements. DNA-PK also functions as a signaling molecule to selectively regulate p53-dependent apoptosis in response to IR. Herein, we demonstrate that the orphan nuclear receptor TR3 suppresses DSB repair by blocking Ku80 DNA-end binding activity and promoting DNA-PK-induced p53 activity in hepatoma cells. We find that TR3 interacts with Ku80 and inhibits its binding to DNA ends, which then suppresses DSB repair. Furthermore, TR3 is a phosphorylation substrate for DNA-PK and interacts with DNA-PK catalytic subunit in a Ku80-independent manner. Phosphorylated TR3, in turn, enhances DNA-PK-induced phosphorylation and p53 transcription activity, thereby enhancing IR-induced apoptosis in hepatoma cells. Together, our findings reveal novel functions for TR3, not only in DSB repair regulation but also in IR-induced hepatoma cell apoptosis, and they suggest that TR3 is a potential target for cancer radiotherapy.

  6. Shape "break-and-repair" strategy and its application to automated medical image segmentation.

    PubMed

    Pu, Jiantao; Paik, David S; Meng, Xin; Roos, Justus E; Rubin, Geoffrey D

    2011-01-01

    In three-dimensional medical imaging, segmentation of specific anatomy structure is often a preprocessing step for computer-aided detection/diagnosis (CAD) purposes, and its performance has a significant impact on diagnosis of diseases as well as objective quantitative assessment of therapeutic efficacy. However, the existence of various diseases, image noise or artifacts, and individual anatomical variety generally impose a challenge for accurate segmentation of specific structures. To address these problems, a shape analysis strategy termed "break-and-repair" is presented in this study to facilitate automated medical image segmentation. Similar to surface approximation using a limited number of control points, the basic idea is to remove problematic regions and then estimate a smooth and complete surface shape by representing the remaining regions with high fidelity as an implicit function. The innovation of this shape analysis strategy is the capability of solving challenging medical image segmentation problems in a unified framework, regardless of the variability of anatomical structures in question. In our implementation, principal curvature analysis is used to identify and remove the problematic regions and radial basis function (RBF) based implicit surface fitting is used to achieve a closed (or complete) surface boundary. The feasibility and performance of this strategy are demonstrated by applying it to automated segmentation of two completely different anatomical structures depicted on CT examinations, namely human lungs and pulmonary nodules. Our quantitative experiments on a large number of clinical CT examinations collected from different sources demonstrate the accuracy, robustness, and generality of the shape "break-and-repair" strategy in medical image segmentation.

  7. Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene

    PubMed Central

    Hollywood, Jennifer A.; Lee, Ciaran M.; Scallan, Martina F.; Harrison, Patrick T.

    2016-01-01

    To maximise the efficiency of template-dependent gene editing, most studies describe programmable and/or RNA-guided endonucleases that make a double-stranded break at, or close to, the target sequence to be modified. The rationale for this design strategy is that most gene repair tracts will be very short. Here, we describe a CRISPR Cas9/gRNA selection-free strategy which uses deep sequencing to characterise repair tracts from a donor plasmid containing seven nucleotide differences across a 216 bp target region in the human CFTR gene. We found that 90% of the template-dependent repair tracts were >100 bp in length with equal numbers of uni-directional and bi-directional repair tracts. The occurrence of long repair tracts suggests that a single gRNA could be used with variants of the same template to create or correct specific mutations within a 200 bp range, the size of ~80% of human exons. The selection-free strategy used here also allowed detection of non-homologous end joining events in many of the homology-directed repair tracts. This indicates a need to modify the donor, possibly by silent changes in the PAM sequence, to prevent creation of a second double-stranded break in an allele that has already been correctly edited by homology-directed repair. PMID:27557525

  8. Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene.

    PubMed

    Hollywood, Jennifer A; Lee, Ciaran M; Scallan, Martina F; Harrison, Patrick T

    2016-08-25

    To maximise the efficiency of template-dependent gene editing, most studies describe programmable and/or RNA-guided endonucleases that make a double-stranded break at, or close to, the target sequence to be modified. The rationale for this design strategy is that most gene repair tracts will be very short. Here, we describe a CRISPR Cas9/gRNA selection-free strategy which uses deep sequencing to characterise repair tracts from a donor plasmid containing seven nucleotide differences across a 216 bp target region in the human CFTR gene. We found that 90% of the template-dependent repair tracts were >100 bp in length with equal numbers of uni-directional and bi-directional repair tracts. The occurrence of long repair tracts suggests that a single gRNA could be used with variants of the same template to create or correct specific mutations within a 200 bp range, the size of ~80% of human exons. The selection-free strategy used here also allowed detection of non-homologous end joining events in many of the homology-directed repair tracts. This indicates a need to modify the donor, possibly by silent changes in the PAM sequence, to prevent creation of a second double-stranded break in an allele that has already been correctly edited by homology-directed repair.

  9. Contribution of sleep to the repair of neuronal DNA double-strand breaks: evidence from flies and mice

    PubMed Central

    Bellesi, Michele; Bushey, Daniel; Chini, Mattia; Tononi, Giulio; Cirelli, Chiara

    2016-01-01

    Exploration of a novel environment leads to neuronal DNA double-strand breaks (DSBs). These DSBs are generated by type 2 topoisomerase to relieve topological constrains that limit transcription of plasticity-related immediate early genes. If not promptly repaired, however, DSBs may lead to cell death. Since the induction of plasticity-related genes is higher in wake than in sleep, we asked whether it is specifically wake associated with synaptic plasticity that leads to DSBs, and whether sleep provides any selective advantage over wake in their repair. In flies and mice, we find that enriched wake, more than simply time spent awake, induces DSBs, and their repair in mice is delayed or prevented by subsequent wake. In both species the repair of irradiation-induced neuronal DSBs is also quicker during sleep, and mouse genes mediating the response to DNA damage are upregulated in sleep. Thus, sleep facilitates the repair of neuronal DSBs. PMID:27830758

  10. Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks

    PubMed Central

    Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

    2016-01-01

    Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress. PMID:26765540

  11. Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity.

    PubMed

    Oeck, S; Al-Refae, K; Riffkin, H; Wiel, G; Handrick, R; Klein, D; Iliakis, G; Jendrossek, V

    2017-02-17

    The survival kinase Akt has clinical relevance to radioresistance. However, its contributions to the DNA damage response, DNA double strand break (DSB) repair and apoptosis remain poorly defined and often contradictory. We used a genetic approach to explore the consequences of genetic alterations of Akt1 for the cellular radiation response. While two activation-associated mutants with prominent nuclear access, the phospho-mimicking Akt1-TDSD and the clinically relevant PH-domain mutation Akt1-E17K, accelerated DSB repair and improved survival of irradiated Tramp-C1 murine prostate cancer cells and Akt1-knockout murine embryonic fibroblasts in vitro, the classical constitutively active membrane-targeted myrAkt1 mutant had the opposite effects. Interestingly, DNA-PKcs directly phosphorylated Akt1 at S473 in an in vitro kinase assay but not vice-versa. Pharmacological inhibition of DNA-PKcs or Akt restored radiosensitivity in tumour cells expressing Akt1-E17K or Akt1-TDSD. In conclusion, Akt1-mediated radioresistance depends on its activation state and nuclear localization and is accessible to pharmacologic inhibition.

  12. RNF4 regulates DNA double-strand break repair in a cell cycle-dependent manner.

    PubMed

    Kuo, Ching-Ying; Li, Xu; Stark, Jeremy M; Shih, Hsiu-Ming; Ann, David K

    2016-01-01

    Both RNF4 and KAP1 play critical roles in the response to DNA double-strand breaks (DSBs), but the functional interplay of RNF4 and KAP1 in regulating DNA damage response remains unclear. We have previously demonstrated the recruitment and degradation of KAP1 by RNF4 require the phosphorylation of Ser824 (pS824) and SUMOylation of KAP1. In this report, we show the retention of DSB-induced pS824-KAP1 foci and RNF4 abundance are inversely correlated as cell cycle progresses. Following irradiation, pS824-KAP1 foci predominantly appear in the cyclin A (-) cells, whereas RNF4 level is suppressed in the G0-/G1-phases and then accumulates during S-/G2-phases. Notably, 53BP1 foci, but not BRCA1 foci, co-exist with pS824-KAP1 foci. Depletion of KAP1 yields opposite effect on the dynamics of 53BP1 and BRCA1 loading, favoring homologous recombination repair. In addition, we identify p97 is present in the RNF4-KAP1 interacting complex and the inhibition of p97 renders MCF7 breast cancer cells relatively more sensitive to DNA damage. Collectively, these findings suggest that combined effect of dynamic recruitment of RNF4 to KAP1 regulates the relative occupancy of 53BP1 and BRCA1 at DSB sites to direct DSB repair in a cell cycle-dependent manner.

  13. Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9.

    PubMed

    Yoder, Kristine E; Bundschuh, Ralf

    2016-07-12

    CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance.

  14. Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks.

    PubMed

    Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

    2016-01-01

    Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.

  15. Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9

    PubMed Central

    Yoder, Kristine E.; Bundschuh, Ralf

    2016-01-01

    CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance. PMID:27404981

  16. RNF4 regulates DNA double-strand break repair in a cell cycle-dependent manner

    PubMed Central

    Kuo, Ching-Ying; Li, Xu; Stark, Jeremy M.; Shih, Hsiu-Ming; Ann, David K.

    2016-01-01

    Abstract Both RNF4 and KAP1 play critical roles in the response to DNA double-strand breaks (DSBs), but the functional interplay of RNF4 and KAP1 in regulating DNA damage response remains unclear. We have previously demonstrated the recruitment and degradation of KAP1 by RNF4 require the phosphorylation of Ser824 (pS824) and SUMOylation of KAP1. In this report, we show the retention of DSB-induced pS824-KAP1 foci and RNF4 abundance are inversely correlated as cell cycle progresses. Following irradiation, pS824-KAP1 foci predominantly appear in the cyclin A (-) cells, whereas RNF4 level is suppressed in the G0-/G1-phases and then accumulates during S-/G2-phases. Notably, 53BP1 foci, but not BRCA1 foci, co-exist with pS824-KAP1 foci. Depletion of KAP1 yields opposite effect on the dynamics of 53BP1 and BRCA1 loading, favoring homologous recombination repair. In addition, we identify p97 is present in the RNF4-KAP1 interacting complex and the inhibition of p97 renders MCF7 breast cancer cells relatively more sensitive to DNA damage. Collectively, these findings suggest that combined effect of dynamic recruitment of RNF4 to KAP1 regulates the relative occupancy of 53BP1 and BRCA1 at DSB sites to direct DSB repair in a cell cycle-dependent manner. PMID:26766492

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

    PubMed Central

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

    1996-01-01

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

  18. Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity

    PubMed Central

    Oeck, S.; Al-Refae, K.; Riffkin, H.; Wiel, G.; Handrick, R.; Klein, D.; Iliakis, G.; Jendrossek, V.

    2017-01-01

    The survival kinase Akt has clinical relevance to radioresistance. However, its contributions to the DNA damage response, DNA double strand break (DSB) repair and apoptosis remain poorly defined and often contradictory. We used a genetic approach to explore the consequences of genetic alterations of Akt1 for the cellular radiation response. While two activation-associated mutants with prominent nuclear access, the phospho-mimicking Akt1-TDSD and the clinically relevant PH-domain mutation Akt1-E17K, accelerated DSB repair and improved survival of irradiated Tramp-C1 murine prostate cancer cells and Akt1-knockout murine embryonic fibroblasts in vitro, the classical constitutively active membrane-targeted myrAkt1 mutant had the opposite effects. Interestingly, DNA-PKcs directly phosphorylated Akt1 at S473 in an in vitro kinase assay but not vice-versa. Pharmacological inhibition of DNA-PKcs or Akt restored radiosensitivity in tumour cells expressing Akt1-E17K or Akt1-TDSD. In conclusion, Akt1-mediated radioresistance depends on its activation state and nuclear localization and is accessible to pharmacologic inhibition. PMID:28209968

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

    PubMed

    Savolainen, Linda; Helleday, Thomas

    2009-02-01

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

  20. Defective DNA Ligation during Short-Patch Single-Strand Break Repair in Ataxia Oculomotor Apraxia 1 ▿

    PubMed Central

    Reynolds, John J.; El-Khamisy, Sherif F.; Katyal, Sachin; Clements, Paula; McKinnon, Peter J.; Caldecott, Keith W.

    2009-01-01

    Ataxia oculomotor apraxia 1 (AOA1) results from mutations in aprataxin, a component of DNA strand break repair that removes AMP from 5′ termini. Despite this, global rates of chromosomal strand break repair are normal in a variety of AOA1 and other aprataxin-defective cells. Here we show that short-patch single-strand break repair (SSBR) in AOA1 cell extracts bypasses the point of aprataxin action at oxidative breaks and stalls at the final step of DNA ligation, resulting in the accumulation of adenylated DNA nicks. Strikingly, this defect results from insufficient levels of nonadenylated DNA ligase, and short-patch SSBR can be restored in AOA1 extracts, independently of aprataxin, by the addition of recombinant DNA ligase. Since adenylated nicks are substrates for long-patch SSBR, we reasoned that this pathway might in part explain the apparent absence of a chromosomal SSBR defect in aprataxin-defective cells. Indeed, whereas chemical inhibition of long-patch repair did not affect SSBR rates in wild-type mouse neural astrocytes, it uncovered a significant defect in Aptx−/− neural astrocytes. These data demonstrate that aprataxin participates in chromosomal SSBR in vivo and suggest that short-patch SSBR arrests in AOA1 because of insufficient nonadenylated DNA ligase. PMID:19103743

  1. The effects of radioprotectors on DNA polymerase I-directed repair synthesis and DNA strand breaks in toluene-treated and X-irradiated Escherichia coli

    SciTech Connect

    Billen, D.

    1983-07-01

    In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is induced by exposure to X rays. This repair synthesis may be amplified and easily measured through inhibition of DNA ligase action. In an effort to learn more of the relationship between X-ray-induced strand breaks in cellular DNA and the extent of this repair synthesis, experiments designed to compare the influence of radioprotectors on both strand-break production and repair synthesis have been carried out. The results show that cysteamine, sodium formate, and glycerol not only protect against strand breaks but also reduce DNA polymerase I-directed repair synthesis. However, I-, an efficient hydroxyl radical scavenger, is not as effective a protective agent against strand breaks and does not measurably affect repair synthesis in our system.

  2. Effects of radioprotectors on DNA polymerase I-directed repair synthesis and DNA strand breaks in toluene-treated and x-irradiated Escherichia coli

    SciTech Connect

    Billen, D.

    1983-07-01

    In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is induced by exposure to x rays. This repair synthesis may be amplified and easily measured through inhibition of DNA ligase action. In an effort to learn more of the relationship between x-ray-induced strand breaks in cellular DNA and the extent of this repair synthesis, experiments designed to compare the influence of radioprotectors on both strand-break production and repair synthesis have been carried out. The results show that cysteamine, sodium formate, and glycerol not only protect against strand breaks but also reduce DNA polymerase I-directed repair synthesis. However, I/sup -/, an efficient hydroxyl radical scavenger, is not as effective a protective agent against strand breaks and does not measurably affect repair synthesis in our system.

  3. The Contribution of Alu Elements to Mutagenic DNA Double-Strand Break Repair

    PubMed Central

    Streva, Vincent A.; DeFreece, Cecily B.; Hedges, Dale J.; Deininger, Prescott L.

    2015-01-01

    Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both

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

    PubMed Central

    Boulton, S J; Jackson, S P

    1996-01-01

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

  5. A role for the HOXB7 homeodomain protein in DNA repair.

    PubMed

    Rubin, Ethel; Wu, Xinyan; Zhu, Tao; Cheung, Joyce C Y; Chen, Hexin; Lorincz, Annaka; Pandita, Raj K; Sharma, Girdhar G; Ha, Hyo Chol; Gasson, Judith; Hanakahi, Les A; Pandita, Tej K; Sukumar, Saraswati

    2007-02-15

    Homeobox genes encode transcription factors which function in body axis patterning in the developing embryo. Recent evidence suggests that the maintenance of specific HOX expression patterns is necessary for regulating the homeostasis of adult tissues as well. In this study, HOXB7 transformed human mammary epithelial cells, MCF10A, to grow in minimally supplemented medium, to form colonies in Matrigel, and display resistance to ionizing radiation. Searching for protein partners of HOXB7 that might contribute to resistance to ionizing radiation, we identified four HOXB7-binding proteins by GST pull-down/affinity chromatography and confirmed their interactions by coimmunoprecipitation in vivo. Interestingly, all four HOXB7-binding proteins shared functions as genomic caretakers and included members of the DNA-dependent protein kinase holoenzyme (Ku70, Ku80, DNA-PK(cs)) responsible for DNA double-strand break repair by nonhomologous end joining pathway and poly(ADP) ribose polymerase. Exogenous and endogenous expression of HOXB7 enhanced nonhomologous end joining and DNA repair functions in vitro and in vivo, which were reversed by silencing HOXB7. This is the first mechanistic study providing definitive evidence for the involvement of any HOX protein in DNA double-strand break repair.

  6. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins.

    PubMed

    Lisby, Michael; Barlow, Jacqueline H; Burgess, Rebecca C; Rothstein, Rodney

    2004-09-17

    DNA repair is an essential process for preserving genome integrity in all organisms. In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into centers (foci). Here, we analyze the cellular response to DNA double-strand breaks (DSBs) and replication stress in Saccharomyces cerevisiae. The Mre11 nuclease and the ATM-related Tel1 kinase are the first proteins detected at DSBs. Next, the Rfa1 single-strand DNA binding protein relocalizes to the break and recruits other key checkpoint proteins. Later and only in S and G2 phase, the homologous recombination machinery assembles at the site. Unlike the response to DSBs, Mre11 and recombination proteins are not recruited to hydroxyurea-stalled replication forks unless the forks collapse. The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting and processing DNA ends in conjunction with Sae2 and by RP-A recognizing single-stranded DNA and recruiting additional checkpoint and repair proteins.

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

    SciTech Connect

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

    2009-01-01

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

  8. BMI1-mediated histone ubiquitylation promotes DNA double-strand break repair

    PubMed Central

    Ismail, Ismail Hassan; Andrin, Christi; McDonald, Darin

    2010-01-01

    Polycomb group (PcG) proteins are major determinants of cell identity, stem cell pluripotency, and epigenetic gene silencing during development. The polycomb repressive complex 1, which contains BMI1, RING1, and RING2, functions as an E3-ubuiquitin ligase. We found that BMI1 and RING2 are recruited to sites of DNA double-strand breaks (DSBs) where they contribute to the ubiquitylation of γ-H2AX. In the absence of BMI1, several proteins dependent on ubiquitin signaling, including 53BP1, BRCA1, and RAP80, are impaired in recruitment to DSBs. Loss of BMI1 sensitizes cells to ionizing radiation to the same extent as loss of RNF8. The simultaneous depletion of both proteins revealed an additive increase in radiation sensitivity. These data uncover an unexpected link between the polycomb and the DNA damage response pathways, and suggest a novel function for BMI1 in maintaining genomic stability. PMID:20921134

  9. How protein-making machine bends without breaking

    SciTech Connect

    2011-01-01

    Scientists from several institutions including the U.S. Department of Energy's Lawrence Berkeley National Laboratory. They derived atomic-scale resolution structures of the cell's protein-making machine, the ribosome, at key stages of its job. The ability to bend but not break comes from this hinge within transfer RNA, which allows it to bend as much as 70 degrees when it passes through the ribosome during protein synthesis. The structures, developed primarily at Berkeley Lab's Advanced Light Source, reveal that the ribosome's ability to rotate an incredible amount without falling apart is due to the never-before-seen springiness of molecular widgets that hold it together.

  10. Double-strand break repair based on short-homology regions is suppressed under terminal deoxynucleotidyltransferase expression, as revealed by a novel vector system for analysing DNA repair by nonhomologous end joining.

    PubMed

    Maezawa, So; Nakano, Saori; Kuniya, Takaaki; Koiwai, Osamu; Koiwai, Kotaro

    2016-01-01

    We have constructed a novel, nonhomologous end-joining (NHEJ) assay vector (NAV), containing mKate2, Venus and ccdB genes. Cotransfection of NAV with a construct expressing the restriction enzyme I-SceI generated a double-strand break (DSB) in NAV that excised mKate2 and ccdB. Repair of this DSB produced an intact vector that expressed Venus, a green fluorescent protein. Because cells bearing the repaired NAV lacked the ccdB gene which slows cell proliferation, the cultures were enriched in cells containing repaired DSBs. DNA sequence analysis of the DSB junctions indicated that the repair was carried out mainly by using the closest homology sequence. Use of the NAV yielded rapid results within 3 days after transfection. We then used the NAV to analyse NHEJ in cells overexpressing terminal deoxynucleotidyltransferase (TdT). The results indicated that TdT suppresses DNA repair that is based on short (one- or two-base) homology regions, to efficiently add deoxynucleotides during VDJ recombination in lymphoid cells.

  11. Histone deacetylase inhibitors decrease NHEJ both by acetylation of repair factors and trapping of PARP1 at DNA double-strand breaks in chromatin.

    PubMed

    Robert, Carine; Nagaria, Pratik K; Pawar, Nisha; Adewuyi, Adeoluwa; Gojo, Ivana; Meyers, David J; Cole, Philip A; Rassool, Feyruz V

    2016-06-01

    Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and modulate the acetylation of proteins involved in DNA double-strand break (DSB) repair. Non-homologous end-joining (NHEJ) is one of the main pathways for repairing DSBs. Decreased NHEJ activity has been reported with HDACi treatment. However, mechanisms through which these effects are regulated in the context of chromatin are unclear. We show that pan-HDACi, trichostatin A (TSA), causes differential acetylation of DNA repair factors Ku70/Ku80 and poly ADP-ribose polymerase-1 (PARP1), and impairs NHEJ. Repair effects are reversed by treatments with p300/CBP inhibitor C646, with significantly decreased acetylation of PARP1. In keeping with these findings, TSA treatment significantly increases PARP1 binding to DSBs in chromatin. Notably, AML patients treated with HDACi entinostat (MS275) in vivo also show increased formation of poly ADP-ribose (PAR) that co-localizes with DSBs. Further, we demonstrate that PARP1 bound to chromatin increases with duration of TSA exposure, resembling PARP "trapping". Knockdown of PARP1 inhibits trapping and mitigates HDACi effects on NHEJ. Finally, combination of HDACi with potent PARP inhibitor talazoparib (BMN673) shows a dose-dependent increase in PARP "trapping", which correlates with increased apoptosis. These results provide a mechanism through which HDACi inhibits deacetylation and increases binding of PARP1 to DSBs, leading to decreased NHEJ and cytotoxicity of leukemia cells.

  12. APE1 overexpression in XRCC1-deficient cells complements the defective repair of oxidative single strand breaks but increases genomic instability

    PubMed Central

    Sossou, Marguerite; Flohr-Beckhaus, Claudia; Schulz, Ina; Daboussi, Fayza; Epe, Bernd; Radicella, J. Pablo

    2005-01-01

    XRCC1 protein is essential for mammalian viability and is required for the efficient repair of single strand breaks (SSBs) and damaged bases in DNA. XRCC1-deficient cells are genetically unstable and sensitive to DNA damaging agents. XRCC1 has no known enzymatic activity and is thought to act as a scaffold protein for both SSB and base excision repair activities. To further define the defects leading to genetic instability in XRCC1-deficient cells, we overexpressed the AP endonuclease APE1, shown previously to interact with and be stimulated by XRCC1. Here, we report that the overexpression of APE1 can compensate for the impaired capability of XRCC1-deficient cells to repair SSBs induced by oxidative DNA damage, both in vivo and in whole-cell extracts. We show that, for this kind of damage, the repair of blocked DNA ends is rate limiting and can be performed by APE1. Conversely, APE1 overproduction resulted in a 3-fold increase in the sensitivity of XRCC1-deficient cells to an alkylating agent, most probably due to the accumulation of SSBs. Finally, the overproduction of APE1 results in increases of 40% in the frequency of micronuclei and 33% in sister chromatid exchanges of XRCC1− cells. These data suggest that the spontaneous generation of AP sites could be at the origin of the SSBs responsible for the spontaneous genetic instability characteristic of XRCC1-deficient cells. PMID:15647512

  13. Inhibition of X-ray-induced DNA strand break repair in polyamine-depleted HeLa cells.

    PubMed

    Snyder, R D

    1989-05-01

    Treatment of HeLa cells with the polyamine biosynthesis inhibitors, alpha-difluoromethylornithine (DFMO) or methylglyoxal bis(guanylhydrazone) (MGBG), results in, depending on the conditions, partial or complete depletion of the cellular polyamines: putrescine, spermidine and spermine. In this compromised state cells exhibited a distinct deficiency in repair of X-ray-induced DNA strand breaks. The half-time for return of normal DNA sedimentation following 1.6 Gy was 9.5 min for untreated control cells and 22, 32 and 50 min for cells treated with MGBG, DFMO + MGBG and DFMO, respectively. Normal repair kinetics were restored to these cells upon a short incubation in media containing all three polyamines. The rapid early phase of repair following higher X-ray doses (16 Gy) was also delayed in polyamine-depleted cells but later repair occurring 1-4 h post-irradiation, representing chromatin reconstitution, was apparently normal.

  14. The non-homologous end-joining (NHEJ) mathematical model for the repair of double-strand breaks: II. Application to damage induced by ultrasoft X rays and low-energy electrons.

    PubMed

    Taleei, Reza; Girard, Peter M; Sankaranarayanan, Krishnaswami; Nikjoo, Hooshang

    2013-05-01

    We investigated the kinetics of simple and complex types of double-strand breaks (DSB) using our newly proposed mechanistic mathematical model for NHEJ DSB repair. For this purpose the simulated initial spectrum of DNA DSB, induced in an atomistic canonical model of B-DNA by low-energy single electron tracks, 100 eV to 4.55 keV, and the electrons generated by ultrasoft X rays (CK, AlK and TiK), were subjected to NHEJ repair processes. The activity elapsed time of sequentially independent steps of repair performed by proteins involved in NHEJ repair process were calculated for separate DSB. The repair kinetics of DSBs were computed and compared with published data on repair kinetics obtained by pulsed-field gel electrophoresis method. The comparison shows good agreement for V79-4 cells irradiated with ultrasoft X rays. The average times for the repair of simple and complex DSB confirm that double-strand break complexity is a potential explanation for the slow component of DSB repair observed in V79-4 cells irradiated by ultrasoft X rays.

  15. Inhibition of Poly(ADP-Ribose) Polymerase Enhances Radiochemosensitivity in Cancers Proficient in DNA Double-Strand Break Repair.

    PubMed

    Shunkwiler, Lauren; Ferris, Gina; Kunos, Charles

    2013-02-08

    Pharmacologic inhibitors of poly(ADP-ribose) polymerase (PARP) putatively enhance radiation toxicity in cancer cells. Although there is considerable information on the molecular interactions of PARP and BRCA1- and BRCA2-deficient cancers, very little is known of the PARP inhibition effect upon cancers proficient in DNA double-strand break repair after ionizing radiation or after stalled replication forks. In this work, we investigate whether PARP inhibition by ABT-888 (veliparib) augments death-provoking effects of ionizing radiation, or of the topoisomerase I poison topotecan, within uterine cervix cancers cells harboring an unfettered, overactive ribonucleotide reductase facilitating DNA double-strand break repair and contrast these findings with ovarian cancer cells whose regulation of ribonucleotide reductase is relatively intact. Cell lethality of a radiation-ABT-888 combination is radiation and drug dose dependent. Data particularly highlight an enhanced topotecan-ABT-888 cytotoxicity, and corresponds to an increased number of unrepaired DNA double-strand breaks. Overall, our findings support enhanced radiochemotherapy toxicity in cancers proficient in DNA double-strand break repair when PARP is inhibited by ABT-888.

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

    SciTech Connect

    Bradley, M.O.; Dysart, G. )

    1985-06-01

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

  17. DNA repair by thiols in air shows two radicals make a double-strand break

    SciTech Connect

    Milligan, J.R.; Ng J.Y.Y.; Wu, C.C.L.

    1995-09-01

    Using agarose gel electrophoresis, we have measured the yields of DNA single- and double-strand breaks (SSBs and DSBs) for plasmid DNA {gamma}-irradiated in aerobic aqueous solution. The presence during irradiation of either of the thiols cysteamine or N-(2-thioethyl)-1,3-diaminopropane (WR-1065) resulted in a concentration-dependent decrease in the yield of SSBs and a much greater decrease in the yield of DSBs. This large differential protective effect was not produced by thioethers or an alcohol of structural similarity to the two thiols, suggesting that repair of DSB radical precursors by thiols is more efficient than for SSB precursors. These observations suggest the existence of a diradical intermediate in the formation of DSBs. The results argue against a major contribution by a single radical mechanism involving interstrand radical transfer via hydrogen abstraction by a peroxyl intermediate, since the half-life of this radical transfer reaction appears to be significantly greater than the lifetime of the intermediate. 35 refs., 7 figs.

  18. Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: a role for SAE2/COM1.

    PubMed Central

    Rattray, A J; McGill, C B; Shafer, B K; Strathern, J N

    2001-01-01

    Errors associated with the repair of DNA double-strand breaks (DSBs) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ). We previously demonstrated that DNA synthesis is approximately 100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants that affect the fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants. Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants. In wild-type cells the recombinational repair process is efficient and fairly accurate. Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations. We isolated several mutant strains with altered fidelity of recombination. Here we characterize one of these mutants that revealed an approximately 10-fold elevation in the frequency of can1 mutants among TRP1 recombinants. The gene was cloned by complementation of a coincident sporulation defect and proved to be an allele of SAE2/COM1. Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Delta strain. Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex. PMID:11333222

  19. Endonucleolytic processing of covalent protein-linked DNA double-strand breaks.

    PubMed

    Neale, Matthew J; Pan, Jing; Keeney, Scott

    2005-08-18

    DNA double-strand breaks (DSBs) with protein covalently attached to 5' strand termini are formed by Spo11 to initiate meiotic recombination. The Spo11 protein must be removed for the DSB to be repaired, but the mechanism for removal is unclear. Here we show that meiotic DSBs in budding yeast are processed by endonucleolytic cleavage that releases Spo11 attached to an oligonucleotide with a free 3'-OH. Two discrete Spo11-oligonucleotide complexes were found in equal amounts, differing with respect to the length of the bound DNA. We propose that these forms arise from different spacings of strand cleavages flanking the DSB, with every DSB processed asymmetrically. Thus, the ends of a single DSB may be biochemically distinct at or before the initial processing step-much earlier than previously thought. SPO11-oligonucleotide complexes were identified in extracts of mouse testis, indicating that this mechanism is evolutionarily conserved. Oligonucleotide-topoisomerase II complexes were also present in extracts of vegetative yeast, although not subject to the same genetic control as for generating Spo11-oligonucleotide complexes. Our findings suggest a general mechanism for repair of protein-linked DSBs.

  20. Distinct Functions of Human Cohesin-SA1 and Cohesin-SA2 in Double-Strand Break Repair

    PubMed Central

    Kong, Xiangduo; Ball, Alexander R.; Pham, Hoang Xuan; Zeng, Weihua; Chen, Hsiao-Yuan; Schmiesing, John A.; Kim, Jong-Soo; Berns, Michael

    2014-01-01

    Cohesin is an essential multiprotein complex that mediates sister chromatid cohesion critical for proper segregation of chromosomes during cell division. Cohesin is also involved in DNA double-strand break (DSB) repair. In mammalian cells, cohesin is involved in both DSB repair and the damage checkpoint response, although the relationship between these two functions is unclear. Two cohesins differing by one subunit (SA1 or SA2) are present in somatic cells, but their functional specificities with regard to DNA repair remain enigmatic. We found that cohesin-SA2 is the main complex corecruited with the cohesin-loading factor NIPBL to DNA damage sites in an S/G2-phase-specific manner. Replacing the diverged C-terminal region of SA1 with the corresponding region of SA2 confers this activity on SA1. Depletion of SA2 but not SA1 decreased sister chromatid homologous recombination repair and affected repair pathway choice, indicating that DNA repair activity is specifically associated with cohesin recruited to damage sites. In contrast, both cohesin complexes function in the intra-S checkpoint, indicating that cell cycle-specific damage site accumulation is not a prerequisite for cohesin's intra-S checkpoint function. Our findings reveal the unique ways in which cohesin-SA1 and cohesin-SA2 participate in the DNA damage response, coordinately protecting genome integrity in human cells. PMID:24324008

  1. Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair

    PubMed Central

    Zapotoczny, Grzegorz; Sekelsky, Jeff

    2017-01-01

    DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila. To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells. PMID:28179392

  2. Double strand break repair by homologous recombination is regulated by cell cycle-independent signaling via ATM in human glioma cells.

    PubMed

    Golding, Sarah E; Rosenberg, Elizabeth; Khalil, Ashraf; McEwen, Alison; Holmes, Matthew; Neill, Steven; Povirk, Lawrence F; Valerie, Kristoffer

    2004-04-09

    To investigate double strand break (DSB) repair and signaling in human glioma cells, we stably transfected human U87 (ATM(+), p53(+)) glioma cells with a plasmid having a single I-SceI site within an inactive green fluorescent protein (GFP) expression cassette, allowing for the detection of homologous recombination repair (HRR) by GFP expression. HRR and nonhomologous end joining (NHEJ) were also determined by PCR. DSB repair was first detected at 12 h postinfection with an adenovirus expressing I-SceI with repair reaching plateau levels between 24 and 48 h. Within this time frame, NHEJ predominated over HRR in the range of 3-50-fold. To assess the involvement of ATM in DSB repair, we first examined whether ATM was associated with the DSB. Chromatin immunoprecipitation showed that ATM was present at the site of the DSB as early as 18 h postinfection. In cells treated with caffeine, an inhibitor of ATM, HRR was reduced, whereas NHEJ was not. In support of this finding, GFP flow cytometry demonstrated that caffeine reduced HRR by 90% under conditions when ATM kinase activity was inhibited. Dominant-negative ATM expressed from adenovirus inhibited HRR by 45%, also having little to no effect on NHEJ. Furthermore, HRR was inhibited by caffeine in serum-starved cells arrested in G(0)/G(1), suggesting that ATM is also important for HRR outside of the S and G(2) cell cycle phases. Altogether, these results demonstrate that HRR contributes substantially to DSB repair in human glioma cells, and, importantly, ATM plays a critical role in regulating HRR but not NHEJ throughout the cell cycle.

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

    PubMed

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

    2013-11-01

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

  4. Polycomb repressive complex 2 contributes to DNA double-strand break repair

    PubMed Central

    Campbell, Stuart; Ismail, Ismail Hassan; Young, Leah C; Poirier, Guy G; Hendzel, Michael J

    2013-01-01

    Polycomb protein histone methyltransferase, enhancer of Zeste homolog 2 (EZH2), is frequently overexpressed in human malignancy and is implicated in cancer cell proliferation and invasion. However, it is largely unknown whether EZH2 has a role in modulating the DNA damage response. Here, we show that polycomb repressive complex 2 (PRC2) is recruited to sites of DNA damage. This recruitment is independent of histone 2A variant X (H2AX) and the PI-3-related kinases ATM and DNA-PKcs. We establish that PARP activity is required for retaining PRC2 at sites of DNA damage. Furthermore, depletion of EZH2 in cells decreases the efficiency of DSB repair and increases sensitivity of cells to gamma-irradiation. These data unravel a crucial role of PRC2 in determining cancer cellular sensitivity following DNA damage and suggest that therapeutic targeting of EZH2 activity might serve as a strategy for improving conventional chemotherapy in a given malignancy. PMID:23907130

  5. Detection of DNA single-strand breaks during the repair of UV damage in xeroderma pigmentosum cells

    SciTech Connect

    Fornace, A.J. Jr.; Seres, D.S.

    1983-01-01

    In this investigation, xeroderma pigmentosum (XP) fibroblasts, XP12BE, were uv-irradiated and then incubated with cytosine arabinoside and hydroxyurea for 4 hr to inhibit the polymerase step of DNA excision repair. By alkaline elution, DNA single-strand breaks (SSB) were detected in XP cells with this regimen with an efficiency of 0.1-0.2 SSB per 10/sup 9/ daltons of DNA per J m/sup -2/. There was an approximately linear relation between the SSB frequency and uv dose over a range of 0.2 to 25 J m/sup -2/. This effect was approximately two orders of magnitude greater in excision-proficient normal human fibroblasts than in XP cells. These results support the conclusion that a low residual level of DNA excision repair occurs in XP group A cells and that the SSB generated during this repair can be accumulated with this polymerase inhibitor.

  6. Characterization of Cardiac Glycoside Natural Products as Potent Inhibitors of DNA Double-Strand Break Repair by a Whole-Cell Double Immunofluorescence Assay.

    PubMed

    Surovtseva, Yulia V; Jairam, Vikram; Salem, Ahmed F; Sundaram, Ranjini K; Bindra, Ranjit S; Herzon, Seth B

    2016-03-23

    Small-molecule inhibitors of DNA repair pathways are being intensively investigated as primary and adjuvant chemotherapies. We report the discovery that cardiac glycosides, natural products in clinical use for the treatment of heart failure and atrial arrhythmia, are potent inhibitors of DNA double-strand break (DSB) repair. Our data suggest that cardiac glycosides interact with phosphorylated mediator of DNA damage checkpoint protein 1 (phospho-MDC1) or E3 ubiquitin-protein ligase ring finger protein 8 (RNF8), two factors involved in DSB repair, and inhibit the retention of p53 binding protein 1 (53BP1) at the site of DSBs. These observations provide an explanation for the anticancer activity of this class of compounds, which has remained poorly understood for decades, and provide guidance for their clinical applications. This discovery was enabled by the development of the first high-throughput unbiased cellular assay to identify new small-molecule inhibitors of DSB repair. Our assay is based on the fully automated, time-resolved quantification of phospho-SER139-H2AX (γH2AX) and 53BP1 foci, two factors involved in the DNA damage response network, in cells treated with small molecules and ionizing radiation (IR). This primary assay is supplemented by robust secondary assays that establish lead compound potencies and provide further insights into their mechanisms of action. Although the cardiac glycosides were identified in an evaluation of 2366 small molecules, the assay is envisioned to be adaptable to larger compound libraries. The assay is shown to be compatible with small-molecule DNA cleaving agents, such as bleomycin, neocarzinostatin chromophore, and lomaiviticin A, in place of IR.

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

    PubMed Central

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

    2012-01-01

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

  8. The involvement of c-Myc in the DNA double-strand break repair via regulating radiation-induced phosphorylation of ATM and DNA-PKcs activity.

    PubMed

    Cui, Fengmei; Fan, Rong; Chen, Qiu; He, Yongming; Song, Man; Shang, Zengfu; Zhang, Shimeng; Zhu, Wei; Cao, Jianping; Guan, Hua; Zhou, Ping-Kun

    2015-08-01

    Deregulation of c-Myc often occurs in various human cancers, which not only contributes to the genesis and progression of cancers but also affects the outcomes of cancer radio- or chemotherapy. In this study, we have investigated the function of c-Myc in the repair of DNA double-strand break (DSB) induced by γ-ray irradiation. A c-Myc-silenced Hela-630 cell line was generated from HeLa cells using RNA interference technology. The DNA DSBs were detected by γ-H2AX foci, neutral comet assay and pulsed-field gel electrophoresis. We found that the capability of DNA DSB repair in Hela-630 cells was significantly reduced, and the repair kinetics of DSB was delayed as compared to the control Hela-NC cells. Silence of c-myc sensitized the cellular sensitivity to ionizing radiation. The phosphorylated c-Myc (Thr58/pSer62) formed the consistent co-localisation foci with γ-H2AX as well as the phosphorylated DNA-PKcs/S2056 in the irradiated cells. Moreover, depression of c-Myc largely attenuated the ionizing radiation-induced phosphorylation of the ataxia telangiectasia mutated (ATM) and decreased the in vitro kinase activity of DNA-PKcs. Taken together, our results demonstrated that c-Myc protein functions in the process of DNA double-strand break repair, at least partially, through affecting the ATM phosphorylation and DNA-PKcs kinase activity. The overexpression of c-Myc in tumours can account for the radioresistance of some tumour cell types.

  9. CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants

    PubMed Central

    Shen, Hexi; Strunks, Gary D.; Klemann, Bart J. P. M.; Hooykaas, Paul J. J.; de Pater, Sylvia

    2016-01-01

    Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants. PMID:27866150

  10. Reduced Activity of Double-Strand Break Repair Genes in Prostate Cancer Patients With Late Normal Tissue Radiation Toxicity

    SciTech Connect

    Oorschot, Bregje van; Hovingh, Suzanne E.; Moerland, Perry D.; Medema, Jan Paul; Stalpers, Lukas J.A.; Vrieling, Harry; Franken, Nicolaas A.P.

    2014-03-01

    Purpose: To investigate clinical parameters and DNA damage response as possible risk factors for radiation toxicity in the setting of prostate cancer. Methods and Materials: Clinical parameters of 61 prostate cancer patients, 34 with (overresponding, OR) and 27 without (non-responding, NR) severe late radiation toxicity were assembled. In addition, for a matched subset the DNA damage repair kinetics (γ-H2AX assay) and expression profiles of DNA repair genes were determined in ex vivo irradiated lymphocytes. Results: Examination of clinical data indicated none of the considered clinical parameters to be correlated with the susceptibility of patients to develop late radiation toxicity. Although frequencies of γ-H2AX foci induced immediately after irradiation were similar (P=.32), significantly higher numbers of γ-H2AX foci were found 24 hours after irradiation in OR compared with NR patients (P=.03). Patient-specific γ-H2AX foci decay ratios were significantly higher in NR patients than in OR patients (P<.0001). Consequently, NR patients seem to repair DNA double-strand breaks (DSBs) more efficiently than OR patients. Moreover, gene expression analysis indicated several genes of the homologous recombination pathway to be stronger induced in NR compared with OR patients (P<.05). A similar trend was observed in genes of the nonhomologous end-joining repair pathway (P=.09). This is congruent with more proficient repair of DNA DSBs in patients without late radiation toxicity. Conclusions: Both gene expression profiling and DNA DSB repair kinetics data imply that less-efficient repair of radiation-induced DSBs may contribute to the development of late normal tissue damage. Induction levels of DSB repair genes (eg, RAD51) may potentially be used to assess the risk for late radiation toxicity.

  11. SFPQ•NONO and XLF function separately and together to promote DNA double-strand break repair via canonical nonhomologous end joining.

    PubMed

    Jaafar, Lahcen; Li, Zhentian; Li, Shuyi; Dynan, William S

    2016-12-06

    A complex of two related mammalian proteins, SFPQ and NONO, promotes DNA double-strand break repair via the canonical nonhomologous end joining (c-NHEJ) pathway. However, its mechanism of action is not fully understood. Here we describe an improved SFPQ•NONO-dependent in vitro end joining assay. We use this system to demonstrate that the SFPQ•NONO complex substitutes in vitro for the core c-NHEJ factor, XLF. Results are consistent with a model where SFPQ•NONO promotes sequence-independent pairing of DNA substrates, albeit in a way that differs in detail from XLF. Although SFPQ•NONO and XLF function redundantly in vitro, shRNA-mediated knockdown experiments indicate that NONO and XLF are both required for efficient end joining and radioresistance in cell-based assays. In addition, knockdown of NONO sensitizes cells to the interstrand crosslinking agent, cisplatin, whereas knockdown of XLF does not, and indeed suppresses the effect of NONO deficiency. These findings suggest that each protein has one or more unique activities, in addition to the DNA pairing revealed in vitro, that contribute to DNA repair in the more complex cellular milieu. The SFPQ•NONO complex contains an RNA binding domain, and prior work has demonstrated diverse roles in RNA metabolism. It is thus plausible that the additional repair function of NONO, revealed in cell-based assays, could involve RNA interaction.

  12. Functions of disordered regions in mammalian early base excision repair proteins

    PubMed Central

    Hegde, Muralidhar L.; Hazra, Tapas K.

    2010-01-01

    Reactive oxygen species, generated endogenously and induced as a toxic response, produce several dozen oxidized or modified bases and/or single-strand breaks in mammalian and other genomes. These lesions are predominantly repaired via the conserved base excision repair (BER) pathway. BER is initiated with excision of oxidized or modified bases by DNA glycosylases leading to formation of abasic (AP) site or strand break at the lesion site. Structural analysis by experimental and modeling approaches shows the presence of a disordered segment commonly localized at the N- or C-terminus as a characteristic signature of mammalian DNA glycosylases which is absent in their bacterial prototypes. Recent studies on unstructured regions in DNA metabolizing proteins have indicated their essential role in interaction with other proteins and target DNA recognition. In this review, we have discussed the unique presence of disordered segments in human DNA glycosylases, and AP endonuclease involved in the processing of glycosylase products, and their critical role in regulating repair functions. These disordered segments also include sites for posttranslational modifications and nuclear localization signal. The teleological basis for their structural flexibility is discussed. PMID:20714778

  13. Purification of mammalian DNA repair protein XRCC1

    SciTech Connect

    Chen, I.

    1995-11-01

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

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

    PubMed

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

    2013-10-01

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

  15. β-HPV 5 and 8 E6 Disrupt Homology Dependent Double Strand Break Repair by Attenuating BRCA1 and BRCA2 Expression and Foci Formation

    PubMed Central

    Wallace, Nicholas A.; Robinson, Kristin; Howie, Heather L.; Galloway, Denise A.

    2015-01-01

    Recent work has explored a putative role for the E6 protein from some β-human papillomavirus genus (β-HPVs) in the development of non-melanoma skin cancers, specifically β-HPV 5 and 8 E6. Because these viruses are not required for tumor maintenance, they are hypothesized to act as co-factors that enhance the mutagenic capacity of UV-exposure by disrupting the repair of the resulting DNA damage. Supporting this proposal, we have previously demonstrated that UV damage signaling is hindered by β-HPV 5 and 8 E6 resulting in an increase in both thymine dimers and UV-induced double strand breaks (DSBs). Here we show that β-HPV 5 and 8 E6 further disrupt the repair of these DSBs and provide a mechanism for this attenuation. By binding and destabilizing a histone acetyltransferase, p300, β-HPV 5 and 8 E6 reduce the enrichment of the transcription factor at the promoter of two genes critical to the homology dependent repair of DSBs (BRCA1 and BRCA2). The resulting diminished BRCA1/2 transcription not only leads to lower protein levels but also curtails the ability of these proteins to form repair foci at DSBs. Using a GFP-based reporter, we confirm that this reduced foci formation leads to significantly diminished homology dependent repair of DSBs. By deleting the p300 binding domain of β-HPV 8 E6, we demonstrate that the loss of robust repair is dependent on viral-mediated degradation of p300 and confirm this observation using a combination of p300 mutants that are β-HPV 8 E6 destabilization resistant and p300 knock-out cells. In conclusion, this work establishes an expanded ability of β-HPV 5 and 8 E6 to attenuate UV damage repair, thus adding further support to the hypothesis that β-HPV infections play a role in skin cancer development by increasing the oncogenic potential of UV exposure. PMID:25803638

  16. Androgen receptor in Sertoli cells regulates DNA double-strand break repair and chromosomal synapsis of spermatocytes partially through intercellular EGF-EGFR signaling.

    PubMed

    Chen, Su-Ren; Hao, Xiao-Xia; Zhang, Yan; Deng, Shou-Long; Wang, Zhi-Peng; Wang, Yu-Qian; Wang, Xiu-Xia; Liu, Yi-Xun

    2016-04-05

    Spermatogenesis does not progress beyond the pachytene stages of meiosis in Sertoli cell-specific AR knockout (SCARKO) mice. However, further evidence of meiotic arrest and underlying paracrine signals in SCARKO testes is still lacking. We utilized co-immunostaining of meiotic surface spreads to examine the key events during meiotic prophase I. SCARKO spermatocytes exhibited a failure in chromosomal synapsis observed by SCP1/SCP3 double-staining and CREST foci quantification. In addition, DNA double-strand breaks (DSBs) were formed but were not repaired in the mutant spermatocytes, as revealed by γ-H2AX staining and DNA-dependent protein kinase (DNA-PK) activity examination. The later stages of DSB repair, such as the accumulation of the RAD51 strand exchange protein and the localization of mismatch repair protein MLH1, were correspondingly altered in SCARKO spermatocytes. Notably, the expression of factors that guide RAD51 loading onto sites of DSBs, including TEX15, BRCA1/2 and PALB2, was severely impaired when either AR was down-regulated or EGF was up-regulated. We observed that some ligands in the epidermal growth factor (EGF) family were over-expressed in SCARKO Sertoli cells and that some receptors in the EGF receptor (EGFR) family were ectopically activated in the mutant spermatocytes. When EGF-EGFR signaling was repressed to approximately normal by the specific inhibitor AG1478 in the cultured SCARKO testis tissues, the arrested meiosis was partially rescued, and functional haploid cells were generated. Based on these data, we propose that AR in Sertoli cells regulates DSB repair and chromosomal synapsis of spermatocytes partially through proper intercellular EGF-EGFR signaling.

  17. Heat effects on DNA repair after ionising radiation: hyperthermia commonly increases the number of non-repaired double-strand breaks and structural rearrangements

    PubMed Central

    El-Awady, R. A.; Dikomey, E.; Dahm-Daphi, J.

    2001-01-01

    After ionising radiation double-strand breaks (dsb) are lethal if not repaired or misrepaired. Cell killing is greatly enhanced by hyperthermia and it is questioned here whether heat not only affects dsb repair capacity but also fidelity in a chromosomal context. dsb repair experiments were designed so as to mainly score non-homologous end joining, while homologous recombination was largely precluded. Human male G0 fibroblasts were either preheated (45°C, 20 min) or not before X-irradiation. dsb induction and repair were measured by conventional gel electrophoresis and an assay combining restriction digestion using a rare cutting enzyme (NotI) and Southern hybridisation, which detects large chromosomal rearrangements (>100 kb). dsb induction rate in an X-chromosomal NotI fragment was 4.8 × 10–3 dsb/Gy/Mb. Similar values were found for the genome overall and also when cells were preheated. After 50 Gy, fibroblasts were competent to largely restore the original restriction fragment size. Five per cent of dsb remained non-rejoined and 14% were misrejoined. Correct restitution of restriction fragments occurred preferably during the first hour but continued at a slow rate for 12–16 h. In addition, dsb appeared to misrejoin throughout the entire repair period. After hyperthermia the fractions of non-rejoined and misrejoined dsb were similarly increased to 13 and 51%, respectively. It is suggested that heat increases the probability of dsb being incorrectly rejoined but it is not likely to interfere with one dsb repair pathway in particular. PMID:11328880

  18. The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.

    PubMed

    Jasin, Maria; Haber, James E

    2016-08-01

    DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution.

  19. Protein damage and repair controlling seed vigor and longevity.

    PubMed

    Ogé, Laurent; Broyart, Caroline; Collet, Boris; Godin, Béatrice; Jallet, Denis; Bourdais, Gildas; Job, Dominique; Grappin, Philippe

    2011-01-01

    The formation of abnormal isoaspartyl residues derived from aspartyl or asparaginyl residues is a major source of spontaneous protein misfolding in cells. The repair enzyme protein L: -isoaspartyl methyltransferase (PIMT) counteracts such damage by catalyzing the conversion of abnormal isoaspartyl residues to their normal aspartyl forms. Thus, this enzyme contributes to the survival of many organisms, including plants. Analysis of the accumulation of isoaspartyl-containing proteins and its modulation by the PIMT repair pathway, using germination tests, immunodetection, enzymatic assays, and HPLC analysis, gives new insights in understanding controlling mechanisms of seed longevity and vigor.

  20. Proteins for breaking barriers in lignocellulosic bioethanol production.

    PubMed

    Ulaganathan, Kandasamy; Goud, Burragoni S; Reddy, Mettu M; Kumar, Vanaparthi P; Balsingh, Jatoth; Radhakrishna, Surabhi

    2015-01-01

    Reduction in fossil fuel consumption by using alternate sources of energy is a major challenge facing mankind in the coming decades. Bioethanol production using lignocellulosic biomass is the most viable option for addressing this challenge. Industrial bioconversion of lignocellulosic biomass, though possible now, is not economically viable due to presence of barriers that escalate the cost of production. As cellulose and hemicellulose are the major constituents of terrestrial biomass, which is available in massive quantities, hydrolysis of cellulose and hemicellulose by the microorganisms are the most prominent biochemical processes happening in the earth. Microorganisms possess different categories of proteins associated with different stages of bioethanol production and a number of them are already found and characterized. Many more of these proteins need to be identified which suit the specificities needed for the bioethanol production process. Discovery of proteins with novel specificities and application of genetic engineering technologies to harvest the synergies existing between them with the aim to develop consolidated bioprocess is the major direction of research in the future. In this review, we discuss the different categories of proteins used for bioethanol production in the context of breaking the barriers existing for the economically feasible lignocellulosic bioethanol production.

  1. Rad5-dependent DNA repair functions of the Saccharomyces cerevisiae FANCM protein homolog Mph1.

    PubMed

    Daee, Danielle L; Ferrari, Elisa; Longerich, Simonne; Zheng, Xiao-feng; Xue, Xiaoyu; Branzei, Dana; Sung, Patrick; Myung, Kyungjae

    2012-08-03

    Interstrand cross-links (ICLs) covalently link complementary DNA strands, block DNA replication, and transcription and must be removed to allow cell survival. Several pathways, including the Fanconi anemia (FA) pathway, can faithfully repair ICLs and maintain genomic integrity; however, the precise mechanisms of most ICL repair processes remain enigmatic. In this study we genetically characterized a conserved yeast ICL repair pathway composed of the yeast homologs (Mph1, Chl1, Mhf1, Mhf2) of four FA proteins (FANCM, FANCJ, MHF1, MHF2). This pathway is epistatic with Rad5-mediated DNA damage bypass and distinct from the ICL repair pathways mediated by Rad18 and Pso2. In addition, consistent with the FANCM role in stabilizing ICL-stalled replication forks, we present evidence that Mph1 prevents ICL-stalled replication forks from collapsing into double-strand breaks. This unique repair function of Mph1 is specific for ICL damage and does not extend to other types of damage. These studies reveal the functional conservation of the FA pathway and validate the yeast model for future studies to further elucidate the mechanism of the FA pathway.

  2. Relative rates of repair of single-strand breaks and postirradiation DNA degradation in normal and induced cells of Escherichia coli.

    PubMed Central

    Pollard, E C; Fugate, J K

    1978-01-01

    Labeled DNA from irradiated Excherichia coli cells has been studied on an alkaline sucrose gradient without acid precipitation of the DNA. This enables the observation of both DNA repair and DNA degradation. The use of a predose of ultraviolet light (UV) causes induction of an inhibitor of postirradiation DNA degradation in lex+ strains. The effect of this induction on both the repair of single-strand breaks and DNA degradation has been followed in strains WU3610 (uvr+) and WU3610-89 (uvr-). The repair process is more rapid than the degradation, and when degradation is inhibited more repair is apparent. Cells that are lex- (Bs-1 and AB2474) cannot be induced for inhibition of degradation. Nevertheless, by observation at short times repair can be seen clearly. This repaired DNA is degraded, suggesting that the signal for DNA degradation is not a single-strand break. PMID:365253

  3. Double-strand break repair and colorectal cancer: gene variants within 3′ UTRs and microRNAs binding as modulators of cancer risk and clinical outcome

    PubMed Central

    Naccarati, Alessio; Rosa, Fabio; Vymetalkova, Veronika; Barone, Elisa; Jiraskova, Katerina; Di Gaetano, Cornelia; Novotny, Jan; Levy, Miroslav; Vodickova, Ludmila; Gemignani, Federica; Buchler, Tomas; Landi, Stefano

    2016-01-01

    Genetic variations in 3′ untranslated regions of target genes may affect microRNA binding, resulting in differential protein expression. microRNAs regulate DNA repair, and single-nucleotide polymorphisms in miRNA binding sites (miRSNPs) may account for interindividual differences in the DNA repair capacity. Our hypothesis is that miRSNPs in relevant DNA repair genes may ultimately affect cancer susceptibility and impact prognosis. In the present study, we analysed the association of polymorphisms in predicted microRNA target sites of double-strand breaks (DSBs) repair genes with colorectal cancer (CRC) risk and clinical outcome. Twenty-one miRSNPs in non-homologous end-joining and homologous recombination pathways were assessed in 1111 cases and 1469 controls. The variant CC genotype of rs2155209 in MRE11A was strongly associated with decreased cancer risk when compared with the other genotypes (OR 0.54, 95% CI 0.38–0.76, p = 0.0004). A reduced expression of the reporter gene was observed for the C allele of this polymorphism by in vitro assay, suggesting a more efficient interaction with potentially binding miRNAs. In colon cancer patients, the rs2155209 CC genotype was associated with shorter survival while the TT genotype of RAD52 rs11226 with longer survival when both compared with their respective more frequent genotypes (HR 1.63, 95% CI 1.06-2.51, p = 0.03 HR 0.60, 95% CI 0.41–0.89, p = 0.01, respectively). miRSNPs in DSB repair genes involved in the maintenance of genomic stability may have a role on CRC susceptibility and clinical outcome. PMID:26735576

  4. Down-Regulation of Rad51 Activity during Meiosis in Yeast Prevents Competition with Dmc1 for Repair of Double-Strand Breaks

    PubMed Central

    Liu, Yan; Gaines, William A.; Callender, Tracy; Busygina, Valeria; Oke, Ashwini; Sung, Patrick; Fung, Jennifer C.; Hollingsworth, Nancy M.

    2014-01-01

    Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a preference for repairing double strand breaks (DSBs) using sister chromatids, in contrast to the conserved, meiosis-specific recombinase, Dmc1, which preferentially repairs programmed DSBs using homologs. Despite the different preferences for repair templates, both Rad51 and Dmc1 are required for interhomolog recombination during meiosis. This paradox has recently been explained by the finding that Rad51 protein, but not its strand exchange activity, promotes Dmc1 function in budding yeast. Rad51 activity is inhibited in dmc1Δ mutants, where the failure to repair meiotic DSBs triggers the meiotic recombination checkpoint, resulting in prophase arrest. The question remains whether inhibition of Rad51 activity is important during wild-type meiosis, or whether inactivation of Rad51 occurs only as a result of the absence of DMC1 or checkpoint activation. This work shows that strains in which mechanisms that down-regulate Rad51 activity are removed exhibit reduced numbers of interhomolog crossovers and noncrossovers. A hypomorphic mutant, dmc1-T159A, makes less stable presynaptic filaments but is still able to mediate strand exchange and interact with accessory factors. Combining dmc1-T159A with up-regulated Rad51 activity reduces interhomolog recombination and spore viability, while increasing intersister joint molecule formation. These results support the idea that down-regulation of Rad51 activity is important during meiosis to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs. PMID:24465215

  5. QUANTITATION OF INTRACELLULAR NAD(P)H IN LIVING CELLS CAN MONITOR AN IMBALANCE OF DNA SINGLE STRAND BREAK REPAIR IN REAL TIME

    EPA Science Inventory

    Quantitation of intracellular NAD(P)H in living cells can monitor an imbalance of DNA single strand break repair in real time.

    ABSTRACT

    DNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or du...

  6. The phosphatase activity of mammalian polynucleotide kinase takes precedence over its kinase activity in repair of single strand breaks.

    PubMed

    Dobson, Caroline J; Allinson, Sarah L

    2006-01-01

    The dual function mammalian DNA repair enzyme, polynucleotide kinase (PNK), facilitates strand break repair through catalysis of 5'-hydroxyl phosphorylation and 3'-phosphate dephosphorylation. We have examined the relative activities of the kinase and phosphatase functions of PNK using a novel assay, which allows the simultaneous characterization of both activities in processing nicks and gaps containing both 3'-phosphate and 5'-hydroxyl. Under multiple turnover conditions the phosphatase activity of the purified enzyme is significantly more active than its kinase activity. Consistent with this result, phosphorylation of the 5'-hydroxyl is rate limiting in cell extract mediated-repair of a nicked substrate. On characterizing the effects of individually mutating the two active sites of PNK we find that while site-directed mutagenesis of the kinase domain of PNK does not affect its phosphatase activity, disruption of the phosphatase domain also abrogates kinase function. This loss of kinase function requires the presence of a 3'-phosphate, but it need not be present in the same strand break as the 5'-hydroxyl. PNK preferentially binds 3'-phosphorylated substrates and DNA binding to the phosphatase domain blocks further DNA binding by the kinase domain.

  7. Mismatch Repair Proteins Are Activators of Toxic Responses to Chromium-DNA Damage

    PubMed Central

    Peterson-Roth, Elizabeth; Reynolds, Mindy; Quievryn, George; Zhitkovich, Anatoly

    2005-01-01

    Chromium(VI) is a toxic and carcinogenic metal that causes the formation of DNA phosphate-based adducts. Cr-DNA adducts are genotoxic in human cells, although they do not block replication in vitro. Here, we report that induction of cytotoxicity in Cr(VI)-treated human colon cells and mouse embryonic fibroblasts requires the presence of all major mismatch repair (MMR) proteins. Cr-DNA adducts lost their ability to block replication of Cr-modified plasmids in human colon cells lacking MLH1 protein. The presence of functional mismatch repair caused induction of p53-independent apoptosis associated with activation of caspases 2 and 7. Processing of Cr-DNA damage by mismatch repair resulted in the extensive formation of γ-H2AX foci in G2 phase, indicating generation of double-stranded breaks as secondary toxic lesions. Induction of γ-H2AX foci was observed at 6 to 12 h postexposure, which was followed by activation of apoptosis in the absence of significant G2 arrest. Our results demonstrate that mismatch repair system triggers toxic responses to Cr-DNA backbone modifications through stress mechanisms that are significantly different from those for other forms of DNA damage. Selection for Cr(VI) resistant, MMR-deficient cells may explain the very high frequency of lung cancers with microsatellite instability among chromate workers. PMID:15831465

  8. New Insights into the Post-Translational Regulation of DNA Damage Response and Double-Strand Break Repair in Caenorhabditis elegans.

    PubMed

    Kim, Hyun-Min; Colaiácovo, Monica P

    2015-06-01

    Although a growing number of studies have reported the importance of SUMOylation in genome maintenance and DNA double-strand break repair (DSBR), relevant target proteins and how this modification regulates their functions are yet to be clarified. Here, we analyzed SUMOylation of ZTF-8, the homolog of mammalian RHINO, to test the functional significance of this protein modification in the DSBR and DNA damage response (DDR) pathways in the Caenorhabditis elegans germline. We found that ZTF-8 is a direct target for SUMOylation in vivo and that its modification is required for DNA damage checkpoint induced apoptosis and DSBR. Non-SUMOylatable mutants of ZTF-8 mimic the phenotypes observed in ztf-8 null mutants, including reduced fertility, impaired DNA damage repair, and defective DNA damage checkpoint activation. However, while mutants for components acting in the SUMOylation pathway fail to properly localize ZTF-8, its localization is not altered in the ZTF-8 non-SUMOylatable mutants. Taken together, these data show that direct SUMOylation of ZTF-8 is required for its function in DSBR as well as DDR but not its localization. ZTF-8's human ortholog is enriched in the germline, but its meiotic role as well as its post-translational modification has never been explored. Therefore, our discovery may assist in understanding the regulatory mechanism of this protein in DSBR and DDR in the germline.

  9. Non-histone chromosomal proteins HMG1 and 2 enhance ligation reaction of DNA double-strand breaks.

    PubMed

    Nagaki, S; Yamamoto, M; Yumoto, Y; Shirakawa, H; Yoshida, M; Teraoka, H

    1998-05-08

    DNA ligase IV in a complex with XRCC4 is responsible for DNA end-joining in repair of DNA double-strand breaks (DSB) and V(D)J recombination. We found that non-histone chromosomal high mobility group (HMG) proteins 1 and 2 enhanced the ligation of linearized pUC119 DNA with DNA ligase IV from rat liver nuclear extract. Intra-molecular and inter-molecular ligations of cohesive-ended and blunt-ended DNA were markedly stimulated by HMG1 and 2. Recombinant HMG2-domain A, B, and (A + B) polypeptides were similarly, but non-identically, effective for the stimulation of DSB ligation reaction. Ligation of single-strand breaks (nicks) was only slightly activated by the HMG proteins. The DNA end-binding Ku protein singly or in combination with the catalytic component of DNA-dependent protein kinase (DNA-PK) as the DNA-PK holoenzyme was ineffective for the ligation of linearized pUC119 DNA. Although the stimulatory effect of HMG1 and 2 on ligation of DSB in vitro was not specific to DNA ligase IV, these results suggest that HMG1 and 2 are involved in the final ligation step in DNA end-joining processes of DSB repair and V(D)J recombination.

  10. Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis

    PubMed Central

    Kwon, Taegun; Huq, Enamul; Herrin, David L.

    2010-01-01

    Chloroplast DNA (cpDNA) is under great photooxidative stress, yet its evolution is very conservative compared with nuclear or mitochondrial genomes. It can be expected that DNA repair mechanisms play important roles in cpDNA survival and evolution, but they are poorly understood. To gain insight into how the most severe form of DNA damage, a double-strand break (DSB), is repaired, we have developed an inducible system in Arabidopsis that employs a psbA intron endonuclease from Chlamydomonas, I-CreII, that is targeted to the chloroplast using the rbcS1 transit peptide. In Chlamydomonas, an I-CreII-induced DSB in psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20–60 bp) abundant in that genome; Arabidopsis cpDNA is very repeat poor, however. Phenotypically strong and weak transgenic lines were examined and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of DNA at the psbA locus, but not cpDNA as a whole, in the strongly expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site indicative of DSB repair using microhomology (6–12 bp perfect repeats, or 10–16 bp with mismatches) and no homology. These results provide evidence of alternative DSB repair pathways in the Arabidopsis chloroplast that resemble the nuclear, microhomology-mediated and nonhomologous end joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, the data suggest an evolutionary relationship may exist between the repeat structure of the genome and the organelle's ability to repair broken chromosomes. PMID:20643920

  11. ATM-mediated phosphorylation of the chromatin remodeling enzyme BRG1 modulates DNA double-strand break repair.

    PubMed

    Kwon, S-J; Park, J-H; Park, E-J; Lee, S-A; Lee, H-S; Kang, S W; Kwon, J

    2015-01-15

    ATP-dependent chromatin remodeling complexes such as SWI/SNF (SWItch/Sucrose NonFermentable) have been implicated in DNA double-strand break (DSB) repair and damage responses. However, the regulatory mechanisms that control the function of chromatin remodelers in DNA damage response are largely unknown. Here, we show that ataxia telangiectasia mutated (ATM) mediates the phosphorylation of BRG1, the catalytic ATPase of the SWI/SNF complex that contributes to DSB repair by binding γ-H2AX-containing nucleosomes via interaction with acetylated histone H3 and stimulating γ-H2AX formation, at Ser-721 in response to DNA damage. ATM-mediated phosphorylation of BRG1 occurs rapidly and transiently after DNA damage. Phosphorylated BRG1 binds γ-H2AX-containing nucleosomes to form the repair foci. The Ser-721 phosphorylation of BRG1 is critical for binding γ-H2AX-containing nucleosomes and stimulating γ-H2AX formation and DSB repair. BRG1 binds to acetylated H3 peptides much better after phosphorylation at Ser-721 by DNA damage. However, the phosphorylation of Ser-721 does not significantly affect the ATPase and transcriptional activities of BRG1. These results, establishing BRG1 as a novel and functional ATM substrate, suggest that the ATM-mediated phosphorylation of BRG1 facilitates DSB repair by stimulating the association of this remodeler with γ-H2AX nucleosomes via enhancing the affinity to acetylated H3. Our work also suggests that the mechanism of BRG1 stimulation of DNA repair is independent of the remodeler's enzymatic or transcriptional activities.

  12. MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end-resection

    PubMed Central

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

    2015-01-01

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

  13. BCL10 is recruited to sites of DNA damage to facilitate DNA double-strand break repair

    PubMed Central

    Ismail, Ismail Hassan; Dronyk, Ashley; Hu, Xiuying; Hendzel, Michael J.; Shaw, Andrew R.

    2016-01-01

    ABSTRACT Recent studies have found BCL10 can localize to the nucleus and that this is linked to tumor aggression and poorer prognosis. These studies suggest that BCL10 localization plays a novel role in the nucleus that may contribute to cellular transformation and carcinogenesis. In this study, we show that BCL10 functions as part of the DNA damage response (DDR). We found that BCL10 facilitates the rapid recruitment of RPA, BRCA1 and RAD51 to sites of DNA damage. Furthermore, we also found that ATM phosphorylates BCL10 in response to DNA damage. Functionally, BCL10 promoted DNA double-strand breaks repair, enhancing cell survival after DNA damage. Taken together our results suggest a novel role for BCL10 in the repair of DNA lesions. PMID:26771713

  14. Sodium azide-induced DNA single-strand breaks and DNA-protein crosslinks in barley embryos

    SciTech Connect

    Veleminsky, J.; Kleinhofs, A.; Nilan, R.A.

    1982-01-01

    Barley (Hordeum vulgare L., cv. Himalaya) embryos germinated for two days in sterile culture were exposed to 5 mM and 10 mM NaN/sub 3/ in pH 3 buffer for 2 h. (/sup 3/H) DNA from the isolated nuclei was analyzed by alkaline elution from PVC filters. DNA single-strand breaks and DNA-protein crosslinks, detected by proteinase K digestion, were detected both immediately and 24 h after the azide treatment. Repair of these lesions during 24 h of posttreatment incubation of embryos in nutrient medium was not observed.

  15. Double strand breaks in DNA inhibit nucleotide excision repair in vitro.

    PubMed

    Calsou, P; Frit, P; Salles, B

    1996-11-01

    Nucleotide excision repair (NER) was measured in human cell extracts incubated with either supercoiled or linearized damaged plasmid DNA as repair substrate. NER, as quantified by the extent of repair synthesis activity, was reduced by up to 80% in the case of linearized plasmid DNA when compared with supercoiled DNA. An excess of undamaged linearized plasmid in the repair mixture did not interfere with DNA repair synthesis activity on a supercoiled damaged plasmid, indicating a cis-acting inhibiting effect. In contrast, gaps on circular or linearized plasmids were filled in identically by the DNA polymerases operating in the extracts. When the extent of damage-dependent incision activity was measured, a approximately 70% reduction of repair incision activity by human cell extract was observed on linearized damaged plasmids. Recessed, protruding, or blunt ends were similarly inhibitory. NER activity was partly restored when the extracts were preincubated with autoimmune human sera containing antibodies against the nuclear DNA end-binding heterodimer Ku. In addition, the inhibition of repair activity on linear damaged plasmids was released in extracts from rodent cells deficient in Ku activity but not in extracts from murine scid cells devoid of Ku-associated DNA-dependent kinase activity.

  16. HYDROGEL-BASED NANOCOMPOSITES OF THERAPEUTIC PROTEINS FOR TISSUE REPAIR

    PubMed Central

    Zhu, Suwei; Segura, Tatiana

    2014-01-01

    The ability to design artificial extracellular matrices as cell instructive scaffolds has opened the door to technologies capable of studying cell fates in vitro and to guide tissue repair in vivo. One main component of the design of artificial extracellular matrices is the incorporation of protein-based biochemical cues to guide cell phenotypes and multicellular organizations. However, promoting the long-term bioactivity, controlling the bioavailability and understanding how the physical presentations of these proteins impacts cellular fates are among the challenges of the field. Nanotechnolgy has advanced to meet the challenges of protein therapeutics. For example, the approaches to incorporating proteins into tissue repairing scaffolds have ranged from bulk encapsulations to smart nanodepots that protect proteins from degradations and allow opportunities for controlled release. PMID:24778979

  17. HYDROGEL-BASED NANOCOMPOSITES OF THERAPEUTIC PROTEINS FOR TISSUE REPAIR.

    PubMed

    Zhu, Suwei; Segura, Tatiana

    2014-05-01

    The ability to design artificial extracellular matrices as cell instructive scaffolds has opened the door to technologies capable of studying cell fates in vitro and to guide tissue repair in vivo. One main component of the design of artificial extracellular matrices is the incorporation of protein-based biochemical cues to guide cell phenotypes and multicellular organizations. However, promoting the long-term bioactivity, controlling the bioavailability and understanding how the physical presentations of these proteins impacts cellular fates are among the challenges of the field. Nanotechnolgy has advanced to meet the challenges of protein therapeutics. For example, the approaches to incorporating proteins into tissue repairing scaffolds have ranged from bulk encapsulations to smart nanodepots that protect proteins from degradations and allow opportunities for controlled release.

  18. Identification and Analysis of MS5(d): A Gene That Affects Double-Strand Break (DSB) Repair during Meiosis I in Brassica napus Microsporocytes.

    PubMed

    Zeng, Xinhua; Yan, Xiaohong; Yuan, Rong; Li, Keqi; Wu, Yuhua; Liu, Fang; Luo, Junling; Li, Jun; Wu, Gang

    2016-01-01

    Here, we report the identification of the Brassica-specific gene MS5(d), which is responsible for male sterility in Brassica napus. The MS5(d) gene is highly expressed in the microsporocyte and encodes a protein that localizes to the nucleus. Light microscopy analyses have demonstrated that the MS5(d) gene affects microsporocyte meiosis in the thermosensitive genic male sterility line TE5A. Sequence comparisons and genetic complementation revealed a C-to-T transition in MS5(d), encoding a Leu-to-Phe (L281F) substitution and causing abnormal male meiosis in TE5A. These findings suggest arrested meiotic chromosome dynamics at pachytene. Furthermore, immunofluorescence analyses showed that double-strand break (DSB) formation and axial elements were normal but that DSB repair and spindle behavior were aberrant in TE5A meiocytes. Collectively, our results indicate that MS5(d) likely encodes a protein required for chromosomal DSB repair at early stages of meiosis in B. napus.

  19. Identification and Analysis of MS5d: A Gene That Affects Double-Strand Break (DSB) Repair during Meiosis I in Brassica napus Microsporocytes

    PubMed Central

    Zeng, Xinhua; Yan, Xiaohong; Yuan, Rong; Li, Keqi; Wu, Yuhua; Liu, Fang; Luo, Junling; Li, Jun; Wu, Gang

    2017-01-01

    Here, we report the identification of the Brassica-specific gene MS5d, which is responsible for male sterility in Brassica napus. The MS5d gene is highly expressed in the microsporocyte and encodes a protein that localizes to the nucleus. Light microscopy analyses have demonstrated that the MS5d gene affects microsporocyte meiosis in the thermosensitive genic male sterility line TE5A. Sequence comparisons and genetic complementation revealed a C-to-T transition in MS5d, encoding a Leu-to-Phe (L281F) substitution and causing abnormal male meiosis in TE5A. These findings suggest arrested meiotic chromosome dynamics at pachytene. Furthermore, immunofluorescence analyses showed that double-strand break (DSB) formation and axial elements were normal but that DSB repair and spindle behavior were aberrant in TE5A meiocytes. Collectively, our results indicate that MS5d likely encodes a protein required for chromosomal DSB repair at early stages of meiosis in B. napus. PMID:28101089

  20. Collision of Trapped Topoisomerase 2 with Transcription and Replication: Generation and Repair of DNA Double-Strand Breaks with 5′ Adducts

    PubMed Central

    Yan, Hong; Tammaro, Margaret; Liao, Shuren

    2016-01-01

    Topoisomerase 2 (Top2) is an essential enzyme responsible for manipulating DNA topology during replication, transcription, chromosome organization and chromosome segregation. It acts by nicking both strands of DNA and then passes another DNA molecule through the break. The 5′ end of each nick is covalently linked to the tyrosine in the active center of each of the two subunits of Top2 (Top2cc). In this configuration, the two sides of the nicked DNA are held together by the strong protein-protein interactions between the two subunits of Top2, allowing the nicks to be faithfully resealed in situ. Top2ccs are normally transient, but can be trapped by cancer drugs, such as etoposide, and subsequently processed into DSBs in cells. If not properly repaired, these DSBs would lead to genome instability and cell death. Here, I review the current understanding of the mechanisms by which DSBs are induced by etoposide, the unique features of such DSBs and how they are repaired. Implications for the improvement of cancer therapy will be discussed. PMID:27376333

  1. The Exonuclease Homolog OsRAD1 Promotes Accurate Meiotic Double-Strand Break Repair by Suppressing Nonhomologous End Joining1[OPEN

    PubMed Central

    Tang, Ding; Shen, Yi; Chen, Xiaojun; Ji, Jianhui; Du, Guijie; Li, Yafei; Cheng, Zhukuan

    2016-01-01

    During meiosis, programmed double-strand breaks (DSBs) are generated to initiate homologous recombination, which is crucial for faithful chromosome segregation. In yeast, Radiation sensitive1 (RAD1) acts together with Radiation sensitive9 (RAD9) and Hydroxyurea sensitive1 (HUS1) to facilitate meiotic recombination via cell-cycle checkpoint control. However, little is known about the meiotic functions of these proteins in higher eukaryotes. Here, we characterized a RAD1 homolog in rice (Oryza sativa) and obtained evidence that O. sativa RAD1 (OsRAD1) is important for meiotic DSB repair. Loss of OsRAD1 led to abnormal chromosome association and fragmentation upon completion of homologous pairing and synapsis. These aberrant chromosome associations were independent of OsDMC1. We found that classical nonhomologous end-joining mediated by Ku70 accounted for most of the ectopic associations in Osrad1. In addition, OsRAD1 interacts directly with OsHUS1 and OsRAD9, suggesting that these proteins act as a complex to promote DSB repair during rice meiosis. Together, these findings suggest that the 9-1-1 complex facilitates accurate meiotic recombination by suppressing nonhomologous end-joining during meiosis in rice. PMID:27512017

  2. Methods to alter levels of a DNA repair protein

    DOEpatents

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

    2006-10-17

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

  3. Increased DNA double-strand break was associated with downregulation of repair and upregulation of apoptotic factors in rat hippocampus after alcohol exposure.

    PubMed

    Suman, Shubhankar; Kumar, Santosh; N'Gouemo, Prosper; Datta, Kamal

    2016-08-01

    Binge drinking is known to cause damage in critical areas of the brain, including the hippocampus, which is important for relational memory and is reported to be sensitive to alcohol toxicity. However, the roles of DNA double-strand break (DSB) and its repair pathways, homologous recombination (HR), and non-homologous end joining (NHEJ) in alcohol-induced hippocampal injury remain to be elucidated. The purpose of this first study was to assess alcohol-induced DNA DSB and the mechanism by which alcohol affects DSB repair pathways in rat hippocampus. Male Sprague-Dawley rats (8-10 weeks old) were put on a 4-day binge ethanol treatment regimen. Control animals were maintained under similar conditions but were given the vehicle without ethanol. All animals were humanely euthanized 24 h after the last dose of ethanol administration and the hippocampi were dissected for immunoblot and immunohistochemistry analysis. Ethanol exposure caused increased 4-hydroxynonenal (4-HNE) staining as well as elevated γH2AX and 53BP1 foci in hippocampal cells. Immunoblot analysis showed decreased Mre11, Rad51, Rad50, and Ku86 as well as increased Bax and p21 in samples from ethanol-treated rats. Additionally, we also observed increased activated caspase3 staining in hippocampal cells 24 h after ethanol withdrawal. Taken together, our data demonstrated that ethanol concurrently induced DNA DSB, downregulated DSB repair pathway proteins, and increased apoptotic factors in hippocampal cells. We believe these findings will provide the impetus for further research on DNA DSB and its repair pathways in relation to alcohol toxicity in brain.

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

    PubMed

    Srivastava, Niloo; Raman, Mercy J

    2007-01-01

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

  5. Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks.

    PubMed

    Vriend, Lianne E M; Prakash, Rohit; Chen, Chun-Chin; Vanoli, Fabio; Cavallo, Francesca; Zhang, Yu; Jasin, Maria; Krawczyk, Przemek M

    2016-06-20

    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 ((nick)HR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided nuclease Cas9. However, the mechanisms of (nick)HR are largely unexplored. Here, we applied Cas9 nickases to study (nick)HR in mammalian cells. We find that (nick)HR 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.

  6. 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

  7. RecBCD is required to complete chromosomal replication: Implications for double-strand break frequencies and repair mechanisms.

    PubMed

    Courcelle, Justin; Wendel, Brian M; Livingstone, Dena D; Courcelle, Charmain T

    2015-08-01

    Several aspects of the mechanism of homologous double-strand break repair remain unclear. Although intensive efforts have focused on how recombination reactions initiate, far less is known about the molecular events that follow. Based upon biochemical studies, current models propose that RecBCD processes double-strand ends and loads RecA to initiate recombinational repair. However, recent studies have shown that RecBCD plays a critical role in completing replication events on the chromosome through a mechanism that does not involve RecA or recombination. Here, we examine several studies, both early and recent, that suggest RecBCD also operates late in the recombination process - after initiation, strand invasion, and crossover resolution have occurred. Similar to its role in completing replication, we propose a model in which RecBCD is required to resect and resolve the DNA synthesis associated with homologous recombination at the point where the missing sequences on the broken molecule have been restored. We explain how the impaired ability to complete chromosome replication in recBC and recD mutants is likely to account for the loss of viability and genome instability in these mutants, and conclude that spontaneous double-strand breaks and replication fork collapse occur far less frequently than previously speculated.

  8. UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages

    PubMed Central

    Greinert, R.; Volkmer, B.; Henning, S.; Breitbart, E. W.; Greulich, K. O.; Cardoso, M. C.; Rapp, Alexander

    2012-01-01

    UVA (320–400 nm) represents the main spectral component of solar UV radiation, induces pre-mutagenic DNA lesions and is classified as Class I carcinogen. Recently, discussion arose whether UVA induces DNA double-strand breaks (dsbs). Only few reports link the induction of dsbs to UVA exposure and the underlying mechanisms are poorly understood. Using the Comet-assay and γH2AX as markers for dsb formation, we demonstrate the dose-dependent dsb induction by UVA in G1-synchronized human keratinocytes (HaCaT) and primary human skin fibroblasts. The number of γH2AX foci increases when a UVA dose is applied in fractions (split dose), with a 2-h recovery period between fractions. The presence of the anti-oxidant Naringin reduces dsb formation significantly. Using an FPG-modified Comet-assay as well as warm and cold repair incubation, we show that dsbs arise partially during repair of bi-stranded, oxidative, clustered DNA lesions. We also demonstrate that on stretched chromatin fibres, 8-oxo-G and abasic sites occur in clusters. This suggests a replication-independent formation of UVA-induced dsbs through clustered single-strand breaks via locally generated reactive oxygen species. Since UVA is the main component of solar UV exposure and is used for artificial UV exposure, our results shine new light on the aetiology of skin cancer. PMID:22941639

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

    PubMed

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

    2014-11-01

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

  10. The power of DNA double-strand break (DSB) repair testing to predict breast cancer susceptibility.

    PubMed

    Keimling, Marlen; Deniz, Miriam; Varga, Dominic; Stahl, Andreea; Schrezenmeier, Hubert; Kreienberg, Rolf; Hoffmann, Isabell; König, Jochem; Wiesmüller, Lisa

    2012-05-01

    Most presently known breast cancer susceptibility genes have been linked to DSB repair. To identify novel markers that may serve as indicators for breast cancer risk, we performed DSB repair analyses using a case-control design. Thus, we examined 35 women with defined familial history of breast and/or ovarian cancer (first case group), 175 patients with breast cancer (second case group), and 245 healthy women without previous cancer or family history of breast cancer (control group). We analyzed DSB repair in peripheral blood lymphocytes (PBLs) by a GFP-based test system using 3 pathway-specific substrates. We found increases of microhomology-mediated nonhomologous end joining (mmNHEJ) and nonconservative single-strand annealing (SSA) in women with familial risk vs. controls (P=0.0001-0.0022) and patients with breast cancer vs. controls (P=0.0004-0.0042). Young age (<50) at initial diagnosis of breast cancer, which could be indicative of genetic predisposition, was associated with elevated SSA using two different substrates, amounting to similar odds ratios (ORs=2.54-4.46, P=0.0059-0.0095) as for familial risk (ORs=2.61-4.05, P=0.0007-0.0045). These findings and supporting validation data underscore the great potential of detecting distinct DSB repair activities in PBLs as method to estimate breast cancer susceptibility beyond limitations of genotyping and to predict responsiveness to therapeutics targeting DSB repair-dysfunctional tumors.

  11. TRIM proteins in therapeutic membrane repair of muscular dystrophy.

    PubMed

    Alloush, Jenna; Weisleder, Noah

    2013-07-01

    Muscular dystrophy represents a major unmet medical need; only palliative treatments exist for this group of debilitating diseases. Because multiple forms of muscular dystrophy arise from compromised sarcolemmal membrane integrity, a therapeutic approach that can target this loss of membrane function could be applicable to a number of these distinct diseases.One promising therapeutic approach involves the process the cell uses to repair injuries to the plasma membrane. Recent discoveries of genes associated with the membrane repair process provide an opportunity to promote this process as a way to treat muscular dystrophy. One such gene is mitsugumin 53 (MG53), a member of the tripartite motif (TRIM) family of proteins (TRIM72), which is an essential component of the membrane repair pathway in muscle. Recent results indicate that MG53/TRIM72 protein can be directly applied as a therapeutic agent to increase membrane repair capacity of many cell types and treat some aspects of the disease in mouse models of muscular dystrophy. There is great potential for the use of recombinant human MG53 in treating muscular dystrophy and other diseases in which compromised membrane integrity contributes to the disease. Other TRIM family proteins may provide additional targets for therapeutic intervention in similar disease states.

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

    PubMed

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

    2015-02-07

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

  13. The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    PubMed Central

    Liao, Shuren; Tammaro, Margaret; Yan, Hong

    2016-01-01

    The key event in the choice of repair pathways for DNA double-strand breaks (DSBs) is the initial processing of ends. Non-homologous end joining (NHEJ) involves limited processing, but homology-dependent repair (HDR) requires extensive resection of the 5′ strand. How cells decide if an end is channeled to resection or NHEJ is not well understood. We hypothesize that the structure of ends is a major determinant and tested this hypothesis with model DNA substrates in Xenopus egg extracts. While ends with normal nucleotides are efficiently channeled to NHEJ, ends with damaged nucleotides or bulky adducts are channeled to resection. Resection is dependent on Mre11, but its nuclease activity is critical only for ends with 5′ bulky adducts. CtIP is absolutely required for activating the nuclease-dependent mechanism of Mre11 but not the nuclease-independent mechanism. Together, these findings suggest that the structure of ends is a major determinant for the pathway choice of DSB repair and the Mre11 nuclease dependency of resection. PMID:27084932

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

    SciTech Connect

    Protic, M.

    1994-12-31

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

  15. PARP activation regulates the RNA-binding protein NONO in the DNA damage response to DNA double-strand breaks.

    PubMed

    Krietsch, Jana; Caron, Marie-Christine; Gagné, Jean-Philippe; Ethier, Chantal; Vignard, Julien; Vincent, Michel; Rouleau, Michèle; Hendzel, Michael J; Poirier, Guy G; Masson, Jean-Yves

    2012-11-01

    After the generation of DNA double-strand breaks (DSBs), poly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins to be recruited and activated through its binding to the free DNA ends. Upon activation, PARP-1 uses NAD+ to generate large amounts of poly(ADP-ribose) (PAR), which facilitates the recruitment of DNA repair factors. Here, we identify the RNA-binding protein NONO, a partner protein of SFPQ, as a novel PAR-binding protein. The protein motif being primarily responsible for PAR-binding is the RNA recognition motif 1 (RRM1), which is also crucial for RNA-binding, highlighting a competition between RNA and PAR as they share the same binding site. Strikingly, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR, generated by activated PARP-1. Furthermore, we show that upon PAR-dependent recruitment, NONO stimulates nonhomologous end joining (NHEJ) and represses homologous recombination (HR) in vivo. Our results therefore place NONO after PARP activation in the context of DNA DSB repair pathway decision. Understanding the mechanism of action of proteins that act in the same pathway as PARP-1 is crucial to shed more light onto the effect of interference on PAR-mediated pathways with PARP inhibitors, which have already reached phase III clinical trials but are until date poorly understood.

  16. Investigation of the repair of single-strand breaks in human DNA using alkaline gel electrophoresis

    SciTech Connect

    Kovacs, E.; Langemann, H. )

    1990-11-01

    Unstimulated lymphocytes from eight healthy persons were exposed to 10-, 30-, and 100-Gy doses of 60Co gamma radiation. The repair of damaged DNA was measured by (1) alkaline gel electrophoresis (extracted DNA loaded on 0.25% agarose gel, run at 1 V/cm for 39-44 h) at 0, 1, and 2 h after exposure and (2) incorporation of (3H)thymidine into unstimulated lymphocytes in the presence of 2 mM hydroxyurea 1 and 2 h after exposure. Both methods--alkaline gel electrophoresis and thymidine incorporation--showed that repair was completed within 2 h.

  17. Biomolecular Simulation of Base Excision Repair and Protein Signaling

    SciTech Connect

    Straatsma, TP; McCammon, J A; Miller, John H; Smith, Paul E; Vorpagel, Erich R; Wong, Chung F; Zacharias, Martin W

    2006-03-03

    The goal of the Biomolecular Simulation of Base Excision Repair and Protein Signaling project is to enhance our understanding of the mechanism of human polymerase-β, one of the key enzymes in base excision repair (BER) and the cell-signaling enzymes cyclic-AMP-dependent protein kinase. This work used molecular modeling and simulation studies to specifically focus on the • dynamics of DNA and damaged DNA • dynamics and energetics of base flipping in DNA • mechanism and fidelity of nucleotide insertion by BER enzyme human polymerase-β • mechanism and inhibitor design for cyclic-AMP-dependent protein kinase. Molecular dynamics simulations and electronic structure calculations have been performed using the computer resources at the Molecular Science Computing Facility at the Environmental Molecular Sciences Laboratory.

  18. A Novel Way Of Repair Of Insulation Breaks During Pacemaker Generator Replacement

    PubMed Central

    Manzoor Ali, Syed; Iqbal, Khurshid; Tramboo, Nisar A; Lone, Aijaz A; Kaul, Suresh; Kaul, Neelam; Hafiz, Imran

    2009-01-01

    Minor abrasions can occur while mobilising old lead during pacemaker generator replacement necesittating placement of additional lead adding to the financial burden and junk in heart. We describe a novel way of repair of old pacemaker lead preventing additional lead placement. PMID:19763196

  19. Stress-induced condensation of bacterial genomes results in re-pairing of sister chromosomes: implications for double strand DNA break repair.

    PubMed

    Shechter, Nelia; Zaltzman, Liron; Weiner, Allon; Brumfeld, Vlad; Shimoni, Eyal; Fridmann-Sirkis, Yael; Minsky, Abraham

    2013-08-30

    Genome condensation is increasingly recognized as a generic stress response in bacteria. To better understand the physiological implications of this response, we used fluorescent markers to locate specific sites on Escherichia coli chromosomes following exposure to cytotoxic stress. We find that stress-induced condensation proceeds through a nonrandom, zipper-like convergence of sister chromosomes, which is proposed to rely on the recently demonstrated intrinsic ability of identical double-stranded DNA molecules to specifically identify each other. We further show that this convergence culminates in spatial proximity of homologous sites throughout chromosome arms. We suggest that the resulting apposition of homologous sites can explain how repair of double strand DNA breaks might occur in a mechanism that is independent of the widely accepted yet physiologically improbable genome-wide search for homologous templates. We claim that by inducing genome condensation and orderly convergence of sister chromosomes, diverse stress conditions prime bacteria to effectively cope with severe DNA lesions such as double strand DNA breaks.

  20. Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes

    PubMed Central

    Chakraborty, Anirban; Tapryal, Nisha; Venkova, Tatiana; Horikoshi, Nobuo; Pandita, Raj K.; Sarker, Altaf H.; Sarkar, Partha S.; Pandita, Tej K.; Hazra, Tapas K.

    2016-01-01

    DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome. PMID:27703167

  1. CFTR protein repair therapy in cystic fibrosis.

    PubMed

    Quintana-Gallego, Esther; Delgado-Pecellín, Isabel; Calero Acuña, Carmen

    2014-04-01

    Cystic fibrosis is a single gene, autosomal recessive disorder, in which more than 1,900 mutations grouped into 6 classes have been described. It is an example a disease that could be well placed to benefit from personalised medicine. There are currently 2 very different approaches that aim to correct the basic defect: gene therapy, aimed at correcting the genetic alteration, and therapy aimed at correcting the defect in the CFTR protein. The latter is beginning to show promising results, with several molecules under development. Ataluren (PTC124) is a molecule designed to make the ribosomes become less sensitive to the premature stop codons responsible for class i mutations. Lumacaftor (VX-809) is a CFTR corrector directed at class ii mutations, among which Phe508del is the most frequent, with encouraging results. Ivacaftor (VX-770) is a potentiator, the only one marketed to date, which has shown good efficacy for the class iii mutation Gly551Asp in children over the age of 6 and adults. These drugs, or a combination of them, are currently undergoing various clinical trials for other less common genetic mutations. In the last 5 years, CFTR has been designated as a therapeutic target. Ivacaftor is the first drug to treat the basic defect in cystic fibrosis, but only provides a response in a small number of patients. New drugs capable of restoring the CFTR protein damaged by the most common mutations are required.

  2. Escherichia coli radD (yejH) gene: a novel function involved in radiation resistance and double-strand break repair.

    PubMed

    Chen, Stefanie H; Byrne, Rose T; Wood, Elizabeth A; Cox, Michael M

    2015-03-01

    A transposon insertion screen implicated the yejH gene in the repair of ionizing radiation-induced damage. The yejH gene, which exhibits significant homology to the human transcription-coupled DNA repair gene XPB, is involved in the repair of double-strand DNA breaks. Deletion of yejH significantly sensitized cells to agents that cause double-strand breaks (ionizing radiation, UV radiation, ciprofloxacin). In addition, deletion of both yejH and radA hypersensitized the cells to ionizing radiation, UV and ciprofloxacin damage, indicating that these two genes have complementary repair functions. The ΔyejH ΔradA double deletion also showed a substantial decline in viability following an induced double-strand DNA break, of a magnitude comparable with the defect measured when the recA, recB, recG or priA genes are deleted. The ATPase activity and C-terminal zinc finger motif of yejH play an important role in its repair function, as targeted mutant alleles of yejH did not rescue sensitivity. We propose that yejH be renamed radD, reflecting its role in the DNA repair of radiation damage.

  3. Genes Involved in DNA Double-Strand Break Repair: Implications for Breast Cancer.

    DTIC Science & Technology

    1996-10-01

    locus . The disruption of one of 3 the two Ku alleles might lead to a phenotype, since Chinese hamster ovary cells are functionally...significant in-frame deletions. This is the substantial part of the genome in Chinese hamster cell lines is first direct evidence that mutant cells in group 5...Department of the Army, by grant A135763 from DNA repair gene in Chinese hamster ovary cells . Mol. Cell . Biol. 6:2944- the National

  4. Artemis is required to improve the accuracy of repair of double-strand breaks with 5'-blocked termini generated from non-DSB-clustered lesions.

    PubMed

    Malyarchuk, Svitlana; Castore, Reneau; Shi, Runhua; Harrison, Lynn

    2013-05-01

    Clustered DNA lesions are defined as ≥2 damage events within 20 bp. Oxidised bases, abasic (AP) sites, single-strand breaks and double-strand breaks (DSBs) exist in radiation-induced clusters, and these lesions are more difficult to repair and can be more mutagenic than single lesions. Understanding clustered lesion repair is therefore important for the design of complementary treatments to enhance radiotherapy. Non-DSB-clustered lesions consisting of opposing AP sites can be converted to DSBs by base excision repair, and non-homologous end-joining (NHEJ) plays a role in repairing these DSBs. Artemis is an endonuclease that removes blocking groups from DSB termini during NHEJ. Hence, we hypothesised that Artemis plays a role in the processing of DSBs or complex DSBs generated from non-DSB-clustered lesions. We examined the repair of clusters containing two or three lesions in wild-type (WT) or Artemis-deficient (ART(-/-)) mouse fibroblasts using a reporter plasmid. Each cluster contained two opposing tetrahydrofurans (an AP site analogue), which AP endonuclease can convert to a DSB with blocked 5' termini. Loss of Artemis did not decrease plasmid survival, but did result in more mutagenic repair with plasmids containing larger deletions. This increase in deletions did not occur with ClaI-linearised plasmid. Since Mre11 has been implicated in deletional NHEJ, we used small interfering RNA to reduce Mre11 in WT and ART(-/-) cells, but decreasing Mre11 did not change the size of deletions in the repair products. This work implicates Artemis in limiting the deletions introduced during repair of 5'-blocked termini DSBs generated from non-DSB-clustered lesions. Decreasing repair accuracy without decreasing repair capacity could result in mutated cells surviving irradiation. Inhibiting Artemis in normal cells could promote carcinogenesis, while in tumour cells enhanced mutagenic repair following irradiation could promote tumour recurrence.

  5. Nontoxic concentration of DNA-PK inhibitor NU7441 radio-sensitizes lung tumor cells with little effect on double strand break repair.

    PubMed

    Sunada, Shigeaki; Kanai, Hideki; Lee, Younghyun; Yasuda, Takeshi; Hirakawa, Hirokazu; Liu, Cuihua; Fujimori, Akira; Uesaka, Mitsuru; Okayasu, Ryuichi

    2016-09-01

    High-linear energy transfer (LET) heavy ions have been increasingly employed as a useful alternative to conventional photon radiotherapy. As recent studies suggested that high LET radiation mainly affects the nonhomologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair, we further investigated this concept by evaluating the combined effect of an NHEJ inhibitor (NU7441) at a non-toxic concentration and carbon ions. NU7441-treated non-small cell lung cancer (NSCLC) A549 and H1299 cells were irradiated with X-rays and carbon ions (290 MeV/n, 50 keV/μm). Cell survival was measured by clonogenic assay. DNA DSB repair, cell cycle distribution, DNA fragmentation and cellular senescence induction were studied using a flow cytometer. Senescence-associated protein p21 was detected by western blotting. In the present study, 0.3 μM of NU7441, nontoxic to both normal and tumor cells, caused a significant radio-sensitization in tumor cells exposed to X-rays and carbon ions. This concentration did not seem to cause inhibition of DNA DSB repair but induced a significant G2/M arrest, which was particularly emphasized in p53-null H1299 cells treated with NU7441 and carbon ions. In addition, the combined treatment induced more DNA fragmentation and a higher degree of senescence in H1299 cells than in A549 cells, indicating that DNA-PK inhibitor contributes to various modes of cell death in a p53-dependent manner. In summary, NSCLC cells irradiated with carbon ions were radio-sensitized by a low concentration of DNA-PK inhibitor NU7441 through a strong G2/M cell cycle arrest. Our findings may contribute to further effective radiotherapy using heavy ions.

  6. SPR-5 is a histone H3K4 demethylase with a role in meiotic double-strand break repair.

    PubMed

    Nottke, Amanda C; Beese-Sims, Sara E; Pantalena, Luiz F; Reinke, Valerie; Shi, Yang; Colaiácovo, Monica P

    2011-08-02

    Regulation of histone methylation levels has long been implicated in multiple cellular processes, many of which involve transcription. Here, however, we report a unique role for the Caenorhabditis elegans histone demethylase SPR-5 in meiotic DNA double-strand break repair (DSBR). SPR-5 shows enzymatic activity toward H3K4me2 both in vitro and in the nematode germline, and spr-5 mutants show several phenotypes indicating a perturbation of DSBR, including increased p53-dependent germ cell apoptosis, increased levels of the DSBR marker RAD-51, and sensitivity toward DSB-inducing treatments. spr-5 mutants show no transcriptional misregulation of known DSBR involved genes. Instead, SPR-5 shows a rapid subcellular relocalization upon DSB-inducing treatment, which suggests that SPR-5 may function directly in DSBR.

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

    PubMed

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

    2007-10-12

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

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

    PubMed

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

    2012-02-01

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

  9. 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.

  10. Elastin-like protein matrix reinforced with collagen microfibers for soft tissue repair

    PubMed Central

    Caves, Jeffrey M.; Cui, Wanxing; Wen, Jing; Kumar, Vivek A.; Haller, Carolyn A.; Chaikof, Elliot L.

    2011-01-01

    Artificial composites designed to mimic the structure and properties of native extracellular matrix may lead to acellular materials for soft tissue repair and replacement, which display mechanical strength, stiffness, and resilience resembling native tissue. We describe the fabrication of thin lamellae consisting of continuous collagen microfiber embedded at controlled orientations and densities in a recombinant elastin-like protein polymer matrix. Multilamellar stacking affords flexible, protein-based composite sheets whose properties are dependent upon both the elastomeric matrix and collagen content and organization. Sheets are produced with properties that range over 13-fold in elongation to break (23 – 314%), six-fold in Young’s modulus (5.3 to 33.1 MPa), and more than two-fold in tensile strength (1.85 to 4.08 MPa), exceeding that of a number of native human tissues, including urinary bladder, pulmonary artery, and aorta. A sheet approximating the mechanical response of human abdominal wall fascia is investigated as a fascial substitute for ventral hernia repair. Protein-based composite patches prevent hernia recurrence in Wistar rats over an 8-week period with new tissue formation and sustained structural integrity. PMID:21550111

  11. Distinct roles for the RSC and Swi/Snf ATP-dependent chromatin remodelers in DNA double-strand break repair.

    PubMed

    Chai, Bob; Huang, Jian; Cairns, Bradley R; Laurent, Brehon C

    2005-07-15

    The failure of cells to repair damaged DNA can result in genomic instability and cancer. To efficiently repair chromosomal DNA lesions, the repair machinery must gain access to the damaged DNA in the context of chromatin. Here we report that both the RSC and Swi/Snf ATP-dependent chromatin-remodeling complexes play key roles in double-strand break (DSB) repair, specifically by homologous recombination (HR). RSC and Swi/Snf are each recruited to an in vivo DSB site but with distinct kinetics. We show that Swi/Snf is required earlier, at or preceding the strand invasion step of HR, while RSC is required following synapsis for completion of the recombinational repair event.

  12. Mitosis, double strand break repair, and telomeres: a view from the end: how telomeres and the DNA damage response cooperate during mitosis to maintain genome stability.

    PubMed

    Cesare, Anthony J

    2014-11-01

    Double strand break (DSB) repair is suppressed during mitosis because RNF8 and downstream DNA damage response (DDR) factors, including 53BP1, do not localize to mitotic chromatin. Discovery of the mitotic kinase-dependent mechanism that inhibits DSB repair during cell division was recently reported. It was shown that restoring mitotic DSB repair was detrimental, resulting in repair dependent genome instability and covalent telomere fusions. The telomere DDR that occurs naturally during cellular aging and in cancer is known to be refractory to G2/M checkpoint activation. Such DDR-positive telomeres, and those that occur as part of the telomere-dependent prolonged mitotic arrest checkpoint, normally pass through mitosis without covalent ligation, but result in cell growth arrest in G1 phase. The discovery that suppressing DSB repair during mitosis may function primarily to protect DDR-positive telomeres from fusing during cell division reinforces the unique cooperation between telomeres and the DDR to mediate tumor suppression.

  13. Tandem repeat modification during double-strand break repair induced by an engineered TAL effector nuclease in zebrafish genome.

    PubMed

    Huang, Wanxu; Zheng, Jianbo; He, Ying; Luo, Chen

    2013-01-01

    Tandem repeats (TRs) are abundant and widely distributed in eukaryotic genomes. TRs are thought to have various functions in gene transcription, DNA methylation, nucleosome position and chromatin organization. Variation of repeat units in the genome is observed in association with a number of diseases, such as Fragile X Syndrome, Huntington's disease and Friedreich's ataxia. However, the underlying mechanisms involved are poorly understood, largely owing to the technical limitations in modification of TRs at definite sites in the genome in vivo. Transcription activator-like effector nucleases (TALENs) are widely used in recent years in gene targeting for their specific binding to target sequences when engineered in vitro. Here, we show that the repair of a double-strand break (DSB) induced by TALENs adjacent to a TR can produce serial types of mutations in the TR region. Sequencing analysis revealed that there are three types of mutations induced by the DSB repair, including indels only within the TR region or within the flanking TALEN target region or simutaneously within both regions. Therefore, desired TR mutant types can be conveniently obtained by using engineered TALENs. These results demonstrate that TALENs can serve as a convenient tool for modifying TRs in the genome in studying the functions of TRs.

  14. Genetic Requirements for the Single-Strand Annealing Pathway of Double-Strand Break Repair in Saccharomyces Cerevisiae

    PubMed Central

    Ivanov, E. L.; Sugawara, N.; Fishman-Lobell, J.; Haber, J. E.

    1996-01-01

    HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Escherichia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with >80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two lacZ sequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type (MAT) switching. PMID:8849880

  15. Bevacizumab radiosensitizes non-small cell lung cancer xenografts by inhibiting DNA double-strand break repair in endothelial cells.

    PubMed

    Gao, Hui; Xue, Jianxin; Zhou, Lin; Lan, Jie; He, Jiazhuo; Na, Feifei; Yang, Lifei; Deng, Lei; Lu, You

    2015-08-28

    The aims of this study were to evaluate the effects of biweekly bevacizumab administration on a tumor microenvironment and to investigate the mechanisms of radiosensitization that were induced by it. Briefly, bevacizumab was administered intravenously to Balb/c nude mice bearing non-small cell lung cancer (NSCLC) H1975 xenografts; in addition, bevacizumab was added to NSCLC or endothelial cells (ECs) in vitro, followed by irradiation (IR). The anti-tumor efficacy, anti-angiogenic efficacy and repair of DNA double-strand breaks (DSBs) were evaluated. The activation of signaling pathways was determined using immunoprecipitation (IP) and WB analyses. Finally, biweekly bevacizumab administration inhibited the growth of H1975 xenografts and induced vascular normalization periodically. Bevacizumab more significantly increased cellular DSB and EC apoptosis when administered 1 h prior to 12 Gy/1f IR than when administered 5 days prior to IR, thereby inhibiting tumor angiogenesis and growth. In vitro, bevacizumab more effectively increased DSBs and apoptosis prior to IR and inhibited the clonogenic survival of ECs but not NSCLC cells. Using IP and WB analyses, we confirmed that bevacizumab can directly inhibit the phosphorylation of components of the VEGR2/PI3K/Akt/DNA-PKcs signaling pathway that are induced by IR in ECs. In conclusion, bevacizumab radiosensitizes NSCLC xenografts mainly by inhibiting DSB repair in ECs rather than by inducing vascular normalization.

  16. Synthetic lethal targeting of DNA double strand break repair deficient cells by human apurinic/apyrimidinic endonuclease (APE1) inhibitors

    PubMed Central

    Sultana, Rebeka; McNeill, Daniel R.; Abbotts, Rachel; Mohammed, Mohammed Z.; Zdzienicka, Małgorzata Z.; Qutob, Haitham; Seedhouse, Claire; Laughton, Charles A.; Fischer, Peter M.; Patel, Poulam M.; Wilson, David M.; Madhusudan, Srinivasan

    2013-01-01

    An apurinic/apyrimidinic (AP) site is an obligatory cytotoxic intermediate in DNA Base Excision Repair (BER) that is processed by human AP endonuclease 1 (APE1). APE1 is essential for BER and an emerging drug target in cancer. We have isolated novel small molecule inhibitors of APE1. In the current study we have investigated the ability of APE1 inhibitors to induce synthetic lethality in a panel of DNA double strand break (DSB) repair deficient and proficient cells; a) Chinese hamster (CH) cells: BRCA2 deficient (V-C8), ATM deficient (V-E5), wild type (V79) and BRCA2 revertant (V-C8(Rev1)). b) Human cancer cells: BRCA1 deficient (MDA-MB-436), BRCA1 proficient (MCF-7), BRCA2 deficient (CAPAN-1 and HeLa SilenciX cells), BRCA2 proficient (PANC1 and control SilenciX cells). We also tested synthetic lethality (SL) in CH ovary cells expressing a dominant–negative form of APE1 (E8 cells) using ATM inhibitors and DNA-PKcs inhibitors (DSB inhibitors). APE1 inhibitors are synthetically lethal in BRCA and ATM deficient cells. APE1 inhibition resulted in accumulation of DNA DSBs and G2/M cell cycle arrest. Synthetic lethality was also demonstrated in CH cells expressing a dominant–negative form of APE1 treated with ATM or DNA-PKcs inhibitors. We conclude that APE1 is a promising synthetic lethality target in cancer. PMID:22377908

  17. Phosphorylation-regulated binding of Ctp1 to Nbs1 is critical for repair of DNA double-strand breaks

    PubMed Central

    Dodson, Gerald E.; Limbo, Oliver; Nieto, Devon; Russell, Paul

    2011-01-01

    Repair of DNA double-strand breaks (DSBs) is critical for cell survival and for maintaining genome stability in eukaryotes. In Schizosaccharomyces pombe, the Mre11-Rad50-Nbs1 (MRN) complex and Ctp1 cooperate to perform the initial steps that process and repair these DNA lesions via homologous recombination (HR). While Ctp1 is recruited to DSBs in an MRN-dependent manner, the specific mechanism of this process remained unclear. We recently found that Ctp1 is phosphorylated on a domain rich in putative Casein kinase 2 (CK2) phosphoacceptor sites that resembles the SDTD repeats of Mdc1. Furthermore, phosphorylation of this motif is required for interaction with the FHA domain of Nbs1 that localizes Ctp1 to DSB sites. Here, we review and discuss these findings, and we present new data that further characterize the cellular consequences of mutating CK2 phosphorylation motifs of Ctp1, including data showing that these sites are critical for meiosis. PMID:20421724

  18. Defective DNA single-strand break repair is responsible for senescence and neoplastic escape of epithelial cells

    PubMed Central

    Nassour, Joe; Martien, Sébastien; Martin, Nathalie; Deruy, Emeric; Tomellini, Elisa; Malaquin, Nicolas; Bouali, Fatima; Sabatier, Laure; Wernert, Nicolas; Pinte, Sébastien; Gilson, Eric; Pourtier, Albin; Pluquet, Olivier; Abbadie, Corinne

    2016-01-01

    The main characteristic of senescence is its stability which relies on the persistence of DNA damage. We show that unlike fibroblasts, senescent epithelial cells do not activate an ATM-or ATR-dependent DNA damage response (DDR), but accumulate oxidative-stress-induced DNA single-strand breaks (SSBs). These breaks remain unrepaired because of a decrease in PARP1 expression and activity. This leads to the formation of abnormally large and persistent XRCC1 foci that engage a signalling cascade involving the p38MAPK and leading to p16 upregulation and cell cycle arrest. Importantly, the default in SSB repair also leads to the emergence of post-senescent transformed and mutated precancerous cells. In human-aged skin, XRCC1 foci accumulate in the epidermal cells in correlation with a decline of PARP1, whereas DDR foci accumulate mainly in dermal fibroblasts. These findings point SSBs as a DNA damage encountered by epithelial cells with aging which could fuel the very first steps of carcinogenesis. PMID:26822533

  19. Targeting DNA double strand break repair with hyperthermia and DNA-PKcs inhibition to enhance the effect of radiation treatment.

    PubMed

    van Oorschot, Bregje; Granata, Giovanna; Di Franco, Simone; Ten Cate, Rosemarie; Rodermond, Hans M; Todaro, Matilde; Medema, Jan Paul; Franken, Nicolaas A P

    2016-10-04

    Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 μM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

  20. Targeting DNA double strand break repair with hyperthermia and DNA-PKcs inhibition to enhance the effect of radiation treatment

    PubMed Central

    van Oorschot, Bregje; Granata, Giovanna; Di Franco, Simone; Cate, Rosemarie ten; Rodermond, Hans M.; Todaro, Matilde; Medema, Jan Paul; Franken, Nicolaas A.P.

    2016-01-01

    Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation. The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 μM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found. In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome. PMID:27602767

  1. 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.

  2. High energy exchange: proteins that make or break phosphoramidate bonds.

    PubMed

    Robinson, V L; Stock, A M

    1999-03-15

    Several proteins that catalyze phosphoryl transfer reactions involving phosphohistidine residues have recently been structurally characterized. The architecture of two histidine kinases defines a new protein kinase fold. The diverse folds of several phosphotransfer proteins appear to be designed to foster protein-protein interactions between transfer partners.

  3. Repair of DNA double-strand breaks is not modulated by low-dose gamma radiation in C57BL/6J mice.

    PubMed

    Blimkie, Melinda S J; Fung, Luke C W; Petoukhov, Eugenia S; Girard, Cyrielle; Klokov, Dmitry

    2014-05-01

    In this study, we sought to determine whether low-dose ionizing radiation, previously shown to induce a systemic adaptive response in C57BL/6J mice, is capable of enhancing the rate of DNA double-strand break repair. Repair capacity was determined by measuring γ-H2AX levels in splenic and thymic lymphocytes, using flow cytometry, at different times after a challenge irradiation (2 Gy, (60)Co). Irradiation with low doses (20 and 100 mGy) was conducted in vivo, whereas the challenge dose was applied to primary cultures of splenocytes and thymocytes in vitro 24 h later. Obtained kinetics curves of formation and loss of γ-H2AX indicated that cells from low-dose irradiated mice did not express more efficient DNA double-strand break repair compared to controls. Immunoblot analysis of γ-H2AX and Phospho-Ser-1981 ATM confirmed that DNA damage signaling was not modulated by preliminary low-dose radiation. Mouse embryonic fibroblasts of C57BL genetic background failed to show clonogenic survival radioadaptive response or enhanced repair of DNA double-strand breaks as evaluated by immunofluorescence microscopy of γ-H2AX foci. Our results indicate that radiation adaptive responses at systemic levels, such as increases in the tumor latency times in aging mice, may not be mediated by modulated DNA repair, and that the genetic background may affect expression of a radioadaptive response.

  4. End-processing nucleases and phosphodiesterases: An elite supporting cast for the non-homologous end joining pathway of DNA double-strand break repair.

    PubMed

    Menon, Vijay; Povirk, Lawrence F

    2016-07-01

    Nonhomologous end joining (NHEJ) is an error-prone DNA double-strand break repair pathway that is active throughout the cell cycle. A substantial fraction of NHEJ repair events show deletions and, less often, insertions in the repair joints, suggesting an end-processing step comprising the removal of mismatched or damaged nucleotides by nucleases and other phosphodiesterases, as well as subsequent strand extension by polymerases. A wide range of nucleases, including Artemis, Metnase, APLF, Mre11, CtIP, APE1, APE2 and WRN, are biochemically competent to carry out such double-strand break end processing, and have been implicated in NHEJ by at least circumstantial evidence. Several additional DNA end-specific phosphodiesterases, including TDP1, TDP2 and aprataxin are available to resolve various non-nucleotide moieties at DSB ends. This review summarizes the biochemical specificities of these enzymes and the evidence for their participation in the NHEJ pathway.

  5. Meiotic DNA double-strand break repair requires two nucleases, MRN and Ctp1, to produce a single size class of Rec12 (Spo11)-oligonucleotide complexes.

    PubMed

    Milman, Neta; Higuchi, Emily; Smith, Gerald R

    2009-11-01

    Programmed DNA double-strand breaks (DSBs) in meiosis are formed by Spo11 (Rec12 in fission yeast), a topoisomerase II-like protein, which becomes covalently attached to DNA 5' ends. For DSB repair through homologous recombination, the protein must be removed from these DNA ends. We show here that Rec12 is endonucleolytically removed from DSB ends attached to a short oligonucleotide (Rec12-oligonucleotide complex), as is Spo11 in budding yeast. Fission yeast, however, has only one size class of Rec12-oligonucleotide complexes, whereas budding yeast has two size classes, suggesting different endonucleolytic regulatory mechanisms. Rec12-oligonucleotide generation strictly requires Ctp1 (Sae2 nuclease homolog), the Rad32 (Mre11) nuclease domain, and Rad50 of the MRN complex. Surprisingly, Nbs1 is not strictly required, indicating separable roles for the MRN subunits. On the basis of these and other data, we propose that Rad32 nuclease has the catalytic site for Rec12-oligonucleotide generation and is activated by Ctp1, which plays an additional role in meiotic recombination.

  6. Characterization of DNA binding and pairing activities associated with the native SFPQ·NONO DNA repair protein complex.

    PubMed

    Udayakumar, Durga; Dynan, William S

    2015-08-07

    Nonhomologous end joining (NHEJ) is a major pathway for repair of DNA double-strand breaks. We have previously shown that a complex of SFPQ (PSF) and NONO (p54(nrb)) cooperates with Ku protein at an early step of NHEJ, forming a committed preligation complex and stimulating end-joining activity by 10-fold or more. SFPQ and NONO show no resemblance to other repair factors, and their mechanism of action is uncertain. Here, we use an optimized microwell-based assay to characterize the in vitro DNA binding behavior of the native SFPQ·NONO complex purified from human (HeLa) cells. SFPQ·NONO and Ku protein bind independently to DNA, with little evidence of cooperativity and only slight mutual interference at high concentration. Whereas Ku protein requires free DNA ends for binding, SFPQ·NONO does not. Both Ku and SFPQ·NONO have pairing activity, as measured by the ability of DNA-bound protein to capture a second DNA fragment in a microwell-based assay. Additionally, SFPQ·NONO stimulates DNA-dependent protein kinase autophosphorylation, consistent with the ability to promote formation of a synaptic complex formation without occluding the DNA termini proper. These findings suggest that SFPQ·NONO promotes end joining by binding to internal DNA sequences and cooperating with other repair proteins to stabilize a synaptic pre-ligation complex.

  7. Triggering of DNA strand breaks by 45/sup 0/C hyperthermia and its influence on the repair of. gamma. -radiation damage in human white blood cells

    SciTech Connect

    Mitchel, R.E.J.; Birnboim, H.C.

    1985-05-01

    Human peripheral white blood cells, freshly isolated from normal individual donors, were exposed to hyperthermia. Heat-generated DNA strand break damage and white blood cell capacity to repair radiation-induced breaks were determined by a fluorometric alkaline unwinding assay. Strand breaks could be readily detected when white blood cells were incubated in a physiological salt solution at temperatures between 41/sup 0/ and 46/sup 0/C, for times up to 90 min. The time course of strand break induction at 45/sup 0/C was characterized by a short initial lag, followed by a period of rapid break induction and subsequently a lower rate. Evidence is presented which suggests that the induction of DNA damage involved a triggering mechanism; a short treatment at 45/sup 0/C (10 to 20 min) initiated a cellular event which led to a rapid increase in the number of stand breaks during subsequent incubation of 37/sup 0/C. Continuous incubation at 45/sup 0/C produced less DNA damage than an initial period at 45/sup 0/C followed by incubation at 37/sup 0/C. This apparent triggering phenomenon was not due to a triggering of the respiratory burst in phagocytic cells, since no O/sub 2//sup -/ could be detected; in fact, a 30-min treatment at 45/sup 0/C largely blocked the capacity of the cells to respond normally to a soluble stimulator of the respiratory burst. Unlike ..gamma..-ray-induced breaks, 45/sup 0/C hyperthermia-induced breaks did not rejoin during subsequent incubation for up to 1 h at 37/sup 0/C. Additionally, 45/sup 0/C hyperthermia treatment progressively inhibited the ability of the cells to repair subsequent ..gamma..-ray-induced breaks (4 Gy). 17 references, 1 table.

  8. Bisdemethoxycurcumin induces DNA damage and inhibits DNA repair associated protein expressions in NCI-H460 human lung cancer cells.

    PubMed

    Yu, Chien-Chih; Yang, Su-Tso; Huang, Wen-Wen; Peng, Shu-Fen; Huang, An-Cheng; Tang, Nou-Ying; Liu, Hsin-Chung; Yang, Mei-Due; Lai, Kuang-Chi; Chung, Jing-Gung

    2015-08-30

    Nonsmall cell lung carcinoma (NSCLC) is a devastating primary lung tumor resistant to conventional therapies. Bisdemethoxycurcumin (BDMC) is one of curcumin derivate from Turmeric and has been shown to induce NSCLC cell death. Although there is one report to show BDMC induced DNA double strand breaks, however, no available information to show BDMC induced DNA damage action with inhibited DNA repair protein in lung cancer cells in detail. In this study, we tested BDMC-induced DNA damage and condensation in NCI-H460 cells by using Comet assay and DAPI staining examinations, respectively and we found BDMC induced DNA damage and condension. Western blotting was used to examine the effects of BDMC on protein expression associated with DNA damage and repair and results indicated that BDMC suppressed the protein levels associated with DNA damage and repair, such as 14-3-3σ (an important checkpoint keeper of DDR), O6-methylguanine-DNA methyltransferase, DNA repair proteins breast cancer 1, early onset, mediator of DNA damage checkpoint 1 but activate phosphorylated p53 and p-H2A.X (phospho Ser140) in NCI-H460 cells. Confocal laser systems microscopy was used for examining the protein translocation and results show that BDMC increased the translocation of p-p53 and p-H2A.X (phospho Ser140) from cytosol to nuclei in NCI-H460 cells. In conclusion, BDMC induced DNA damage and condension and affect DNA repair proteins in NCI-H460 cells in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol, 2015.

  9. Mismatch repair system proteins in oral benign and malignant lesions.

    PubMed

    Amaral-Silva, Gleyson Kleber do; Martins, Manoela Domingues; Pontes, Hélder Antônio Rebelo; Fregnani, Eduardo Rodrigues; Lopes, Márcio Ajudarte; Fonseca, Felipe Paiva; Vargas, Pablo Agustin

    2017-04-01

    Different environmental agents may cause DNA mutations by disrupting its double-strand structure; however, even normal DNA polymerase function may synthesize mismatch nucleotide bases, occasionally demonstrating failure in its proofreading activity. To overcome this issue, mismatch repair (MMR) system, a group of proteins specialized in finding mispairing bases and small loops of insertion or deletion, works to avoid the occurrence of mutations that could ultimately lead to innumerous human diseases. In the last decades, the role of MMR proteins in oral carcinogenesis and in the development of other oral cavity neoplasms has grown, but their importance in the pathogenesis and their prognostic potential for patients affected by oral malignancies, especially oral squamous cell carcinoma (OSCC), remain unclear. Therefore, in this manuscript we aimed to review and critically discuss the currently available data on MMR proteins expression in oral potentially malignant lesions, in OSCC, and in other oral neoplasms to better understand their relevance in these lesions.

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

    PubMed Central

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

    1995-01-01

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

  11. Isoaspartyl Protein Damage and Repair in Mouse Retina

    PubMed Central

    Qin, Zhenxia; Yang, Jing; Klassen, Henry J.; Aswad, Dana W.

    2014-01-01

    Purpose. To determine the propensity of retinal proteins for spontaneous damage via formation of isoaspartyl sites, a common type of protein damage that could contribute to retinal disease. Methods. Tissue extracts were obtained from retinas and brains of control mice and from mice in which the gene for protein L-isoaspartate O-methyltransferase (PIMT; an enzyme that repairs isoaspartyl protein damage) was knocked out. PIMT expression in these extracts was measured by Western blot, and its specific activity was assayed by monitoring the rate of [3H]methyl transfer from S-adenosyl-[methyl-3H]L-methionine to γ-globulin. Isoaspartate levels in extracts were measured by their capacity to accept [3H]methyl groups via the PIMT-catalyzed methylation reaction. To compare molecular weight distributions of isoaspartyl-rich proteins in retina versus brain, proteins from PIMT knockout (KO) and control mice were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF). Isoaspartyl proteins were 3H-labeled on-blot using a PIMT overlay and imaged by autoradiography. Results. When normalized to the β-actin content of each tissue, retina was found to be nearly identical to brain with regard to expression and activity of PIMT and its propensity to accumulate isoaspartyl sites when PIMT is absent. The two tissues show distinct differences in the molecular weight distribution of isoaspartyl proteins. Conclusions. The retina is rich in PIMT activity and contains a wide range of proteins that are highly susceptible to this type of protein damage. Recoverin may be one such protein. Isoaspartate formation, along with oxidation, should be considered as a potential source of protein dysfunction and autoimmunity in retinal disease. PMID:24550364

  12. [Double-strand DNA breaks induction and repair in human blood lymphocytes irradiated with adapting dose].

    PubMed

    Osipov, A N; Lizunova, E Iu; Vorob'eva, N Iu; Pelevina, I I

    2009-01-01

    Using a DNA-comet assay was shown that irradiation of human blood lymphocytes at G1 cell cycle with a low conditioning dose (5 cGy) induces an adaptive response (AR) manifested in reduction of the double-strand DNA (DSB) amount induced by challenging dose at 10 Gy. 24 h after conditioning irradiation (48 h after PHA addition) in cells irradiated at both conditioning and challenging doses a relative DBS amount was approximately 24% less in comparison to versus a control irradiated at challenging dose only. 48 h after adapting irradiation this index increased to approximately 35%, while 72 h after was decreased to approximately 29%. AR observed by us during 72 h after its induction did not accompanied by statistically significant changes in DBS repair enhancing. It is possible to assume that basic role in AR forming in lymphocytes under experimental conditions used by us playing the processes preventing radiation-induced DBS formation (antioxidant defense system activation, chromatin conformation changes ets).

  13. The Landscape of Mouse Meiotic Double-Strand Break Formation, Processing, and Repair.

    PubMed

    Lange, Julian; Yamada, Shintaro; Tischfield, Sam E; Pan, Jing; Kim, Seoyoung; Zhu, Xuan; Socci, Nicholas D; Jasin, Maria; Keeney, Scott

    2016-10-20

    Heritability and genome stability are shaped by meiotic recombination, which is initiated via hundreds of DNA double-strand breaks (DSBs). The distribution of DSBs throughout the genome is not random, but mechanisms molding this landscape remain poorly understood. Here, we exploit genome-wide maps of mouse DSBs at unprecedented nucleotide resolution to uncover previously invisible spatial features of recombination. At fine scale, we reveal a stereotyped hotspot structure-DSBs occur within narrow zones between methylated nucleosomes-and identify relationships between SPO11, chromatin, and the histone methyltransferase PRDM9. At large scale, DSB formation is suppressed on non-homologous portions of the sex chromosomes via the DSB-responsive kinase ATM, which also shapes the autosomal DSB landscape at multiple size scales. We also provide a genome-wide analysis of exonucleolytic DSB resection lengths and elucidate spatial relationships between DSBs and recombination products. Our results paint a comprehensive picture of features governing successive steps in mammalian meiotic recombination.

  14. Phototriggered formation and repair of DNA containing a site-specific single strand break of the type produced by ionizing radiation or AP lyase activity.

    PubMed

    Zhang, K; Taylor, J S

    2001-01-09

    DNA strand breaks are produced by a variety of agents and processes such as ionizing radiation, xenobiotics, oxidative metabolism, and enzymatic processing of DNA base damage. One of the major types of strand breaks produced by these processes is a single nucleotide gap terminating in 5'- and 3'-phosphates. Previously, we had developed a method for sequence-specifically producing such phosphate-terminated strand breaks in an oligodeoxynucleotide by way of two photochemically activated (caged) building blocks placed in tandem. We now report the design and synthesis of a single caged building block consisting of 1,3-(2-nitrophenyl)-1,3-propanediol, for producing phosphate-terminated strand breaks, and its use producing such a break at a specific site in a double-stranded circular DNA vector. To produce the site-specific break in a duplex vector, a primer containing the caged single strand break was extended opposite the single strand form of a circular DNA vector followed by enzymatic ligation and purification. The single strand break could then be formed in quantitative yield by irradiation of the vector with 365 nm light. In contrast to a previous study, it was found that the strand break can be repaired by Escherichia coli DNA polymerase I and E. coli DNA ligase alone, though less efficiently than in the presence of the 3'-phosphate processing enzyme E. coli endonuclease IV. Repair in the absence of endonuclease IV could be attributed to hydrolysis of the 3'-phosphate in the presence of dNTP and to a lesser extent to exonucleolytic removal of the 3'-phosphate-bearing terminal nucleotide by way of the 3' --> 5' exonuclease activity of polymerase I. This work demonstrates that specialized 3'-end processing enzymes such as endonuclease IV or exonuclease III are not absolutely required for repair of phosphate-terminated gaps. In addition to preparing single strand breaks, the caged building block described should also be useful for preparing double strand breaks and

  15. Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications

    PubMed Central

    Polo, Sophie E.; Jackson, Stephen P.

    2011-01-01

    Genome integrity is constantly monitored by sophisticated cellular networks, collectively termed the DNA damage response (DDR). A common feature of DDR proteins is their mobilization in response to genotoxic stress. Here, we outline how the development of various complementary methodologies has provided valuable insights into the spatiotemporal dynamics of DDR protein assembly/disassembly at sites of DNA strand breaks in eukaryotic cells. Considerable advances have also been made in understanding the underlying molecular mechanisms for these events, with post-translational modifications of DDR factors being shown to play prominent roles in controlling the formation of foci in response to DNA-damaging agents. We review these regulatory mechanisms and discuss their biological significance to the DDR. PMID:21363960

  16. Phenothiazine Inhibitors of TLKs Affect Double-Strand Break Repair and DNA Damage Response Recovery and Potentiate Tumor Killing with Radiomimetic Therapy.

    PubMed

    Ronald, Sharon; Awate, Sanket; Rath, Abhijit; Carroll, Jennifer; Galiano, Floyd; Dwyer, Donard; Kleiner-Hancock, Heather; Mathis, J Michael; Vigod, Simone; De Benedetti, Arrigo

    2013-01-01

    The Tousled-like kinases (TLKs) are involved in chromatin assembly, DNA repair, and transcription. Two TLK genes exist in humans, and their expression is often dysregulated in cancer. TLKs phosphorylate Asf1 and Rad9, regulating double-strand break (DSB) repair and the DNA damage response (DDR). TLKs maintain genomic stability and are important therapeutic intervention targets. We identified specific inhibitors of TLKs from several compound libraries, some of which belong to the family of phenothiazine antipsychotics. The inhibitors prevented the TLK-mediated phosphorylation of Rad9(S328) and impaired checkpoint recovery and DSB repair. The inhibitor thioridazine (THD) potentiated tumor killing with chemotherapy and also had activity alone. Staining for γ-H2AX revealed few positive cells in untreated tumors, but large numbers in mice treated with low doxorubicin or THD alone, possibly the result of the accumulation of DSBs that are not promptly repaired as they may occur in the harsh tumor growth environment.

  17. Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate

    NASA Technical Reports Server (NTRS)

    Winters, T. A.; Russell, P. S.; Kohli, M.; Dar, M. E.; Neumann, R. D.; Jorgensen, T. J.

    1999-01-01

    Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.

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

    PubMed

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

    2012-12-01

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

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

    PubMed Central

    Brown, JB; Akutsu, Tatsuya

    2009-01-01

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

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

    SciTech Connect

    Prakash, L.

    1992-04-01

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

  1. Development of a novel rapid assay to assess the fidelity of DNA double-strand-break repair in human tumour cells

    PubMed Central

    Collis, S. J.; Sangar, V. K.; Tighe, A.; Roberts, S. A.; Clarke, N. W.; Hendry, J. H.; Margison, G. P.

    2002-01-01

    Cellular survival following ionising radiation-mediated damage is primarily a function of the ability to successfully detect and repair DNA double-strand breaks (DSBs). Previous studies have demonstrated that radiosensitivity, determined as a reduction in colony forming ability in vitro, may be related to the incorrect repair (misrepair) of DSBs. The novel rapid dual fluorescence (RDF) assay is a plasmid-based reporter system that rapidly assesses the correct rejoining of a restriction-enzyme produced DSBs within transfected cells. We have utilised this novel assay to determine the fidelity of DSB repair in the prostate tumour cell line LNCaP, the bladder tumour cell line MGH-U1 and a radiosensitive subclone S40b. The two bladder cell lines have been shown in previous studies to differ in their ability to correctly repair plasmids containing a single DSB. Using the RDF assay we found that a substantial portion of LNCaP cells [80.4 ± 5.3(standard error)%] failed to reconstitute reporter gene expression; however, there was little difference in this measure of DSB repair fidelity between the two bladder cell lines (48.3 ± 3.5% for MGH-U1; 39.9 ± 8.2% for S40b). The RDF assay has potential to be developed to study the relationship between DSB repair fidelity and radiosensitivity as well as the mechanisms associated with this type of repair defect. PMID:11788727

  2. Human INO80 chromatin-remodelling complex contributes to DNA double-strand break repair via the expression of Rad54B and XRCC3 genes.

    PubMed

    Park, Eun-Jung; Hur, Shin-Kyoung; Kwon, Jongbum

    2010-10-15

    Recent studies have shown that the SWI/SNF family of ATP-dependent chromatin-remodelling complexes play important roles in DNA repair as well as in transcription. The INO80 complex, the most recently described member of this family, has been shown in yeast to play direct role in DNA DSB (double-strand break) repair without affecting the expression of the genes involved in this process. However, whether this function of the INO80 complex is conserved in higher eukaryotes has not been investigated. In the present study, we found that knockdown of hINO80 (human INO80) confers DNA-damage hypersensitivity and inefficient DSB repair. Microarray analysis and other experiments have identified the Rad54B and XRCC3 (X-ray repair complementing defective repair in Chinese-hamster cells 3) genes, implicated in DSB repair, to be repressed by hINO80 deficiency. Chromatin immunoprecipitation studies have shown that hINO80 binds to the promoters of the Rad54B and XRCC3 genes. Re-expression of the Rad54B and XRCC3 genes rescues the DSB repair defect in hINO80-deficient cells. These results suggest that hINO80 assists DSB repair by positively regulating the expression of the Rad54B and XRCC3 genes. Therefore, unlike yeast INO80, hINO80 can contribute to DSB repair indirectly via gene expression, suggesting that the mechanistic role of this chromatin remodeller in DSB repair is evolutionarily diversified.

  3. Molecular Process Producing Oncogene Fusion in Lung Cancer Cells by Illegitimate Repair of DNA Double-Strand Breaks

    PubMed Central

    Seki, Yoshitaka; Mizukami, Tatsuji; Kohno, Takashi

    2015-01-01

    Constitutive activation of oncogenes by fusion to partner genes, caused by chromosome translocation and inversion, is a critical genetic event driving lung carcinogenesis. Fusions of the tyrosine kinase genes ALK (anaplastic lymphoma kinase), ROS1 (c-ros oncogene 1), or RET (rearranged during transfection) occur in 1%–5% of lung adenocarcinomas (LADCs) and their products constitute therapeutic targets for kinase inhibitory drugs. Interestingly, ALK, RET, and ROS1 fusions occur preferentially in LADCs of never- and light-smokers, suggesting that the molecular mechanisms that cause these rearrangements are smoking-independent. In this study, using previously reported next generation LADC genome sequencing data of the breakpoint junction structures of chromosome rearrangements that cause oncogenic fusions in human cancer cells, we employed the structures of breakpoint junctions of ALK, RET, and ROS1 fusions in 41 LADC cases as “traces” to deduce the molecular processes of chromosome rearrangements caused by DNA double-strand breaks (DSBs) and illegitimate joining. We found that gene fusion was produced by illegitimate repair of DSBs at unspecified sites in genomic regions of a few kb through DNA synthesis-dependent or -independent end-joining pathways, according to DSB type. This information will assist in the understanding of how oncogene fusions are generated and which etiological factors trigger them. PMID:26437441

  4. The mismatch repair system modulates curcumin sensitivity through induction of DNA strand breaks and activation of G2-M checkpoint.

    PubMed

    Jiang, Zhihua; Jin, ShunQian; Yalowich, Jack C; Brown, Kevin D; Rajasekaran, Baskaran

    2010-03-01

    The highly conserved mismatch (MMR) repair system corrects postreplicative errors and modulates cellular responses to genotoxic agents. Here, we show that the MMR system strongly influences cellular sensitivity to curcumin. Compared with MMR-proficient cells, isogenically matched MMR-deficient cells displayed enhanced sensitivity to curcumin. Similarly, cells suppressed for MLH1 or MSH2 expression by RNA interference displayed increased curcumin sensitivity. Curcumin treatment generated comparable levels of reactive oxygen species and the mutagenic adduct 8-oxo-guanine in MMR-proficient and MMR-deficient cells; however, accumulation of gammaH2AX foci, a marker for DNA double-strand breaks (DSB), occurred only in MMR-positive cells in response to curcumin treatment. Additionally, MMR-positive cells showed activation of Chk1 and induction of G(2)-M cell cycle checkpoint following curcumin treatment and inhibition of Chk1 by UCN-01 abrogated Chk1 activation and heightened apoptosis in MMR-proficient cells. These results indicate that curcumin triggers the accumulation of DNA DSB and induction of a checkpoint response through a MMR-dependent mechanism. Conversely, in MMR-compromised cells, curcumin-induced DSB is significantly blunted, and as a result, cells fail to undergo cell cycle arrest, enter mitosis, and die through mitotic catastrophe. The results have potential therapeutic value, especially in the treatment of tumors with compromised MMR function.

  5. Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

    PubMed Central

    Singh, Satyendra K.; Wang, Minli; Staudt, Christian; Iliakis, George

    2011-01-01

    In cells exposed to ionizing radiation (IR), double-strand breaks (DSBs) form within clustered-damage sites from lesions disrupting the DNA sugar–phosphate backbone. It is commonly assumed that these DSBs form promptly and are immediately detected and processed by the cellular DNA damage response (DDR) apparatus. This assumption is questioned by the observation that after irradiation of naked DNA, a fraction of DSBs forms minutes to hours after exposure as a result of temperature dependent, chemical processing of labile sugar lesions. Excess DSBs also form when IR-exposed cells are processed at 50°C, but have been hitherto considered method-related artifact. Thus, it remains unknown whether DSBs actually develop in cells after IR exposure from chemically labile damage. Here, we show that irradiation of ‘naked’ or chromatin-organized mammalian DNA produces lesions, which evolve to DSBs and add to those promptly induced, after 8–24 h in vitro incubation at 37°C or 50°C. The conversion is more efficient in chromatin-associated DNA, completed within 1 h in cells and delayed in a reducing environment. We conclude that IR generates sugar lesions within clustered-damage sites contributing to DSB formation only after chemical processing, which occurs efficiently at 37°C. This subset of delayed DSBs may challenge DDR, may affect the perceived repair kinetics and requires further characterization. PMID:21745815

  6. I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila.

    PubMed Central

    Bellaiche, Y; Mogila, V; Perrimon, N

    1999-01-01

    As a step toward the development of a homologous recombination system in Drosophila, we have developed a methodology to target double-strand breaks (DSBs) to a specific position in the Drosophila genome. This method uses the mitochondrial endonuclease I-SceI that recognizes and cuts an 18-bp restriction site. We find that >6% of the progeny derived from males that carry a marker gene bordered by two I-SceI sites and that express I-SceI in their germ line lose the marker gene. Southern blot analysis and sequencing of the regions surrounding the I-SceI sites revealed that in the majority of the cases, the introduction of DSBs at the I-SceI sites resulted in the complete deletion of the marker gene; the other events were associated with partial deletion of the marker gene. We discuss a number of applications for this novel technique, in particular its use to study DSB repair mechanisms. PMID:10388822

  7. Nucleotide excision repair-dependent DNA double-strand break formation and ATM signaling activation in mammalian quiescent cells.

    PubMed

    Wakasugi, Mitsuo; Sasaki, Takuma; Matsumoto, Megumi; Nagaoka, Miyuki; Inoue, Keiko; Inobe, Manabu; Horibata, Katsuyoshi; Tanaka, Kiyoji; Matsunaga, Tsukasa

    2014-10-10

    Histone H2A variant H2AX is phosphorylated at Ser(139) in response to DNA double-strand break (DSB) and single-stranded DNA (ssDNA) formation. UV light dominantly induces pyrimidine photodimers, which are removed from the mammalian genome by nucleotide excision repair (NER). We previously reported that in quiescent G0 phase cells, UV induces ATR-mediated H2AX phosphorylation plausibly caused by persistent ssDNA gap intermediates during NER. In this study, we have found that DSB is also generated following UV irradiation in an NER-dependent manner and contributes to an earlier fraction of UV-induced H2AX phosphorylation. The NER-dependent DSB formation activates ATM kinase and triggers the accumulation of its downstream factors, MRE11, NBS1, and MDC1, at UV-damaged sites. Importantly, ATM-deficient cells exhibited enhanced UV sensitivity under quiescent conditions compared with asynchronously growing conditions. Finally, we show that the NER-dependent H2AX phosphorylation is also observed in murine peripheral T lymphocytes, typical nonproliferating quiescent cells in vivo. These results suggest that in vivo quiescent cells may suffer from NER-mediated secondary DNA damage including ssDNA and DSB.

  8. Nucleosomes Suppress the Formation of Double-strand DNA Breaks during Attempted Base Excision Repair of Clustered Oxidative Damages*

    PubMed Central

    Cannan, Wendy J.; Tsang, Betty P.; Wallace, Susan S.; Pederson, David S.

    2014-01-01

    Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling. PMID:24891506

  9. Haploid meiosis in Arabidopsis: double-strand breaks are formed and repaired but without synapsis and crossovers.

    PubMed

    Cifuentes, Marta; Rivard, Maud; Pereira, Lucie; Chelysheva, Liudmila; Mercier, Raphael

    2013-01-01

    Two hallmark features of meiosis are i) the formation of crossovers (COs) between homologs and ii) the production of genetically-unique haploid spores that will fuse to restore the somatic ploidy level upon fertilization. In this study we analysed meiosis in haploid Arabidopsis thaliana plants and a range of haploid mutants to understand how meiosis progresses without a homolog. Extremely low chiasma frequency and very limited synapsis occurred in wild-type haploids. The resulting univalents segregated in two uneven groups at the first division, and sister chromatids segregated to opposite poles at the second division, leading to the production of unbalanced spores. DNA double-strand breaks that initiate meiotic recombination were formed, but in half the number compared to diploid meiosis. They were repaired in a RAD51- and REC8-dependent manner, but independently of DMC1, presumably using the sister chromatid as a template. Additionally, turning meiosis into mitosis (MiMe genotype) in haploids resulted in the production of balanced haploid gametes and restoration of fertility. The variability of the effect on meiosis of the absence of homologous chromosomes in different organisms is then discussed.

  10. XRCC3 is essential for proper double-strand break repair and homologous recombination in rice meiosis.

    PubMed

    Zhang, Bingwei; Wang, Mo; Tang, Ding; Li, Yafei; Xu, Meng; Gu, Minghong; Cheng, Zhukuan; Yu, Hengxiu

    2015-09-01

    RAD51 paralogues play important roles in the assembly and stabilization of RAD51 nucleoprotein filaments, which promote homologous pairing and strand exchange reactions in organisms ranging from yeast to vertebrates. XRCC3, a RAD51 paralogue, has been characterized in budding yeast, mouse, and Arabidopsis. In the present study, XRCC3 in rice was identified and characterized. The rice xrcc3 mutant exhibited normal vegetative growth but complete male and female sterility. Cytological investigations revealed that homologous pairing and synapsis were severely disrupted in the mutant. Meiotic chromosomes were frequently entangled from diplotene to metaphase I, resulting in chromosome fragmentation at anaphase I. The immunostaining signals from γH2AX were regular, implying that double-strand break (DSB) formation was normal in xrcc3 meiocytes. However, COM1 was not detected on early prophase I chromosomes, suggesting that the DSB end-processing system was destroyed in the mutant. Moreover, abnormal chromosome localization of RAD51C, DMC1, ZEP1, ZIP4, and MER3 was observed in xrcc3. Taken together, the results suggest that XRCC3 plays critical roles in both DSB repair and homologous chromosome recombination during rice meiosis.

  11. Engagement of Components of DNA-Break Repair Complex and NFκB in Hsp70A1A Transcription Upregulation by Heat Shock

    PubMed Central

    Hazra, Joyita; Mukherjee, Pooja; Ali, Asif; Poddar, Soumita; Pal, Mahadeb

    2017-01-01

    An involvement of components of DNA-break repair (DBR) complex including DNA-dependent protein kinase (DNA-PK) and poly-ADP-ribose polymerase 1 (PARP-1) in transcription regulation in response to distinct cellular signalling has been revealed by different laboratories. Here, we explored the involvement of DNA-PK and PARP-1 in the heat shock induced transcription of Hsp70A1A. We find that inhibition of both the catalytic subunit of DNA-PK (DNA-PKc), and Ku70, a regulatory subunit of DNA-PK holo-enzyme compromises transcription of Hsp70A1A under heat shock treatment. In immunoprecipitation based experiments we find that Ku70 or DNA-PK holoenzyme associates with NFκB. This NFκB associated complex also carries PARP-1. Downregulation of both NFκB and PARP-1 compromises Hsp70A1A transcription induced by heat shock treatment. Alteration of three bases by site directed mutagenesis within the consensus κB sequence motif identified on the promoter affected inducibility of Hsp70A1A transcription by heat shock treatment. These results suggest that NFκB engaged with the κB motif on the promoter cooperates in Hsp70A1A activation under heat shock in human cells as part of a DBR complex including DNA-PK and PARP-1. PMID:28099440

  12. Hop2 and Sae3 Are Required for Dmc1-Mediated Double-Strand Break Repair via Homolog Bias during Meiosis

    PubMed Central

    Cho, Hong-Rae; Kong, Yoon-Ju; Hong, Soo-Gil; Kim, Keun Pil

    2016-01-01

    During meiosis, exchange of DNA segments occurs between paired homologous chromosomes in order to produce recombinant chromosomes, helping to increase genetic diversity within a species. This genetic exchange process is tightly controlled by the eukaryotic RecA homologs Rad51 and Dmc1, which are involved in strand exchange of meiotic recombination, with Rad51 participating specifically in mitotic recombination. Meiotic recombination requires an interaction between homologous chromosomes to repair programmed double-strand breaks (DSBs). In this study, we investigated the budding yeast meiosis-specific proteins Hop2 and Sae3, which function in the Dmc1-dependent pathway. This pathway mediates the homology searching and strand invasion processes. Mek1 kinase participates in switching meiotic recombination from sister bias to homolog bias after DSB formation. In the absence of Hop2 and Sae3, DSBs were produced normally, but showed defects in the DSB-to-single-end invasion transition mediated by Dmc1 and auxiliary factors, and mutant strains failed to complete proper chromosome segregation. However, in the absence of Mek1 kinase activity, Rad51-dependent recombination progressed via sister bias in the hop2Δ or sae3Δ mutants, even in the presence of Dmc1. Thus, Hop2 and Sae3 actively modulate Dmc1-dependent recombination, effectively progressing homolog bias, a process requiring Mek1 kinase activation. PMID:27329041

  13. ER stress suppresses DNA double-strand break repair and sensitizes tumor cells to ionizing radiation by stimulating proteasomal degradation of Rad51.

    PubMed

    Yamamori, Tohru; Meike, Shunsuke; Nagane, Masaki; Yasui, Hironobu; Inanami, Osamu

    2013-10-11

    In this study, we provide evidence that endoplasmic reticulum (ER) stress suppresses DNA double-strand break (DSB) repair and increases radiosensitivity of tumor cells by altering Rad51 levels. We show that the ER stress inducer tunicamycin stimulates selective degradation of Rad51 via the 26S proteasome, impairing DSB repair and enhancing radiosensitivity in human lung cancer A549 cells. We also found that glucose deprivation, which is a physiological inducer of ER stress, triggered similar events. These findings suggest that ER stress caused by the intratumoral environment influences tumor radiosensitivity, and that it has potential as a novel target to improve cancer radiotherapy.

  14. Connecting by breaking and repairing: mechanisms of DNA strand exchange in meiotic recombination.

    PubMed

    Sansam, Christopher L; Pezza, Roberto J

    2015-07-01

    During prophase of meiosis I, homologous chromosomes interact and undergo recombination. Successful completion of these processes is required in order for the homologous chromosomes to mount the meiotic spindle as a pair. The organization of the chromosomes into pairs ensures orderly segregation to opposite poles of the dividing cell, such that each gamete receives one copy of each chromosome. Chiasmata, the cytological manifestation of crossover products of recombination, physically connect the homologs in pairs, providing a linkage that facilitates their segregation. Consequently, mutations that reduce the level of recombination are invariably associated with increased errors in meiotic chromosome segregation. In this review, we focus on recent biochemical and genetic advances in elucidating the mechanisms of meiotic DNA strand exchange catalyzed by the Dmc1 protein. We also discuss the mode by which two recombination mediators, Hop2 and Mnd1, facilitate rate-limiting steps of DNA strand exchange catalyzed by Dmc1.

  15. Connecting by breaking and repairing: mechanisms of DNA strand exchange in meiotic recombination

    PubMed Central

    Sansam, Christopher L; Pezza, Roberto J

    2015-01-01

    During prophase of meiosis I, homologous chromosomes interact and undergo recombination. Successful completion of these processes is required in order for the homologous chromosomes to mount the meiotic spindle as a pair. The organization of the chromosomes into pairs ensures orderly segregation to opposite poles of the dividing cell, such that each gamete receives one copy of each chromosome. Chiasmata, the cytological manifestation of crossover products of recombination, physically connect the homologs in pairs, providing a linkage that facilitates their segregation. Consequently, mutations that reduce the level of recombination are invariably associated with increased errors in meiotic chromosome segregation. In this review, we focus on recent biochemical and genetic advances in elucidating the mechanisms of meiotic DNA strand exchange catalyzed by the Dmc1 protein. We also discuss the mode by which two recombination mediators, Hop2 and Mnd1, facilitate rate-limiting steps of DNA strand exchange catalyzed by Dmc1. PMID:25953379

  16. Functions of Human Rad51 and Other Recombination Factors in DNA Double-Strand Break Repair

    DTIC Science & Technology

    2002-06-01

    cells express five Rad5l-like proteins - XRCC2, XRCC3 , Rad51B, Rad51C and Rad51D. These Rad51 paralogs are important for homologous recombination and...complex with 42-kD Rad5 IC species was detected. The observed sizes XRCC3 (Schild et al. 2000; Kurumizaka et al. 2001; Mas- A kIa a-Rad51B kDa a-RadSIC...RadS1C XRCC3 or that the XRCC3 -Rad51C complex was not A x-Rad5l B retained on the Q column. 07a 0- The results above indicated that Rad5lB and Rad5lC

  17. DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferation-dependent regulation.

    PubMed

    Mladenov, Emil; Magin, Simon; Soni, Aashish; Iliakis, George

    2016-06-01

    Eukaryotic cells respond to DNA damage by activating a comprehensive network of biochemical pathways that enable damage recognition and initiate responses leading to repair, apoptosis/autophagy or senescence. This network of responses is commonly described as the "DNA damage response" (DDR). Among the plethora of lesions generated in the DNA from various physical and chemical agents in the environment and in the cell, DNA double strand breaks (DSBs) and DNA replication stress (RS) are the most severe and induce strong DDR, as they bear high risk for cell death, or genomic alterations ultimately causing cancer. Here, we focus on DSBs and provide a state-of-the-art review of the molecular underpinnings of repair pathways that process DSBs in higher eukaryotes, their strengths and limitations, as well as aspects of repair pathway choice and hierarchy. Furthermore, we discuss the regulation of DSB repair pathways throughout the cell cycle and by processes affecting the proliferative state of the cell. We review the role of growth factors and their receptors in the regulation of each DSB repair pathway and discuss aspects of systemic regulation of DNA repair.

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

    PubMed

    Shcherbakov, Victor P; Kudryashova, Elena

    2014-09-01

    The effects of primase and topoisomerase II deficiency on the double-strand break (DSB) repair and genetic recombination in bacteriophage T4 were studied in vivo using focused recombination. Site-specific DSBs were induced by SegC endonuclease in the rIIB gene of one of the parents. The frequency/distance relationship was determined in crosses of the wild-type phage, topoisomerase II mutant amN116 (gene 39), and primase mutant E219 (gene 61). Ordinary two-factor (i×j) and three-factor (i k×j) crosses between point rII mutations were also performed. These data provide information about the frequency and distance distribution of the single-exchange (splice) and double-exchange (patch) events. In two-factor crosses ets1×i, the topoisomerase and primase mutants had similar recombinant frequencies in crosses at ets1-i distances longer than 1000 bp, comprising about 80% of the corresponding wild-type values. They, however, differ remarkably in crosses at shorter distances. In the primase mutant, the recombinant frequencies are similar to those in the wild-type crosses at distances less than 100 bp, being a bit diminished at longer distances. In two-factor crosses ets1×i of the topoisomerase mutant, the recombinant frequencies were reduced ten-fold at the shortest distances. In three-factor crosses a6 ets1×i, where we measure patch-related recombination, the primase mutant was quite proficient across the entire range of distances. The topoisomerase mutant crosses demonstrated virtually complete absence of rII(+) recombinants at distances up to 33 bp, with the frequencies increasing steadily at longer distances. The data were interpreted as follows. The primase mutant is fully recombination-proficient. An obvious difference from the wild-type state is some shortage of EndoVII function leading to prolonged existence of HJs and thus stretched out ds-branch migration. This is also true for the topoisomerase mutant. However, the latter is deficient in the ss

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

    PubMed Central

    Schipler, Agnes; Iliakis, George

    2013-01-01

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

  20. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

    PubMed

    Deniz, Miriam; Kaufmann, Julia; Stahl, Andreea; Gundelach, Theresa; Janni, Wolfgang; Hoffmann, Isabell; Keimling, Marlen; Hampp, Stephanie; Ihle, Michaela; Wiesmüller, Lisa

    2016-11-01

    Dysfunction of homologous recombination is a common denominator of changes associated with breast cancer-predisposing mutations. In our previous work, we identified a functional signature in peripheral blood lymphocytes from women who were predisposed that indicated a shift from homologous recombination to alternative, error-prone DNA double-strand break (DSB) repair pathways. To capture both hereditary and nonhereditary factors, we newly established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial-mesenchymal transition cells (EMTs), and fibroblasts from breast cancer specimens (147 patients). By applying a fluorescence-based test system, we analyzed the error-prone DSB repair pathway microhomology-mediated end joining in these tumor-derived cell types and peripheral blood lymphocytes. In parallel, we investigated DNA lesion processing by quantitative immunofluorescence microscopy of histone H2AX phosphorylated on Ser139 focus after radiomimetic treatment. Our study reveals elevated histone H2AX phosphorylated on Ser139 damage removal in epithelial cells, not EMTs, and poly(ADP-ribose)polymerase inhibitor sensitivities, which suggested a DSB repair pathway shift with increasing patient age. Of interest, we found elevated microhomology-mediated end joining in EMTs, not epithelial cells, from patients who received a treatment recommendation of adjuvant chemotherapy, that is, those with high-risk tumors. Our discoveries of altered DSB repair activities in cells may serve as a method to further classify breast cancer to predict responsiveness to adjuvant chemotherapy and/or therapeutics that target DSB repair-dysfunctional tumors.-Deniz, M., Kaufmann, J., Stahl, A., Gundelach, T., Janni, W., Hoffmann, I., Keimling, M., Hampp, S., Ihle, M., Wiesmüller, L. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

  1. 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.

  2. Influence of Double-Strand Break Repair on Radiation Therapy-Induced Acute Skin Reactions in Breast Cancer Patients

    SciTech Connect

    Mumbrekar, Kamalesh Dattaram; Fernandes, Donald Jerard; Goutham, Hassan Venkatesh; Sharan, Krishna; Vadhiraja, Bejadi Manjunath; Satyamoorthy, Kapaettu; Bola Sadashiva, Satish Rao

    2014-03-01

    Purpose: Curative radiation therapy (RT)-induced toxicity poses strong limitations for efficient RT and worsens the quality of life. The parameter that explains when and to what extent normal tissue toxicity in RT evolves would be of clinical relevance because of its predictive value and may provide an opportunity for personalized treatment approach. Methods and Materials: DNA double-strand breaks and repair were analyzed by microscopic γ-H2AX foci analysis in peripheral lymphocytes from 38 healthy donors and 80 breast cancer patients before RT, a 2 Gy challenge dose of x-ray exposed in vitro. Results: The actual damage (AD) at 0.25, 3, and 6 hours and percentage residual damage (PRD) at 3 and 6 hours were used as parameters to measure cellular radiosensitivity and correlated with RT-induced acute skin reactions in patients stratified as non-overresponders (NOR) (Radiation Therapy Oncology Group [RTOG] grade <2) and overresponders (OR) (RTOG grade ≥2). The results indicated that the basal and induced (at 0.25 and 3 hours) γ-H2AX foci numbers were nonsignificant (P>.05) between healthy control donors and the NOR and OR groups, whereas it was significant between ORs and healthy donors at 6 hours (P<.001). There was a significantly higher PRD in OR versus NOR (P<.05), OR versus healthy donors (P<.001) and NOR versus healthy donors (P<.01), supported further by the trend analysis (r=.2392; P=.0326 at 6 hours). Conclusions: Our findings strongly suggest that the measurement of PRD by performing γ-H2AX foci analysis has the potential to be developed into a clinically useful predictive assay.

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

    PubMed

    Delacôte, Fabien; Lopez, Bernard S

    2008-01-01

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

  4. Triggering of DNA strand breaks by 45 degrees C hyperthermia and its influence on the repair of gamma-radiation damage in human white blood cells

    SciTech Connect

    Mitchel, R.E.; Birnboim, H.C.

    1985-05-01

    Human peripheral white blood cells, freshly isolated from normal individual donors, were exposed to hyperthermia. Heat-generated DNA strand break damage and white blood cell capacity to repair radiation-induced breaks were determined by a fluorometric alkaline unwinding assay. Strand breaks could be readily detected when white blood cells were incubated in a physiological salt solution at temperatures between 41 degrees and 46 degrees C, for times up to 90 min. The time course of strand break induction at 45 degrees C was characterized by a short initial lag, followed by a period of rapid break induction and subsequently a lower rate. Evidence is presented which suggests that the induction of DNA damage involved a triggering mechanism; a short treatment at 45 degrees C (10 to 20 min) initiated a cellular event which led to a rapid increase in the number of strand breaks during subsequent incubation of 37 degrees C. Continuous incubation at 45 degrees C produced less DNA damage than an initial period at 45 degrees C followed by incubation at 37 degrees C. This apparent triggering phenomenon was not due to a triggering of the respiratory burst in phagocytic cells, since no O/sub 2/- could be detected; in fact, a 30-min treatment at 45 degrees C largely blocked the capacity of the cells to respond normally to a soluble stimulator of the respiratory burst. Unlike gamma-ray-induced breaks, 45 degrees C hyperthermia-induced breaks did not rejoin during subsequent incubation for up to 1 h at 37 degrees C.

  5. In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage

    PubMed Central

    Hare, Ian; Gencheva, Marieta; Evans, Rebecca; Fortney, James; Piktel, Debbie; Vos, Jeffrey A.; Howell, David; Gibson, Laura F.

    2016-01-01

    Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation. PMID:26880992

  6. In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage.

    PubMed

    Hare, Ian; Gencheva, Marieta; Evans, Rebecca; Fortney, James; Piktel, Debbie; Vos, Jeffrey A; Howell, David; Gibson, Laura F

    2016-01-01

    Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation.

  7. Can a mixed damage interfere with DNA-protein cross-links repair?

    PubMed

    Marzano, C; Severin, E; Bordin, F

    2001-01-01

    Some photochemical and photobiological properties of 4,5',8-trimethylpsoralen (TMP) have been studied in comparison with 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2 one (FQ) and 8-methoxypsoralen (8-MOP). TMP and FQ can photobind to mammalian cell DNA in vivo, by UVA irradiation, forming DNA-protein cross-links (DPC), but only TMP shows a strong capacity of inducing interstrand cross-links (ISC). The mechanism of DPC formation was studied using the double irradiation method in Chinese hamster ovary (CHO) cells, and DPC were detected by alkaline elution. Both TMP and FQ induce covalent diadducts linking together DNA and proteins. Studying the formation of double strand breaks (DSB) in CHO cells we observed that TMP induced a low amount of DSB, similar to 8-MOP. TMP and 8-MOP induced chromosomal aberrations in CHO cells to the same extent, while FQ appeared to be more active. Our data suggest that the ISC induced by TMP could trap enzymes involved in DPC repair.

  8. The Impact of Individual In Vivo Repair of DNA Double-Strand Breaks on Oral Mucositis in Adjuvant Radiotherapy of Head-and-Neck Cancer

    SciTech Connect

    Fleckenstein, Jochen; Kuehne, Martin; Seegmueller, Katharina; Derschang, Sarah; Melchior, Patrick; Graeber, Stefan; Fricke, Andreas; Ruebe, Claudia E.; Ruebe, Christian

    2011-12-01

    Purpose: To evaluate the impact of individual in vivo DNA double-strand break (DSB) repair capacity on the incidence of severe oral mucositis in patients with head-and-neck cancer undergoing adjuvant radiotherapy (RT) or radiochemotherapy (RCT). Patients and Methods: Thirty-one patients with resected head-and-neck cancer undergoing adjuvant RT or RCT were examined. Patients underwent RT of the primary tumor site and locoregional lymph nodes with a total dose of 60-66 Gy (single dose 2 Gy, five fractions per week). Chemotherapy consisted of two cycles of cisplatin and 5-fluorouracil. To assess DSB repair, {gamma}-H2AX foci in blood lymphocytes were quantified before and 0.5 h, 2.5 h, 5 h, and 24 h after in vivo radiation exposure (the first fraction of RT). World Health Organization scores for oral mucositis were documented weekly and correlated with DSB repair. Results: Sixteen patients received RT alone; 15 patients received RCT. In patients who developed Grade {>=} 3 mucositis (n = 18) the amount of unrepaired DSBs 24 h after radiation exposure and DSB repair half-times did not differ significantly from patients with Grade {<=}2 mucositis (n = 13). Patients with a proportion of unrepaired DSBs after 24 h higher than the mean value + one standard deviation had an increased incidence of severe oral mucositis. Conclusions: Evaluation of in vivo DSB repair by determination of {gamma}-H2AX foci loss is feasible in clinical practice and allows identification of patients with impaired DSB repair. The incidence of oral mucositis is not closely correlated with DSB repair under the evaluated conditions.

  9. Non-covalent interactions between ATP and RecA DNA-repairing proteins: DFT and semiempirical calculations

    NASA Astrophysics Data System (ADS)

    Rodriguez, Jorge

    2015-03-01

    The role of Bacterial RecA in the structural maintenance of genomes and the genetic information they carry has been established. In particular, the RecA DNA-repairing protein from D. Radiodurans, a radiation-resistant bacteria, is crucial for the repair of double strand breaks (DSBs). We have performed semi-empirical free-energy calculations and QM/MM calculations to study their non-covalent interactions with ATP and ADP. Such studies provide insight into the mechanisms of ATP/ADP --> RecA energy transfer and, therefore, about specific functional uses of incoming energy for DNA repairing mechanisms. We present a detailed analysis of the non-covalent interactions which minimize the interaction Gibbs free energies leading to the most stable non-covalent binding sites. Van der Waal, hydrogen bonding and electrostatic interactions has been quantified which provides a detailed insight into the mechanisms of ATP-RecA interaction. Further, possible chemical interactions and functional roles of RecA proteins are explored based on the previously mentioned studies. Acknowledgements: Funded, in part, by DTRA award 106339 (JHR). Dr. Mark C. Palenik and Mrs. Lora Beard are gratefully acknowledged Supported in part by DTRA Award 106339.

  10. Gefitinib Radiosensitizes Stem-Like Glioma Cells: Inhibition of Epidermal Growth Factor Receptor-Akt-DNA-PK Signaling, Accompanied by Inhibition of DNA Double-Strand Break Repair

    SciTech Connect

    Kang, Khong Bee; Zhu Congju; Wong Yinling; Gao Qiuhan; Ty, Albert; Wong, Meng Cheong

    2012-05-01

    Purpose: We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Methods and Materials: Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, {gamma}-H{sub 2}AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, {gamma}-H{sub 2}AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Results: Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G{sub 2}/M arrest and increased {gamma}-H{sub 2}AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased {gamma}-H{sub 2}AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Conclusions: Stem-like gliomaspheres are

  11. Identification of two novel BRCA1-partner genes in the DNA double-strand break repair pathway.

    PubMed

    Guglielmi, Chiara; Cerri, Iacopo; Evangelista, Monica; Collavoli, Anita; Tancredi, Mariella; Aretini, Paolo; Caligo, Maria Adelaide

    2013-10-01

    M1775R and A1789T are two missense variants located within the BRCT domains of BRCA1 gene. The M1775R is a known deleterious variant, while the A1789T is an unclassified variant that has been analyzed and classified as probably deleterious for the first time by our group. In a previous study, we described the expression profile of HeLa G1 cells transfected with the two variants and we found that they altered molecular mechanisms critical for cell proliferation and genome integrity. Considering that the mutations in the BRCA1 C terminus (BRCT) domains are associated to a phenotype with an altered ability in the DNA double-strand break repair, we chose three of the genes previously identified, EEF1E1, MRE11A, and OBFC2B, to be tested for an homologous recombination (HR) in vitro assay. For our purpose, we performed a gene expression knockdown by siRNA transfection in HeLa cells, containing an integrated recombination substrate (hprtDRGFP), for each of the target genes included BRCA1. The knockdown of BRCA1, OBFC2B, MRE11A, and EEF1E1 reduces the HR rate, respectively, of 97.6, 28.6, 41.8, and 42.3 % compared to cells transfected with a scrambled negative control duplex and all these differences are statistically significant (P < 0.05; Kruskal-Wallis test). Finally, we analyzed the effect of target gene depletion both on HR that on cell survival after DNA-damage induction by ionizing radiation. The clonogenic assay showed that the down-regulation of the target genes reduced the cell survival, but the effect on the HR, is not evident. Only the BRCA1-siRNA confirmed the inhibition effect on HR. Overall these results confirmed the involvement of MRE11A in the HR pathway and identified two new genes, OBFC2B and EEF1E1, which according to these data and the knowledge obtained from literature, might be involved in BRCA1-pathway.

  12. The DNA-repair Ku70 protein is located in the nucleus and tail of elongating spermatids in grasshoppers.

    PubMed

    Cabrero, Josefa; Palomino-Morales, Rogelio J; Camacho, Juan Pedro M

    2007-01-01

    Fluorescence immunostaining for the phosphorylated H2AX histone (gammaH2AX) in the grasshopper Eyprepocnemis plorans has shown abundance of gammaH2AX in the nuclei of round and elongating spermatids, suggesting that DNA double-strand breaks (DSBs) occur regularly during spermiogenesis. Immunofluorescence patterns for Ku70, a DNA-repair protein participating in the non-homologous end-joining (NHEJ) pathway, showed that this protein is present in round and elongating spermatids, implying that the NHEJ DNA-repair pathway operates during chromatin compaction in spermiogenesis. In addition, during the final stages of spermiogenesis, the Ku70 protein concentrates on the region forming the sperm tail. Since Ku70 was also abundant in spermatid tails, it is reasonable to assume that Ku70 might play a novel function in sperm-tail formation. The analysis of Ku70 immunofluorescence patterns in 13 other grasshopper species also showed the presence of this protein in the nucleus and tail of elongating spermatids, indicating that this is a general characteristic in grasshoppers.

  13. Mismatch repair proteins MSH2, MLH1, and EXO1 are important for class-switch recombination events occurring in B cells that lack nonhomologous end joining.

    PubMed

    Eccleston, Jennifer; Yan, Catherine; Yuan, Karen; Alt, Frederick W; Selsing, Erik

    2011-02-15

    In the absence of core nonhomologous end-joining (NHEJ) factors, Ab gene class-switch recombination (CSR) uses an alternative end-joining (A-EJ) pathway to recombine switch (S) region DNA breaks. Previous reports showing decreased S-junction microhomologies in MSH2-deficient mice and an exonuclease 1 (EXO1) role in yeast microhomology-mediated end joining suggest that mismatch repair (MMR) proteins might influence A-EJ-mediated CSR. We have directly investigated whether MMR proteins collectively or differentially influence the A-EJ mechanism of CSR by analyzing CSR in mice deficient in both XRCC4 and individual MMR proteins. We find CSR is reduced and that Igh locus chromosome breaks are reduced in the MMR/XRCC4 double-deficient B cells compared with B cells deficient in XRCC4 alone, suggesting MMR proteins function upstream of double-strand break formation to influence CSR efficiency in these cells. Our results show that MLH1, EXO1, and MSH2 are all important for efficient A-EJ-mediated CSR, and we propose that MMR proteins convert DNA nicks and point mutations into dsDNA breaks for both C-NHEJ and A-EJ pathways of CSR. We also find Mlh1-XRCC4(-) B cells have an increased frequency of direct S junctions, suggesting that MLH1 proteins may have additional functions that influence A-EJ-mediated CSR.

  14. Estimating the effect of human base excision repair protein variants on the repair of oxidative DNA base damage.

    PubMed

    Sokhansanj, Bahrad A; Wilson, David M

    2006-05-01

    Epidemiologic studies have revealed a complex association between human genetic variance and cancer risk. Quantitative biological modeling based on experimental data can play a critical role in interpreting the effect of genetic variation on biochemical pathways relevant to cancer development and progression. Defects in human DNA base excision repair (BER) proteins can reduce cellular tolerance to oxidative DNA base damage caused by endogenous and exogenous sources, such as exposure to toxins and ionizing radiation. If not repaired, DNA base damage leads to cell dysfunction and mutagenesis, consequently leading to cancer, disease, and aging. Population screens have identified numerous single-nucleotide polymorphism variants in many BER proteins and some have been purified and found to exhibit mild kinetic defects. Epidemiologic studies have led to conflicting conclusions on the association between single-nucleotide polymorphism variants in BER proteins and cancer risk. Using experimental data for cellular concentration and the kinetics of normal and variant BER proteins, we apply a previously developed and tested human BER pathway model to (i) estimate the effect of mild variants on BER of abasic sites and 8-oxoguanine, a prominent oxidative DNA base modification, (ii) identify ranges of variation associated with substantial BER capacity loss, and (iii) reveal nonintuitive consequences of multiple simultaneous variants. Our findings support previous work suggesting that mild BER variants have a minimal effect on pathway capacity whereas more severe defects and simultaneous variation in several BER proteins can lead to inefficient repair and potentially deleterious consequences of cellular damage.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  16. ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates.

    PubMed

    Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M; Bianco, Piero R; Surtees, Jennifer A

    2014-06-01

    In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3' non-homologous tail removal (3'NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3'NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3'NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3'NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype.

  17. Reduced DNA double-strand break repair capacity and risk of squamous cell carcinoma of the head and neck--A case-control study.

    PubMed

    Liu, Zhensheng; Liu, Hongliang; Gao, Fengqin; Dahlstrom, Kristina R; Sturgis, Erich M; Wei, Qingyi

    2016-04-01

    Tobacco smoke and alcohol use play important roles in the etiology of squamous cell carcinoma of the head and neck (SCCHN). Smoking causes DNA damage, including double-strand DNA breaks (DSBs), that leads to carcinogenesis. To test the hypothesis that suboptimal DSB repair capacity is associated with risk of SCCHN, we applied a flow cytometry-based method to detect the DSB repair phenotype first in four EBV-immortalized human lymphoblastoid cell lines and then in human peripheral blood T-lymphocytes (PBTLs). With this blood-based laboratory assay, we conducted a pilot case-control study of 100 patients with newly diagnosed, previously untreated SCCHN and 124 cancer-free controls of non-Hispanic whites. We found that the mean DSB repair capacity level was significantly lower in cases (42.1%) than that in controls (54.4%) (P<0.001). When we used the median DSB repair capacity level in the controls as the cutoff value for calculating the odds ratios (ORs) with adjustment for age, sex, smoking and drinking status, the cases were more likely than the controls to have a reduced DSB repair capacity (adjusted OR=1.93; 95% confidence interval, CI=1.04-3.56, P=0.037), especially for those subjects who were ever drinkers (adjusted OR=2.73; 95% CI=1.17-6.35, P=0.020) and had oropharyngeal tumors (adjusted OR=2.17; 95% CI=1.06-4.45, P=0.035). In conclusion, these findings suggest that individuals with a reduced DSB repair capacity may be at an increased risk of developing SCCHN. Larger studies are warranted to confirm these preliminary findings.

  18. Nej1 recruits the Srs2 helicase to DNA double-strand breaks and supports repair by a single-strand annealing-like mechanism.

    PubMed

    Carter, Sidney D; Vigasová, Dana; Chen, Jiang; Chovanec, Miroslav; Aström, Stefan U

    2009-07-21

    Double-strand breaks (DSBs) represent the most severe DNA lesion a cell can suffer, as they pose the risk of inducing loss of genomic integrity and promote oncogenesis in mammals. Two pathways repair DSBs, nonhomologous end joining (NHEJ) and homologous recombination (HR). With respect to mechanism and genetic requirements, characterization of these pathways has revealed a large degree of functional separation between the two. Nej1 is a cell-type specific regulator essential to NHEJ in Saccharomyces cerevisiae. Srs2 is a DNA helicase with multiple roles in HR. In this study, we show that Nej1 physically interacts with Srs2. Furthermore, mutational analysis of Nej1 suggests that the interaction was strengthened by Dun1-dependent phosphorylation of Nej1 serines 297/298. Srs2 was previously shown to be recruited to replication forks, where it promotes translesion DNA synthesis. We demonstrate that Srs2 was also efficiently recruited to DSBs generated by the HO endonuclease. Additionally, efficient Srs2 recruitment to this DSB was dependent on Nej1, but independent of mechanisms facilitating Srs2 recruitment to replication forks. Functionally, both Nej1 and Srs2 were required for efficient repair of DSBs with 15-bp overhangs, a repair event reminiscent of a specific type of HR called single-strand annealing (SSA). Moreover, absence of Rad51 suppressed the SSA-defect in srs2 and nej1 strains. We suggest a model in which Nej1 recruits Srs2 to DSBs to promote NHEJ/SSA-like repair by dismantling inappropriately formed Rad51 nucleoprotein filaments. This unexpected link between NHEJ and HR components may represent cross-talk between DSB repair pathways to ensure efficient repair.

  19. Nej1 recruits the Srs2 helicase to DNA double-strand breaks and supports repair by a single-strand annealing-like mechanism

    PubMed Central

    Carter, Sidney D.; Vigašová, Dana; Chen, Jiang; Chovanec, Miroslav; Åström, Stefan U.

    2009-01-01

    Double-strand breaks (DSBs) represent the most severe DNA lesion a cell can suffer, as they pose the risk of inducing loss of genomic integrity and promote oncogenesis in mammals. Two pathways repair DSBs, nonhomologous end joining (NHEJ) and homologous recombination (HR). With respect to mechanism and genetic requirements, characterization of these pathways has revealed a large degree of functional separation between the two. Nej1 is a cell-type specific regulator essential to NHEJ in Saccharomyces cerevisiae. Srs2 is a DNA helicase with multiple roles in HR. In this study, we show that Nej1 physically interacts with Srs2. Furthermore, mutational analysis of Nej1 suggests that the interaction was strengthened by Dun1-dependent phosphorylation of Nej1 serines 297/298. Srs2 was previously shown to be recruited to replication forks, where it promotes translesion DNA synthesis. We demonstrate that Srs2 was also efficiently recruited to DSBs generated by the HO endonuclease. Additionally, efficient Srs2 recruitment to this DSB was dependent on Nej1, but independent of mechanisms facilitating Srs2 recruitment to replication forks. Functionally, both Nej1 and Srs2 were required for efficient repair of DSBs with 15-bp overhangs, a repair event reminiscent of a specific type of HR called single-strand annealing (SSA). Moreover, absence of Rad51 suppressed the SSA-defect in srs2 and nej1 strains. We suggest a model in which Nej1 recruits Srs2 to DSBs to promote NHEJ/SSA-like repair by dismantling inappropriately formed Rad51 nucleoprotein filaments. This unexpected link between NHEJ and HR components may represent cross-talk between DSB repair pathways to ensure efficient repair. PMID:19571008

  20. Benzo[a]pyrene increases DNA double strand break repair in vitro and in vivo: a possible mechanism for benzo[a]pyrene-induced toxicity.

    PubMed

    Tung, Emily W Y; Philbrook, Nicola A; Belanger, Christine L; Ansari, Saad; Winn, Louise M

    2014-01-15

    Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon and carcinogen that is released into the environment through natural and anthropogenic sources. BaP toxicity is dependent on its metabolism by cytochrome P450s to the reactive metabolite benzo[a]pyrene diol epoxide (BPDE), which is strongly associated with increased mutation frequency. BaP can also be metabolized to benzo[a]pyrene quinones that can undergo redox cycling and induce oxidative stress. The purpose of this study was to examine if BaP exposure induces DNA double strand breaks (DSBs) and subsequently activate DNA DSB repair pathways in the CHO 3-6 cell line and pKZ1 mouse model. In vitro assessment of homologous recombination (HR) showed significantly increased HR frequency following exposure to 10μM of BaP. In vivo evaluations of BaP-induced DNA DSB repair demonstrated positive staining for intrachromosomal recombination events, which are associated with non-homologous end joining (NHEJ), in the lung and thymus of exposed animals that were statistically significant in the thymus when quantified by Western blotting. Gene expression analyses from mouse tissues showed significantly decreased expression of ATM and Xrcc6 in BaP-treated liver and lung. In addition, BaP exposure significantly reduced the expression of Xrcc5, p53, and DNA-PKcs in lung. Taken together, our results demonstrate that BaP increases DNA DSB repair in vitro and in vivo, and induces expression changes in DNA repair pathway genes. As repair of DNA DSBs is not error-free, aberrant DNA repair may be contributing to the mechanism of BaP-induced toxicity.

  1. DNA breaks caused by monochromatic 365 nm ultraviolet-A radiation or hydrogen peroxide and their repair in human epithelioid and xeroderma pigmentosum cells.

    PubMed

    Peak, J G; Pilas, B; Dudek, E J; Peak, M J

    1991-08-01

    The induction and repair of DNA single-strand breaks (SSB) assayed by alkaline filter elution was compared in human epithelioid P3 and xeroderma pigmentosum (XP) cells exposed to monochromatic 365-nm UV-A radiation and H2O2. Initial yields of SSB were measured with the cells held at 0.5 degrees C during exposure. The yield from exposure to 365-nm radiation was slightly greater in XP than in P3 cells, whereas H2O2 produced more than three times as many SSB in P3 compared with XP cells. o-Phenanthroline (50 mM) markedly inhibited the yields of SSB induced in XP cells by H2O2, but had no effect on those produced by 365-nm UV-A. These results are consistent with the fact that P3 cells, unlike XP cells, have undetectable levels of catalase. The measured production of trace amounts of H2O2 by the actual 365-nm UV-A exposures was not sufficient to account for the numbers of breaks that were observed. Single-strand breaks produced by both agents were completely repaired after 50 min in P3 cells, as were H2O2-induced SSB in XP cells. However, 25% of the 365-nm UV-A-induced SSB in XP cells remained refractory to repair after 60 min. The results show that SSB produced by these two agents are different and that 365 nm radiation produces most SSB in cells by mechanisms other than by production of H2O2.

  2. G9a inhibition potentiates the anti-tumour activity of DNA double-strand break inducing agents by impairing DNA repair independent of p53 status.

    PubMed

    Agarwal, Pallavi; Jackson, Stephen P

    2016-10-01

    Cancer cells often exhibit altered epigenetic signatures that can misregulate genes involved in processes such as transcription, proliferation, apoptosis and DNA repair. As regulation of chromatin structure is crucial for DNA repair processes, and both DNA repair and epigenetic controls are deregulated in many cancers, we speculated that simultaneously targeting both might provide new opportunities for cancer therapy. Here, we describe a focused screen that profiled small-molecule inhibitors targeting epigenetic regulators in combination with DNA double-strand break (DSB) inducing agents. We identify UNC0638, a catalytic inhibitor of histone lysine N-methyl-transferase G9a, as hypersensitising tumour cells to low doses of DSB-inducing agents without affecting the growth of the non-tumorigenic cells tested. Similar effects are also observed with another, structurally distinct, G9a inhibitor A-366. We also show that small-molecule inhibition of G9a or siRNA-mediated G9a depletion induces tumour cell death under low DNA damage conditions by impairing DSB repair in a p53 independent manner. Furthermore, we establish that G9a promotes DNA non-homologous end-joining in response to DSB-inducing genotoxic stress. This study thus highlights the potential for using G9a inhibitors as anti-cancer therapeutic agents in combination with DSB-inducing chemotherapeutic drugs such as etoposide.

  3. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes.

    PubMed

    Ono, Ryuichi; Ishii, Masayuki; Fujihara, Yoshitaka; Kitazawa, Moe; Usami, Takako; Kaneko-Ishino, Tomoko; Kanno, Jun; Ikawa, Masahito; Ishino, Fumitoshi

    2015-07-28

    The CRISPR/Cas system efficiently introduces double strand breaks (DSBs) at a genomic locus specified by a single guide RNA (sgRNA). The DSBs are subsequently repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Here, we demonstrate that DSBs introduced into mouse zygotes by the CRISPR/Cas system are repaired by the capture of DNA sequences deriving from retrotransposons, genomic DNA, mRNA and sgRNA. Among 93 mice analysed, 57 carried mutant alleles and 22 of them had long de novo insertion(s) at DSB-introduced sites; two were spliced mRNAs of Pcnt and Inadl without introns, indicating the involvement of reverse transcription (RT). Fifteen alleles included retrotransposons, mRNAs, and other sequences without evidence of RT. Two others were sgRNAs with one containing T7 promoter-derived sequence suggestive of a PCR product as its origin. In conclusion, RT-product-mediated DSB repair (RMDR) and non-RMDR repair were identified in the mouse zygote. We also confirmed that both RMDR and non-RMDR take place in CRISPR/Cas transfected NIH-3T3 cells. Finally, as two de novo MuERV-L insertions in C57BL/6 mice were shown to have characteristic features of RMDR in natural conditions, we hypothesize that RMDR contributes to the emergence of novel DNA sequences in the course of evolution.

  4. The role of the Mre11-Rad50-Nbs1 complex in double-strand break repair-facts and myths.

    PubMed

    Takeda, Shunichi; Hoa, Nguyen Ngoc; Sasanuma, Hiroyuki

    2016-08-01

    Homologous recombination (HR) initiates double-strand break (DSB) repair by digesting 5'-termini at DSBs, the biochemical reaction called DSB resection, during which DSBs are processed by nucleases to generate 3' single-strand DNA. Rad51 recombinase polymerizes along resected DNA, and the resulting Rad51-DNA complex undergoes homology search. Although DSB resection by the Mre11 nuclease plays a critical role in HR in Saccharomyces cerevisiae, it remains elusive whether DSB resection by Mre11 significantly contributes to HR-dependent DSB repair in mammalian cells. Depletion of Mre11 decreases the efficiency of DSB resection only by 2- to 3-fold in mammalian cells. We show that although Mre11 is required for efficient HR-dependent repair of ionizing-radiation-induced DSBs, Mre11 is largely dispensable for DSB resection in both chicken DT40 and human TK6 B cell lines. Moreover, a 2- to 3-fold decrease in DSB resection has virtually no impact on the efficiency of HR. Thus, although a large number of researchers have reported the vital role of Mre11-mediated DSB resection in HR, the role may not explain the very severe defect in HR in Mre11-deficient cells, including their lethality. We here show experimental evidence for the additional roles of Mre11 in (i) elimination of chemical adducts from DSB ends for subsequent DSB repair, and (ii) maintaining HR intermediates for their proper resolution.

  5. The Vitamin A Derivative All-Trans Retinoic Acid Repairs Amyloid-β-Induced Double-Strand Breaks in Neural Cells and in the Murine Neocortex.

    PubMed

    Gruz-Gibelli, Emmanuelle; Chessel, Natacha; Allioux, Clélia; Marin, Pascale; Piotton, Françoise; Leuba, Geneviève; Herrmann, François R; Savioz, Armand

    2016-01-01

    The amyloid-β peptide or Aβ is the key player in the amyloid-cascade hypothesis of Alzheimer's disease. Aβ appears to trigger cell death but also production of double-strand breaks (DSBs) in aging and Alzheimer's disease. All-trans retinoic acid (RA), a derivative of vitamin A, was already known for its neuroprotective effects against the amyloid cascade. It diminishes, for instance, the production of Aβ peptides and their oligomerisation. In the present work we investigated the possible implication of RA receptor (RAR) in repair of Aβ-induced DSBs. We demonstrated that RA, as well as RAR agonist Am80, but not AGN 193109 antagonist, repair Aβ-induced DSBs in SH-SY5Y cells and an astrocytic cell line as well as in the murine cortical tissue of young and aged mice. The nonhomologous end joining pathway and the Ataxia Telangiectasia Mutated kinase were shown to be involved in RA-mediated DSBs repair in the SH-SY5Y cells. Our data suggest that RA, besides increasing cell viability in the cortex of young and even of aged mice, might also result in targeted DNA repair of genes important for cell or synaptic maintenance. This phenomenon would remain functional up to a point when Aβ increase and RA decrease probably lead to a pathological state.

  6. DNA-PK triggers histone ubiquitination and signaling in response to DNA double-strand breaks produced during the repair of transcription-blocking topoisomerase I lesions.

    PubMed

    Cristini, Agnese; Park, Joon-Hyung; Capranico, Giovanni; Legube, Gaëlle; Favre, Gilles; Sordet, Olivier

    2016-02-18

    Although defective repair of DNA double-strand breaks (DSBs) leads to neurodegenerative diseases, the processes underlying their production and signaling in non-replicating cells are largely unknown. Stabilized topoisomerase I cleavage complexes (Top1cc) by natural compounds or common DNA alterations are transcription-blocking lesions whose repair depends primarily on Top1 proteolysis and excision by tyrosyl-DNA phosphodiesterase-1 (TDP1). We previously reported that stabilized Top1cc produce transcription-dependent DSBs that activate ATM in neurons. Here, we use camptothecin (CPT)-treated serum-starved quiescent cells to induce transcription-blocking Top1cc and show that those DSBs are generated during Top1cc repair from Top1 peptide-linked DNA single-strand breaks generated after Top1 proteolysis and before excision by TDP1. Following DSB induction, ATM activates DNA-PK whose inhibition suppresses H2AX and H2A ubiquitination and the later assembly of activated ATM into nuclear foci. Inhibition of DNA-PK also reduces Top1 ubiquitination and proteolysis as well as resumption of RNA synthesis suggesting that DSB signaling further enhances Top1cc repair. Finally, we show that co-transcriptional DSBs kill quiescent cells. Together, these new findings reveal that DSB production and signaling by transcription-blocking Top1 lesions impact on non-replicating cell fate and provide insights on the molecular pathogenesis of neurodegenerative diseases such as SCAN1 and AT syndromes, which are caused by TDP1 and ATM deficiency, respectively.

  7. The bacterial DNA repair protein Mfd confers resistance to the host nitrogen immune response

    PubMed Central

    Guillemet, Elisabeth; Leréec, Alain; Tran, Seav-Ly; Royer, Corinne; Barbosa, Isabelle; Sansonetti, Philippe; Lereclus, Didier; Ramarao, Nalini

    2016-01-01

    Production of reactive nitrogen species (NO) is a key step in the immune response following infections. NO induces lesions to bacterial DNA, thus limiting bacterial growth within hosts. Using two pathogenic bacteria, Bacillus cereus and Shigella flexneri, we show that the DNA-repair protein Mfd (Mutation-Frequency-Decline) is required for bacterial resistance to the host-NO-response. In both species, a mutant deficient for mfd does not survive to NO, produced in vitro or by phagocytic cells. In vivo, the ∆mfd mutant is avirulent and unable to survive the NO-stress. Moreover, NO induces DNA-double-strand-breaks and point mutations in the Δmfd mutant. In overall, these observations demonstrate that NO damages bacterial DNA and that Mfd is required to maintain bacterial genomic integrity. This unexpected discovery reveals that Mfd, a typical housekeeping gene, turns out to be a true virulence factor allowing survival and growth of the pathogen in its host, due to its capacity to protect the bacterium against NO, a key molecule of the innate immune defense. As Mfd is widely conserved in the bacterial kingdom, these data highlight a mechanism that may be used by a large spectrum of bacteria to overcome the host immune response and especially the mutagenic properties of NO. PMID:27435260

  8. Roles for mismatch repair family proteins in promoting meiotic crossing over

    PubMed Central

    Manhart, Carol M.; Alani, Eric

    2015-01-01

    The mismatch repair (MMR) family complexes Msh4-Msh5 and Mlh1-Mlh3 act with Exo1 and Sgs1-Top3-Rmi1 in a meiotic double strand break repair pathway that results in the asymmetric cleavage of double Holliday junctions (dHJ) to form crossovers. This review discusses how meiotic roles for Msh4-Msh5 and Mlh1-Mlh3 do not fit paradigms established for post-replicative MMR. We also outline models used to explain how these factors promote the formation of meiotic crossovers required for the accurate segregation of chromosome homologs during the Meiosis I division. PMID:26686657

  9. A role for Saccharomyces cerevisiae Tpa1 protein in direct alkylation repair.

    PubMed

    Shivange, Gururaj; Kodipelli, Naveena; Monisha, Mohan; Anindya, Roy

    2014-12-26

    Alkylating agents induce cytotoxic DNA base adducts. In this work, we provide evidence to suggest, for the first time, that Saccharomyces cerevisiae Tpa1 protein is involved in DNA alkylation repair. Little is known about Tpa1 as a repair protein beyond the initial observation from a high-throughput analysis indicating that deletion of TPA1 causes methyl methane sulfonate sensitivity in S. cerevisiae. Using purified Tpa1, we demonstrate that Tpa1 repairs both single- and double-stranded methylated DNA. Tpa1 is a member of the Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that mutation of the amino acid residues involved in cofactor binding abolishes the Tpa1 DNA repair activity. Deletion of TPA1 along with the base excision repair pathway DNA glycosylase MAG1 renders the tpa1Δmag1Δ double mutant highly susceptible to methylation-induced toxicity. We further demonstrate that the trans-lesion synthesis DNA polymerase Polζ (REV3) plays a key role in tolerating DNA methyl-base lesions and that tpa1Δmag1revΔ3 triple mutant is extremely susceptible to methylation-induced toxicity. Our results indicate a synergism between the base excision repair pathway and direct alkylation repair by Tpa1 in S. cerevisiae. We conclude that Tpa1 is a hitherto unidentified DNA repair protein in yeast and that it plays a crucial role in reverting alkylated DNA base lesions and cytotoxicity.

  10. Order statistics inference for describing topological coupling and mechanical symmetry breaking in multidomain proteins

    NASA Astrophysics Data System (ADS)

    Kononova, Olga; Jones, Lee; Barsegov, V.

    2013-09-01

    Cooperativity is a hallmark of proteins, many of which show a modular architecture comprising discrete structural domains. Detecting and describing dynamic couplings between structural regions is difficult in view of the many-body nature of protein-protein interactions. By utilizing the GPU-based computational acceleration, we carried out simulations of the protein forced unfolding for the dimer WW - WW of the all-β-sheet WW domains used as a model multidomain protein. We found that while the physically non-interacting identical protein domains (WW) show nearly symmetric mechanical properties at low tension, reflected, e.g., in the similarity of their distributions of unfolding times, these properties become distinctly different when tension is increased. Moreover, the uncorrelated unfolding transitions at a low pulling force become increasingly more correlated (dependent) at higher forces. Hence, the applied force not only breaks "the mechanical symmetry" but also couples the physically non-interacting protein domains forming a multi-domain protein. We call this effect "the topological coupling." We developed a new theory, inspired by order statistics, to characterize protein-protein interactions in multi-domain proteins. The method utilizes the squared-Gaussian model, but it can also be used in conjunction with other parametric models for the distribution of unfolding times. The formalism can be taken to the single-molecule experimental lab to probe mechanical cooperativity and domain communication in multi-domain proteins.

  11. CtIP Protein Dimerization Is Critical for Its Recruitment to Chromosomal DNA Double-stranded Breaks*

    PubMed Central

    Wang, Hailong; Shao, Zhengping; Shi, Linda Z.; Hwang, Patty Yi-Hwa; Truong, Lan N.; Berns, Michael W.; Chen, David J.; Wu, Xiaohua

    2012-01-01

    CtIP (CtBP-interacting protein) associates with BRCA1 and the Mre11-Rad50-Nbs1 (MRN) complex and plays an essential role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair. It has been described that CtIP forms dimers in mammalian cells, but the biological significance is not clear. In this study, we identified a conserved motif in the N terminus of CtIP, which is required for dimer formation. We further showed that CtIP mutants impaired in forming dimers are strongly defective in HR, end resection, and activation of the ataxia telangiectasia and Rad3-related pathway, without notable change of CtIP interactions with BRCA1 or Nbs1. In addition to HR, CtIP dimerization is also required for microhomology-mediated end joining. Live cell imaging of enhanced GFP-tagged CtIP demonstrates that the CtIP dimerization mutant fails to be localized to DSBs, whereas placing a heterologous dimerization motif to the dimerization mutant restores CtIP recruitment to DSBs. These studies suggest that CtIP dimer formation is essential for its recruitment to DSBs on chromatin upon DNA damage. Furthermore, DNA damage-induced phosphorylation of CtIP is significantly reduced in the CtIP dimerization mutants. Therefore, in addition to the C-terminal conserved domains critical for CtIP function, the dimerization motif on the N terminus of CtIP is also conserved and essential for its function in DNA damage responses. The severe repair defects of CtIP dimerization mutants are likely due to the failure in localization to chromosomal DSBs upon DNA damage. PMID:22544744

  12. Proteomic identification of hair cell repair proteins in the model sea anemone Nematostella vectensis.

    PubMed

    Tang, Pei-Ciao; Watson, Glen M

    2015-09-01

    Sea anemones have an extraordinary capability to repair damaged hair bundles, even after severe trauma. A group of secreted proteins, named repair proteins (RPs), found in mucus covering sea anemones significantly assists the repair of damaged hair bundle mechanoreceptors both in the sea anemone Haliplanella luciae and the blind cavefish Astyanax hubbsi. The polypeptide constituents of RPs must be identified in order to gain insight into the molecular mechanisms by which repair of hair bundles is accomplished. In this study, several polypeptides of RPs were isolated from mucus using blue native PAGE and then sequenced using LC-MS/MS. Thirty-seven known polypeptides were identified, including Hsp70s, as well as many polypeptide subunits of the 20S proteasome. Other identified polypeptides included those involved in cellular stress responses, protein folding, and protein degradation. Specific inhibitors of Hsp70s and the 20S proteasome were employed in experiments to test their involvement in hair bundle repair. The results of those experiments suggested that repair requires biologically active Hsp70s and 20S proteasomes. A model is proposed that considers the function of extracellular Hsp70s and 20S proteasomes in the repair of damaged hair cells.

  13. Kinetics of chromatid break repair in G2-human fibroblasts exposed to low- and high-LET radiations

    NASA Technical Reports Server (NTRS)

    Kawata, T.; Durante, M.; George, K.; Furusawa, Y.; Gotoh, E.; Takai, N.; Wu, H.; Cucinotta, F. A.

    2001-01-01

    The purpose of this study is to determine the kinetics of chromatid break rejoining following exposure to radiations of different quality. Exponentially growing human fibroblast cells AG1522 were irradiated with gamma-rays, energetic carbon (290 MeV/u), silicon (490 MeV/u) and iron (200 MeV/u, 600 MeV/u). Chromosomes were prematurely condensed using calyculin A. Prematurely condensed chromosomes were collected after several post-irradiation incubation times, ranging from 5 to 600 minutes, and the number of chromatid breaks and exchanges in G2 cells were scored. The relative biological effectiveness (RBE) for initial chromatid breaks per unit dose showed LET dependency having a peak at 55 keV/micrometers silicon (2.4) or 80 keV/micrometers carbon particles (2.4) and then decreased with increasing LET. The kinetics of chromatid break rejoining following low- or high-LET irradiation consisted of two exponential components. Chromatid breaks decreased rapidly after exposure, and then continued to decrease at a slower rate. The rejoining kinetics was similar for exposure to each type of radiation, although the rate of unrejoined breaks was higher for high-LET radiation. Chromatid exchanges were also formed quickly.

  14. The H2B ubiquitin ligase RNF40 cooperates with SUPT16H to induce dynamic changes in chromatin structure during DNA double-strand break repair.

    PubMed

    Kari, Vijayalakshmi; Shchebet, Andrei; Neumann, Heinz; Johnsen, Steven A

    2011-10-15

    Many anticancer therapies function largely by inducing DNA double-strand breaks (DSBs) or altering the ability of cancer cells to repair them. Proper and timely DNA repair requires dynamic changes in chromatin assembly and disassembly characterized by histone H3 lysine 56 acetylation (H3K56ac) and phosphorylation of the variant histone H2AX (γH2AX). Similarly, histone H2B monoubiquitination (H2Bub1) functions in DNA repair, but its role in controlling dynamic changes in chromatin structure following DSBs and the histone chaperone complexes involved remain unknown. Therefore, we investigated the role of the H2B ubiquitin ligase RNF40 in the DSB response. We show that RNF40 depletion results in sustained H2AX phosphorylation and a decrease in rapid cell cycle checkpoint activation. Furthermore, RNF40 knockdown resulted in decreased H3K56ac and decreased recruitment of the facilitates chromatin transcription (FACT) complex to chromatin following DSB. Knockdown of the FACT component suppressor of Ty homolog-16 (SUPT16H) phenocopied the effects of RNF40 knockdown on both γH2AX and H3K56ac following DSB induction. Consistently, both RNF40 and SUPT16H were required for proper DNA end resection and timely DNA repair, suggesting that H2Bub1 and FACT cooperate to increase chromatin dynamics, which facilitates proper checkpoint activation and timely DNA repair. These results provide important mechanistic insights into the tumor suppressor function of H2Bub1 and provide a rational basis for pursuing H2Bub1-based therapies in conjunction with traditional chemo- and radiotherapy.

  15. Common and unique genetic interactions of the poly(ADP-ribose) polymerases PARP1 and PARP2 with DNA double-strand break repair pathways.

    PubMed

    Ghosh, Rajib; Roy, Sanchita; Kamyab, Johan; Dantzer, Francoise; Franco, Sonia

    2016-09-01

    In mammalian cells, chromatin poly(ADP-ribos)ylation (PARylation) at sites of DNA Double-Strand Breaks (DSBs) is mediated by two highly related enzymes, PARP1 and PARP2. However, enzyme-specific genetic interactions with other DSB repair factors remain largely undefined. In this context, it was previously shown that mice lacking PARP1 and H2AX, a histone variant that promotes DSB repair throughout the cell cycle, or the core nonhomologous end-joining (NHEJ) factor Ku80 are not viable, while mice lacking PARP1 and the noncore NHEJ factor DNA-PKcs are severely growth retarded and markedly lymphoma-prone. Here, we have examined the requirement for PARP2 in these backgrounds. We find that, like PARP1, PARP2 is essential for viability in mice lacking H2AX. Moreover, treatment of H2AX-deficient primary fibroblasts or B lymphocytes with PARP inhibitors leads to activation of the G2/M checkpoint and accumulation of chromatid-type breaks in a lineage- and gene-dose dependent manner. In marked contrast to PARP1, loss of PARP2 does not result in additional phenotypes in growth, development or tumorigenesis in mice lacking either Ku80 or DNA-PKcs. Altogether these findings highlight specific nonoverlapping functions of PARP1 and PARP2 at H2AX-deficient chromatin during replicative phases of the cell cycle and uncover a unique requirement for PARP1 in NHEJ-deficient cells.

  16. Either non-homologous ends joining or homologous recombination is required to repair double-strand breaks in the genome of macrophage-internalized Mycobacterium tuberculosis.

    PubMed

    Brzostek, Anna; Szulc, Izabela; Klink, Magdalena; Brzezinska, Marta; Sulowska, Zofia; Dziadek, Jaroslaw

    2014-01-01

    The intracellular pathogen Mycobacterium tuberculosis (Mtb) is constantly exposed to a multitude of hostile conditions and is confronted by a variety of potentially DNA-damaging assaults in vivo, primarily from host-generated antimicrobial toxic radicals. Exposure to reactive nitrogen species and/or reactive oxygen species causes different types of DNA damage, including oxidation, depurination, methylation and deamination, that can result in single- or double-strand breaks (DSBs). These breaks affect the integrity of the whole genome and, when left unrepaired, can lead to cell death. Here, we investigated the role of the DSB repair pathways, homologous recombination (HR) and non-homologous ends joining (NHEJ), in the survival of Mtb inside macrophages. To this end, we constructed Mtb strains defective for HR (ΔrecA), NHEJ [Δ(ku,ligD)], or both DSB repair systems [Δ(ku,ligD,recA)]. Experiments using these strains revealed that either HR or NHEJ is sufficient for the survival and propagation of tubercle bacilli inside macrophages. Inhibition of nitric oxide or superoxide anion production with L-NIL or apocynin, respectively, enabled the Δ(ku,ligD,recA) mutant strain lacking both systems to survive intracellularly. Complementation of the Δ(ku,ligD,recA) mutant with an intact recA or ku-ligD rescued the ability of Mtb to propagate inside macrophages.

  17. Mouse BAZ1A (ACF1) is dispensable for double-strand break repair but is essential for averting improper gene expression during spermatogenesis.

    PubMed

    Dowdle, James A; Mehta, Monika; Kass, Elizabeth M; Vuong, Bao Q; Inagaki, Akiko; Egli, Dieter; Jasin, Maria; Keeney, Scott

    2013-11-01

    ATP-dependent chromatin remodelers control DNA access for transcription, recombination, and other processes. Acf1 (also known as BAZ1A in mammals) is a defining subunit of the conserved ISWI-family chromatin remodelers ACF and CHRAC, first purified over 15 years ago from Drosophila melanogaster embryos. Much is known about biochemical properties of ACF and CHRAC, which move nucleosomes in vitro and in vivo to establish ordered chromatin arrays. Genetic studies in yeast, flies and cultured human cells clearly implicate these complexes in transcriptional repression via control of chromatin structures. RNAi experiments in transformed mammalian cells in culture also implicate ACF and CHRAC in DNA damage checkpoints and double-strand break repair. However, their essential in vivo roles in mammals are unknown. Here, we show that Baz1a-knockout mice are viable and able to repair developmentally programmed DNA double-strand breaks in the immune system and germ line, I-SceI endonuclease-induced breaks in primary fibroblasts via homologous recombination, and DNA damage from mitomycin C exposure in vivo. However, Baz1a deficiency causes male-specific sterility in accord with its high expression in male germ cells, where it displays dynamic, stage-specific patterns of chromosomal localization. Sterility is caused by pronounced defects in sperm development, most likely a consequence of massively perturbed gene expression in spermatocytes and round spermatids in the absence of BAZ1A: the normal spermiogenic transcription program is largely intact but more than 900 other genes are mis-regulated, primarily reflecting inappropriate up-regulation. We propose that large-scale changes in chromatin composition that occur during spermatogenesis create a window of vulnerability to promiscuous transcription changes, with an essential function of ACF and/or CHRAC chromatin remodeling activities being to safeguard against these alterations.

  18. Mouse BAZ1A (ACF1) Is Dispensable for Double-Strand Break Repair but Is Essential for Averting Improper Gene Expression during Spermatogenesis

    PubMed Central

    Dowdle, James A.; Mehta, Monika; Kass, Elizabeth M.; Vuong, Bao Q.; Inagaki, Akiko; Egli, Dieter; Jasin, Maria; Keeney, Scott

    2013-01-01

    ATP-dependent chromatin remodelers control DNA access for transcription, recombination, and other processes. Acf1 (also known as BAZ1A in mammals) is a defining subunit of the conserved ISWI-family chromatin remodelers ACF and CHRAC, first purified over 15 years ago from Drosophila melanogaster embryos. Much is known about biochemical properties of ACF and CHRAC, which move nucleosomes in vitro and in vivo to establish ordered chromatin arrays. Genetic studies in yeast, flies and cultured human cells clearly implicate these complexes in transcriptional repression via control of chromatin structures. RNAi experiments in transformed mammalian cells in culture also implicate ACF and CHRAC in DNA damage checkpoints and double-strand break repair. However, their essential in vivo roles in mammals are unknown. Here, we show that Baz1a-knockout mice are viable and able to repair developmentally programmed DNA double-strand breaks in the immune system and germ line, I-SceI endonuclease-induced breaks in primary fibroblasts via homologous recombination, and DNA damage from mitomycin C exposure in vivo. However, Baz1a deficiency causes male-specific sterility in accord with its high expression in male germ cells, where it displays dynamic, stage-specific patterns of chromosomal localization. Sterility is caused by pronounced defects in sperm development, most likely a consequence of massively perturbed gene expression in spermatocytes and round spermatids in the absence of BAZ1A: the normal spermiogenic transcription program is largely intact but more than 900 other genes are mis-regulated, primarily reflecting inappropriate up-regulation. We propose that large-scale changes in chromatin composition that occur during spermatogenesis create a window of vulnerability to promiscuous transcription changes, with an essential function of ACF and/or CHRAC chromatin remodeling activities being to safeguard against these alterations. PMID:24244200

  19. Cascade of chromosomal rearrangements caused by a heterogeneous T-DNA integration supports the double-strand break repair model for T-DNA integration.

    PubMed

    Hu, Yufei; Chen, Zhiyu; Zhuang, Chuxiong; Huang, Jilei

    2017-02-28

    Transferred DNA (T-DNA) from Agrobacterium tumefaciens can be integrated into the plant genome. The double-strand break repair (DSBR) pathway is a major model for T-DNA integration. From this model, we expect that two ends of a T-DNA molecule would invade into a single DNA double-strand break (DSB) or independent DSBs in the plant genome. We call the later phenomenon a heterogeneous T-DNA integration which has never been observed. In this work, we demonstrated it in an Arabidopsis T-DNA insertion mutant seb19. To resolve the chromosomal structural changes caused by T-DNA integration at both the nucleotide and chromosome levels, we performed inverse PCR, genome resequencing, fluorescence in situ hybridization and linkage analysis. We found, in seb19, a single T-DNA connected two different chromosomal loci and caused complex chromosomal rearrangements. The specific break-junction pattern in seb19 is consistent with the result of heterogeneous T-DNA integration but not of recombination between two T-DNA insertions. We demonstrated that, in seb19, heterogeneous T-DNA integration evoked a cascade of incorrect repair of seven DSBs on chromosome 4 and 5, and then produced translocation, inversion, duplication and deletion. Heterogeneous T-DNA integration supports the DSBR model and suggests that two ends of a T-DNA molecule could be integrated into the plant genome independently. Our results also show a new origin of chromosomal abnormalities. This article is protected by copyright. All rights reserved.

  20. A cooperative activation loop among SWI/SNF, gamma-H2AX and H3 acetylation for DNA double-strand break repair.

    PubMed

    Lee, Han-Sae; Park, Ji-Hye; Kim, So-Jung; Kwon, Su-Jung; Kwon, Jongbum

    2010-04-21

    Although recent studies highlight the importance of histone modifications and ATP-dependent chromatin remodelling in DNA double-strand break (DSB) repair, how these mechanisms cooperate has remained largely unexplored. Here, we show that the SWI/SNF chromatin remodelling complex, earlier known to facilitate the phosphorylation of histone H2AX at Ser-139 (S139ph) after DNA damage, binds to gamma-H2AX (the phosphorylated form of H2AX)-containing nucleosomes in S139ph-dependent manner. Unexpectedly, BRG1, the catalytic subunit of SWI/SNF, binds to gamma-H2AX nucleosomes by interacting with acetylated H3, not with S139ph itself, through its bromodomain. Blocking the BRG1 interaction with gamma-H2AX nucleosomes either by deletion or overexpression of the BRG1 bromodomain leads to defect of S139ph and DSB repair. H3 acetylation is required for the binding of BRG1 to gamma-H2AX nucleosomes. S139ph stimulates the H3 acetylation on gamma-H2AX nucleosomes, and the histone acetyltransferase Gcn5 is responsible for this novel crosstalk. The H3 acetylation on gamma-H2AX nucleosomes is induced by DNA damage. These results collectively suggest that SWI/SNF, gamma-H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DSB repair.

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

    PubMed

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

    2014-05-10

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

  2. DNA repair and radiation sensitivity in mammalian cells

    SciTech Connect

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

    1993-01-01

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

  3. DNA repair and radiation sensitivity in mammalian cells

    SciTech Connect

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

    1993-02-01

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

  4. Differential repair of radiation-induced DNA damage in cells of human squamous cell carcinoma and the effect of caffeine and cysteamine on induction and repair of DNA double-strand breaks

    SciTech Connect

    Smeets, M.F.M.A.; Mooren, E.H.M.; Abdel-Wahab, A.H.A.; Begg, A.C.

    1994-11-01

    The goal of these experiments was to investigate further the relationship between DNA double-strand breaks and cell killing in human tumor cells, first by comparing different cell lines, and second by radiomodification studies. Field-inversion gel electrophoresis was used to quantify double-strand breaks. Two subclones of the radioresistant human squamous cell carcinoma line SQ20B (SQD9 and SQG6) were compared. These subclones differed in DNA index by a factor of 1.7 but showed the same resistance to radiation as cells of the parental cell line. It was found that, although induction of DSBs was not significantly different in the two cell lines, the t{sub 1/2} of the fast component of repair was significantly shorter for SQD9 cells, leading to greater overall repair which was not reflected in increased survival. Caffeine and cysteamine were tested as modifiers of radiosensitivity, using the radioresistant SQ20B line and the radiosensitive SCC61 cell line. No effect of caffeine was seen when the drug was present only during irradiation. Postirradiation incubations with caffeine, however, resulted in a dose reduction factor greater than 2.0 in cell survival for both cell lines. In contrast, induction of DSBs was reduced by caffeine, and no effect on DSB repair was observed. Cysteamine led to a dose protection factor greater than 1.8 in cell survival in both cell lines. A reduction in induced DSBs was found at high doses corresponding approximately with the increase in cell survival. Over the same (low) dose range, however, the correlation between DSB induction and cell killing was poor. These data indicate that DSB induction does not correlate well with cell killing either for different cell lines, for radiochemical modification (cysteamine) or for some other types of modification (caffeine). 31 refs., 8 figs.

  5. 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.

  6. Dual Targeting of Akt and mTORC1 Impairs Repair of DNA Double-Strand Breaks and Increases Radiation Sensitivity of Human Tumor Cells

    PubMed Central

    Holler, Marina; Grottke, Astrid; Mueck, Katharina; Manes, Julia; Jücker, Manfred

    2016-01-01

    Inhibition of mammalian target of rapamycin-complex 1 (mTORC1) induces activation of Akt. Because Akt activity mediates the repair of ionizing radiation-induced DNA double-strand breaks (DNA-DSBs) and consequently the radioresistance of solid tumors, we investigated whether dual targeting of mTORC1 and Akt impairs DNA-DSB repair and induces radiosensitization. Combining mTORC1 inhibitor rapamycin with ionizing radiation in human non-small cell lung cancer (NSCLC) cells (H661, H460, SK-MES-1, HTB-182, A549) and in the breast cancer cell line MDA-MB-231 resulted in radiosensitization of H661 and H460 cells (responders), whereas only a very slight effect was observed in A549 cells, and no effect was observed in SK-MES-1, HTB-182 or MDA-MB-231 cells (non-responders). In responder cells, rapamycin treatment did not activate Akt1 phosphorylation, whereas in non-responders, rapamycin mediated PI3K-dependent Akt activity. Molecular targeting of Akt by Akt inhibitor MK2206 or knockdown of Akt1 led to a rapamycin-induced radiosensitization of non-responder cells. Compared to the single targeting of Akt, the dual targeting of mTORC1 and Akt1 markedly enhanced the frequency of residual DNA-DSBs by inhibiting the non-homologous end joining repair pathway and increased radiation sensitivity. Together, lack of radiosensitization induced by rapamycin was associated with rapamycin-mediated Akt1 activation. Thus, dual targeting of mTORC1 and Akt1 inhibits repair of DNA-DSB leading to radiosensitization of solid tumor cells. PMID:27137757

  7. DNA double-strand break repair is involved in desiccation resistance of Sinorhizobium meliloti, but is not essential for its symbiotic interaction with Medicago truncatula.

    PubMed

    Dupuy, Pierre; Gourion, Benjamin; Sauviac, Laurent; Bruand, Claude

    2016-11-23

    The soil bacterium Sinorhizobium meliloti, a nitrogen-fixing symbiont of legume plants, is exposed to numerous stress conditions in nature, some of which cause the formation of harmful DNA double strand breaks (DSB). In particular, the reactive oxygen (ROS) and nitrogen (RNS) species produced during symbiosis, and the desiccation occurring in dry soils, are conditions which induce DSB. Two major systems of DSB repair are known in S. meliloti: homologous recombination (HR) and non-homologous end-joining (NHEJ). However, their role in the resistance to ROS, RNS and desiccation has never been examined in this bacterial species, and the importance of DSB repair in the symbiotic interaction has not been properly evaluated. Here, we constructed S. meliloti strains deficient in HR (by deleting the recA gene) or in NHEJ (by deleting the four ku genes) or both. Interestingly, we observed that ku and/or recA genes are involved in S. meliloti resistance to ROS and RNS. Nevertheless, a S. meliloti strain deficient in both HR and NHEJ was not altered in its ability to establish and maintain an efficient nitrogen-fixing symbiosis with Medicago truncatula, showing that rhizobial DSB repair is not essential for this process. This result suggests either that DSB formation in S. meliloti is efficiently prevented during symbiosis, or that DSB are not detrimental for symbiosis efficiency. In contrast, we found for the first time that both recA and ku genes are involved in S. meliloti resistance to desiccation, suggesting that DSB repair could be important for rhizobium persistence in the soil.

  8. Bypass of a 5',8-cyclopurine-2'-deoxynucleoside by DNA polymerase β during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks.

    PubMed

    Jiang, Zhongliang; Xu, Meng; Lai, Yanhao; Laverde, Eduardo E; Terzidis, Michael A; Masi, Annalisa; Chatgilialoglu, Chryssostomos; Liu, Yuan

    2015-09-01

    5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase β (pol β) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol β wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol β knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol β plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol β inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol β, indicating a mutagenic effect induced by pol β lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol β generated an intermediate that failed to be extended by pol β, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol β plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol β bypassing of a cdA lesion.

  9. Autologous hematopoietic stem cell transplantation in lymphoma patients is associated with a decrease in the double strand break repair capacity of peripheral blood lymphocytes

    PubMed Central

    Lacoste, Sandrine; Bhatia, Smita; Chen, Yanjun; Bhatia, Ravi; O’Connor, Timothy R.

    2017-01-01

    Patients who undergo autologous hematopoietic stem cell transplantation (aHCT) for treatment of a relapsed or refractory lymphoma are at risk of developing therapy related- myelodysplasia/acute myeloid leukemia (t-MDS/AML). Part of the risk likely resides in inherent interindividual differences in their DNA repair capacity (DRC), which is thought to influence the effect chemotherapeutic treatments have on the patient’s stem cells prior to aHCT. Measuring DRC involves identifying small differences in repair proficiency among individuals. Initially, we investigated the cell model in healthy individuals (primary lymphocytes and/or lymphoblastoid cell lines) that would be appropriate to measure genetically determined DRC using host-cell reactivation assays. We present evidence that interindividual differences in DRC double-strand break repair (by non-homologous end-joining [NHEJ] or single-strand annealing [SSA]) are better preserved in non-induced primary lymphocytes. In contrast, lymphocytes induced to proliferate are required to assay base excision (BER) or nucleotide excision repair (NER). We established that both NHEJ and SSA DRCs in lymphocytes of healthy individuals were inversely correlated with the age of the donor, indicating that DSB repair in lymphocytes is likely not a constant feature but rather something that decreases with age (~0.37% NHEJ DRC/year). To investigate the predictive value of pre-aHCT DRC on outcome in patients, we then applied the optimized assays to the analysis of primary lymphocytes from lymphoma patients and found that individuals who later developed t-MDS/AML (cases) were indistinguishable in their DRC from controls who never developed t-MDS/AML. However, when DRC was investigated shortly after aHCT in the same individuals (21.6 months later on average), aHCT patients (both cases and controls) showed a significant decrease in DSB repair measurements. The average decrease of 6.9% in NHEJ DRC observed among aHCT patients was much

  10. Cell-type specific role of the RNA-binding protein, NONO, in the DNA double-strand break response in the mouse testes.

    PubMed

    Li, Shuyi; Shu, Feng-Jue; Li, Zhentian; Jaafar, Lahcen; Zhao, Shourong; Dynan, William S

    2017-03-01

    The tandem RNA recognition motif protein, NONO, was previously identified as a candidate DNA double-strand break (DSB) repair factor in a biochemical screen for proteins with end-joining stimulatory activity. Subsequent work showed that NONO and its binding partner, SFPQ, have many of the properties expected for bona fide repair factors in cell-based assays. Their contribution to the DNA damage response in intact tissue in vivo has not, however, been demonstrated. Here we compare DNA damage sensitivity in the testes of wild-type mice versus mice bearing a null allele of the NONO homologue (Nono (gt)). In wild-type mice, NONO protein was present in Sertoli, peritubular myoid, and interstitial cells, with an increase in expression following induction of DNA damage. As expected for the product of an X-linked gene, NONO was not detected in germ cells. The Nono (gt/0) mice had at most a mild testis developmental phenotype in the absence of genotoxic stress. However, following irradiation at sublethal, 2-4 Gy doses, Nono (gt/0) mice displayed a number of indicators of radiosensitivity as compared to their wild-type counterparts. These included higher levels of persistent DSB repair foci, increased numbers of apoptotic cells in the seminiferous tubules, and partial degeneration of the blood-testis barrier. There was also an almost complete loss of germ cells at later times following irradiation, evidently arising as an indirect effect reflecting loss of stromal support. Results demonstrate a role for NONO protein in protection against direct and indirect biological effects of ionizing radiation in the whole animal.

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

    PubMed Central

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø.; Rizzo, Carmelo J.; Guengerich, F. Peter; Tudek, Barbara

    2015-01-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno (ε)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ε-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ε-adducts, 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC) and 1,N2-ethenoguanine (1,N2-εG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ε-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed εA and εC from ds and ssDNA but were inactive toward 1,N2-εG. SC-1A repaired only εA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ε-adducts in dsDNA, while only εA and εC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only εC in ssDNA Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N2-εG and that ALKBH3 removes only εC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. PMID:25797601

  12. The Arabidopsis MEI1 gene encodes a protein with five BRCT domains that is involved in meiosis-specific DNA repair events independent of SPO11-induced DSBs.

    PubMed

    Grelon, Mathilde; Gendrot, Ghislaine; Vezon, Daniel; Pelletier, Georges; Mathilde, Grelon; Ghislaine, Gendrot; Daniel, Vezon; Georges, Pelletier

    2003-08-01

    Arabidopsis thaliana MEI1 was first described as a gene involved in male meiosis, encoding a short protein showing homology with a human acrosin-trypsin inhibitor. We have isolated a new allele of mei1, and shown that in both mutants male and female meiosis are affected. In both reproductive pathways, meiosis proceeds while chromosomes become fragmented, resulting in aberrant meiotic products and in a strongly reduced fertility. We have shown that the gene mutated in mei1 mutants actually encodes a protein of 972 amino acids that contains five BRCA1 C-terminus (BRCT) domains and is similar to proteins involved in the response to DNA damage and replication blocks in eukaryotes. During meiosis, recombination is initiated by the formation of DNA double strand breaks (DSBs) induced by the protein SPO11. We analysed meiotic chromosome behaviour of the mei1 mutant in a spo11 mutant background and proved that the meiotic fragmentation observed in mei1 mutants was not the consequence of defects in the repair of meiotic DSBs induced by SPO11. We also analysed the effect of mei1 on the mitotic cell cycle but could not detect any sensitivity of mei1 seedlings to DNA-damaging agents like gamma-rays or UV. Therefore, MEI1 is a BRCT-domain-containing protein that could be specific to the meiotic cell cycle and that plays a crucial role in some DNA repair events independent of SPO11 DSB recombination repair.

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

    PubMed Central

    Singh, K K; Samson, L

    1995-01-01

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

  14. Repair of uv damaged DNA: Genes and proteins of yeast and human. Progress report, November 1, 1991--April 15, 1992

    SciTech Connect

    Prakash, L.

    1992-04-01

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

  15. Atmospheric-pressure plasma jet induces DNA double-strand breaks that require a Rad51-mediated homologous recombination for repair in Saccharomyces cerevisiae.

    PubMed

    Lee, Yoonna; Kim, Kangil; Kang, Kyu-Tae; Lee, Jong-Soo; Yang, Sang Sik; Chung, Woo-Hyun

    2014-10-15

    Non-thermal plasma generated under atmospheric pressure produces a mixture of chemically reactive molecules and has been developed for a number of biomedical applications. Recently, plasma jet has been proposed as novel cancer therapies based on the observation that free radicals generated by plasma jet induce mitochondria-mediated apoptotic cell death. We show here that air plasma jet induces DNA double-strand breaks (DSBs) in yeast chromosomes leading to genomic instability and loss of viability, which are alleviated by Rad51, the yeast homolog of Escherichiacoli RecA recombinase, through DNA damage repair by a homologous recombination (HR) process. Hypersensitivity of rad51 mutant to air plasma was not restored by antioxidant treatment unlike sod1 mutant that was highly sensitive to reactive oxygen species (ROS) challenge, suggesting that plasma jet induces DSB-mediated cell death independent of ROS generation. These results may provide a new insight into the mechanism of air plasma jet-induced cell death.

  16. HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins

    PubMed Central

    Hrecka, Kasia; Hao, Caili; Shun, Ming-Chieh; Kaur, Sarabpreet; Swanson, Selene K.; Florens, Laurence; Washburn, Michael P.; Skowronski, Jacek

    2016-01-01

    HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4DCAF1 E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4DCAF1 E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4DCAF1 E3. Thus, separate functions of HIV-1 Vpr usurp CRL4DCAF1 E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host. PMID:27335459

  17. Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage

    PubMed Central

    Ju, Xiaoming; Vetuschi, Antonella; Sferra, Roberta; Casimiro, Mathew C.; Pompili, Simona; Festuccia, Claudio; Colapietro, Alessandro; Gaudio, Eugenio; Di Cesare, Ernesto; Tombolini, Vincenzo; Pestell, Richard G.

    2016-01-01

    Patients with hormone-resistant prostate cancer (PCa) have higher biochemical failure rates following radiation therapy (RT). Cyclin D1 deregulated expression in PCa is associated with a more aggressive disease: however its role in radioresistance has not been determined. Cyclin D1 levels in the androgen-independent PC3 and 22Rv1 PCa cells were stably inhibited by infecting with cyclin D1-shRNA. Tumorigenicity and radiosensitivity were investigated using in vitro and in vivo experimental assays. Cyclin D1 silencing interfered with PCa oncogenic phenotype by inducing growth arrest in the G1 phase of cell cycle and reducing soft agar colony formation, migration, invasion in vitro and tumor formation and neo-angiogenesis in vivo. Depletion of cyclin D1 significantly radiosensitizes PCa cells by increasing the RT-induced DNA damages by affecting the NHEJ and HR pathways responsible of the DNA double-strand break repair. Following treatment of cells with RT the abundance of a biomarker of DNA damage, γ-H2AX, was dramatically increased in sh-cyclin D1 treated cells compared to shRNA control. Concordant with these observations DNA-PKcs-activation and RAD51-accumulation, part of the DNA double-strand break repair machinery, were reduced in shRNA-cyclin D1 treated cells compared to shRNA control. We further demonstrate the physical interaction between CCND1 with activated-ATM, -DNA-PKcs and RAD51 is enhanced by RT. Finally, siRNA-mediated silencing experiments indicated DNA-PKcs and RAD51 are downstream targets of CCND1-mediated PCa cells radioresistance. In summary, these observations suggest that CCND1 is a key mediator of PCa radioresistance and could represent a potential target for radioresistant hormone-resistant PCa. PMID:26689991

  18. Analysis of Repair Mechanisms following an Induced Double-Strand Break Uncovers Recessive Deleterious Alleles in the Candida albicans Diploid Genome.

    PubMed

    Feri, Adeline; Loll-Krippleber, Raphaël; Commere, Pierre-Henri; Maufrais, Corinne; Sertour, Natacha; Schwartz, Katja; Sherlock, Gavin; Bougnoux, Marie-Elisabeth; d'Enfert, Christophe; Legrand, Mélanie

    2016-10-11

    The diploid genome of the yeast Candida albicans is highly plastic, exhibiting frequent loss-of-heterozygosity (LOH) events. To provide a deeper understanding of the mechanisms leading to LOH, we investigated the repair of a unique DNA double-strand break (DSB) in the laboratory C. albicans SC5314 strain using the I-SceI meganuclease. Upon I-SceI induction, we detected a strong increase in the frequency of LOH events at an I-SceI target locus positioned on chromosome 4 (Chr4), including events spreading from this locus to the proximal telomere. Characterization of the repair events by single nucleotide polymorphism (SNP) typing and whole-genome sequencing revealed a predominance of gene conversions, but we also observed mitotic crossover or break-induced replication events, as well as combinations of independent events. Importantly, progeny that had undergone homozygosis of part or all of Chr4 haplotype B (Chr4B) were inviable. Mining of genome sequencing data for 155 C. albicans isolates allowed the identification of a recessive lethal allele in the GPI16 gene on Chr4B unique to C. albicans strain SC5314 which is responsible for this inviability. Additional recessive lethal or deleterious alleles were identified in the genomes of strain SC5314 and two clinical isolates. Our results demonstrate that recessive lethal alleles in the genomes of C. albicans isolates prevent the occurrence of specific extended LOH events. While these and other recessive lethal and deleterious alleles are likely to accumulate in C. albicans due to clonal reproduction, their occurrence may in turn promote the maintenance of corresponding nondeleterious alleles and, consequently, heterozygosity in the C. albicans species.

  19. Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage.

    PubMed

    Marampon, Francesco; Gravina, Giovanni; Ju, Xiaoming; Vetuschi, Antonella; Sferra, Roberta; Casimiro, Mathew C; Pompili, Simona; Festuccia, Claudio; Colapietro, Alessandro; Gaudio, Eugenio; Di Cesare, Ernesto; Tombolini, Vincenzo; Pestell, Richard G

    2016-02-02

    Patients with hormone-resistant prostate cancer (PCa) have higher biochemical failure rates following radiation therapy (RT). Cyclin D1 deregulated expression in PCa is associated with a more aggressive disease: however its role in radioresistance has not been determined. Cyclin D1 levels in the androgen-independent PC3 and 22Rv1 PCa cells were stably inhibited by infecting with cyclin D1-shRNA. Tumorigenicity and radiosensitivity were investigated using in vitro and in vivo experimental assays. Cyclin D1 silencing interfered with PCa oncogenic phenotype by inducing growth arrest in the G1 phase of cell cycle and reducing soft agar colony formation, migration, invasion in vitro and tumor formation and neo-angiogenesis in vivo. Depletion of cyclin D1 significantly radiosensitizes PCa cells by increasing the RT-induced DNA damages by affecting the NHEJ and HR pathways responsible of the DNA double-strand break repair. Following treatment of cells with RT the abundance of a biomarker of DNA damage, γ-H2AX, was dramatically increased in sh-cyclin D1 treated cells compared to shRNA control. Concordant with these observations DNA-PKcs-activation and RAD51-accumulation, part of the DNA double-strand break repair machinery, were reduced in shRNA-cyclin D1 treated cells compared to shRNA control. We further demonstrate the physical interaction between CCND1 with activated-ATM, -DNA-PKcs and RAD51 is enhanced by RT. Finally, siRNA-mediated silencing experiments indicated DNA-PKcs and RAD51 are downstream targets of CCND1-mediated PCa cells radioresistance. In summary, these observations suggest that CCND1 is a key mediator of PCa radioresistance and could represent a potential target for radioresistant hormone-resistant PCa.

  20. Analysis of Repair Mechanisms following an Induced Double-Strand Break Uncovers Recessive Deleterious Alleles in the Candida albicans Diploid Genome

    PubMed Central

    Feri, Adeline; Loll-Krippleber, Raphaël; Commere, Pierre-Henri; Maufrais, Corinne; Sertour, Natacha; Schwartz, Katja; Sherlock, Gavin; Bougnoux, Marie-Elisabeth

    2016-01-01

    ABSTRACT The diploid genome of the yeast Candida albicans is highly plastic, exhibiting frequent loss-of-heterozygosity (LOH) events. To provide a deeper understanding of the mechanisms leading to LOH, we investigated the repair of a unique DNA double-strand break (DSB) in the laboratory C. albicans SC5314 strain using the I-SceI meganuclease. Upon I-SceI induction, we detected a strong increase in the frequency of LOH events at an I-SceI target locus positioned on chromosome 4 (Chr4), including events spreading from this locus to the proximal telomere. Characterization of the repair events by single nucleotide polymorphism (SNP) typing and whole-genome sequencing revealed a predominance of gene conversions, but we also observed mitotic crossover or break-induced replication events, as well as combinations of independent events. Importantly, progeny that had undergone homozygosis of part or all of Chr4 haplotype B (Chr4B) were inviable. Mining of genome sequencing data for 155 C. albicans isolates allowed the identification of a recessive lethal allele in the GPI16 gene on Chr4B unique to C. albicans strain SC5314 which is responsible for this inviability. Additional recessive lethal or deleterious alleles were identified in the genomes of strain SC5314 and two clinical isolates. Our results demonstrate that recessive lethal alleles in the genomes of C. albicans isolates prevent the occurrence of specific extended LOH events. While these and other recessive lethal and deleterious alleles are likely to accumulate in C. albicans due to clonal reproduction, their occurrence may in turn promote the maintenance of corresponding nondeleterious alleles and, consequently, heterozygosity in the C. albicans species. PMID:27729506

  1. Accumulation of Ku80 proteins at DNA double-strand breaks in living cells

    SciTech Connect

    Koike, Manabu Koike, Aki

    2008-03-10

    Ku plays a key role in multiple nuclear processes, e.g., DNA double-strand break (DSB) repair. The regulation mechanism of the localizations of Ku70 and Ku80 plays a key role in regulating the multiple functions of Ku. Although numerous biochemical studies in vitro have elucidated the DNA binding mechanism of Ku, no accumulation mechanisms of Ku70 and Ku80 at DSBs have been clarified in detail in vivo. In this study, we examined the accumulation mechanism of Ku80 at DSBs in living cells. EGFP-Ku80 accumulation at DSBs began immediately after irradiation. On the other hand, our data show that Ku70 alone, which has DNA binding activity independent of Ku80, cannot accumulate at the DSBs, whereas Ku70 bound to Ku80 can. The deletion of the C-terminal DNA-PKcs-binding domain and the mutation at the SUMOylation site of Ku80 had no effect on Ku80 accumulation. Unexpectedly, N-terminal deletion mutants of Ku80 fully lost their accumulation activity, although the mutants retained their Ku70 binding activity. Altogether, these data demonstrate that Ku80 is essential for Ku70 accumulation at DSBs. Furthermore, three domains of Ku80, i.e., the N-terminal {alpha}/{beta}, the DNA-binding, and Ku70-binding domains, seem to necessary for the accumulation at or recognition of DSBs in the early stage after irradiation.

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

    PubMed

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

    2016-04-20

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

  3. Non-Homologous End Joining and Homology Directed DNA Repair Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents

    PubMed Central

    Zaboikin, Michail; Zaboikina, Tatiana; Freter, Carl

    2017-01-01

    Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents. We also designed TaqMan assays using probes situated across the cut site to discriminate wild type from mutant sequences present after genome editing. The experiments revealed that the sensitivity of the assays to detect NHEJ-mediated DNA repair could be enhanced by selection of transfected cells to reduce the contribution of unmodified genomic DNA from untransfected cells to the DNA melting profile. The presence of donor template DNA lacking the target sequence at the time of genome editing further enhanced the sensitivity of the assays for detection of mutant DNA molecules by excluding the wild-type sequences modified by HDR. A second TaqMan probe that bound to an adjacent site, outside of the primary target cut site, was used to directly determine the contribution of HDR to DNA repair in the presence of the donor template sequence. The TaqMan qPCR assay, designed to measure the contribution of NHEJ and HDR in DNA repair, corroborated the results from HRMA. The data indicated that genome editing reagents can produce DSBs at high efficiency in HEK293T cells but a significant proportion of these are likely masked by reversion to wild type as a result of HDR. Supplying a donor plasmid to provide a template for HDR (that

  4. Non-Homologous End Joining and Homology Directed DNA Repair Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents.

    PubMed

    Zaboikin, Michail; Zaboikina, Tatiana; Freter, Carl; Srinivasakumar, Narasimhachar

    2017-01-01

    Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents. We also designed TaqMan assays using probes situated across the cut site to discriminate wild type from mutant sequences present after genome editing. The experiments revealed that the sensitivity of the assays to detect NHEJ-mediated DNA repair could be enhanced by selection of transfected cells to reduce the contribution of unmodified genomic DNA from untransfected cells to the DNA melting profile. The presence of donor template DNA lacking the target sequence at the time of genome editing further enhanced the sensitivity of the assays for detection of mutant DNA molecules by excluding the wild-type sequences modified by HDR. A second TaqMan probe that bound to an adjacent site, outside of the primary target cut site, was used to directly determine the contribution of HDR to DNA repair in the presence of the donor template sequence. The TaqMan qPCR assay, designed to measure the contribution of NHEJ and HDR in DNA repair, corroborated the results from HRMA. The data indicated that genome editing reagents can produce DSBs at high efficiency in HEK293T cells but a significant proportion of these are likely masked by reversion to wild type as a result of HDR. Supplying a donor plasmid to provide a template for HDR (that

  5. Hypoxia-inducible factor-1alpha regulates the expression of nucleotide excision repair proteins in keratinocytes.

    PubMed

    Rezvani, Hamid Reza; Mahfouf, Walid; Ali, Nsrein; Chemin, Cecile; Ged, Cecile; Kim, Arianna L; de Verneuil, Hubert; Taïeb, Alain; Bickers, David R; Mazurier, Frédéric

    2010-01-01

    The regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotide excision repair factors (XPC and XPD) in human keratinocytes. We show that hypoxia-inducible factor-1alpha (HIF-1alpha) is involved in the regulation of XPC and XPD. Early UVB-induced downregulation of HIF-1alpha increased XPC mRNA expression due to competition between HIF-1alpha and Sp1 for their overlapping binding sites. Late UVB-induced enhanced phosphorylation of HIF-1alpha protein upregulated XPC mRNA expression by direct binding to a separate hypoxia response element (HRE) in the XPC promoter region. HIF-1alpha also regulated XPD expression by binding to a region of seven overlapping HREs in its promoter. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1alpha is a key regulator of the DNA repair machinery. Analysis of the repair kinetics of 6-4 photoproducts and cyclobutane pyrimidine dimers also revealed that HIF-1alpha downregulation led to an increased rate of immediate removal of both photolesions but attenuated their late removal following UVB irradiation, indicating the functional effects of HIF-1alpha in the repair of UVB-induced DNA damage.

  6. DNA ligase IV and artemis act cooperatively to suppress homologous recombination in human cells: implications for DNA double-strand break repair.

    PubMed

    Kurosawa, Aya; Saito, Shinta; So, Sairei; Hashimoto, Mitsumasa; Iwabuchi, Kuniyoshi; Watabe, Haruka; Adachi, Noritaka

    2013-01-01

    Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.

  7. Novel properties of melanins include promotion of DNA strand breaks, impairment of repair, and reduced ability to damage DNA after quenching of singlet oxygen.

    PubMed

    Suzukawa, Andréia Akemi; Vieira, Alessandra; Winnischofer, Sheila Maria Brochado; Scalfo, Alexsandra Cristina; Di Mascio, Paolo; Ferreira, Ana Maria da Costa; Ravanat, Jean-Luc; Martins, Daniela de Luna; Rocha, Maria Eliane Merlin; Martinez, Glaucia Regina

    2012-05-01

    Melanins have been associated with the development of melanoma and its resistance to photodynamic therapy (PDT). Singlet molecular oxygen ((1)O(2)), which is produced by ultraviolet A solar radiation and the PDT system, is also involved. Here, we investigated the effects that these factors have on DNA damage and repair. Our results show that both types of melanin (eumelanin and pheomelanin) lead to DNA breakage in the absence of light irradiation and that eumelanin is more harmful than pheomelanin. Interestingly, melanins were found to bind to the minor grooves of DNA, guaranteeing close proximity to DNA and potentially causing the observed high levels of strand breaks. We also show that the interaction of melanins with DNA can impair the access of repair enzymes to lesions, contributing to the perpetuation of DNA damage. Moreover, we found that after melanins interact with (1)O(2), they exhibit a lower ability to induce DNA breakage; we propose that these effects are due to modifications of their structure. Together, our data highlight the different modes of action of the two types of melanin. Our results may have profound implications for cellular redox homeostasis, under conditions of induced melanin synthesis and irradiation with solar light. These results may also be applied to the development of protocols to sensitize melanoma cells to PDT.

  8. Depletion of Histone Demethylase Jarid1A Resulting in Histone Hyperacetylation and Radiation Sensitivity Does Not Affect DNA Double-Strand Break Repair.

    PubMed

    Penterling, Corina; Drexler, Guido A; Böhland, Claudia; Stamp, Ramona; Wilke, Christina; Braselmann, Herbert; Caldwell, Randolph B; Reindl, Judith; Girst, Stefanie; Greubel, Christoph; Siebenwirth, Christian; Mansour, Wael Y; Borgmann, Kerstin; Dollinger, Günther; Unger, Kristian; Friedl, Anna A

    2016-01-01

    Histone demethylases have recently gained interest as potential targets in cancer treatment and several histone demethylases have been implicated in the DNA damage response. We investigated the effects of siRNA-mediated depletion of histone demethylase Jarid1A (KDM5A, RBP2), which demethylates transcription activating tri- and dimethylated lysine 4 at histone H3 (H3K4me3/me2), on growth characteristics and cellular response to radiation in several cancer cell lines. In unirradiated cells Jarid1A depletion lead to histone hyperacetylation while not affecting cell growth. In irradiated cells, depletion of Jarid1A significantly increased cellular radiosensitivity. Unexpectedly, the hyperacetylation phenotype did not lead to disturbed accumulation of DNA damage response and repair factors 53BP1, BRCA1, or Rad51 at damage sites, nor did it influence resolution of radiation-induced foci or rejoining of reporter constructs. We conclude that the radiation sensitivity observed following depletion of Jarid1A is not caused by a deficiency in repair of DNA double-strand breaks.

  9. The Structural Features of Thousands of T-DNA Insertion Sites Are Consistent with a Double-Strand Break Repair-Based Insertion Mechanism.

    PubMed

    Kleinboelting, Nils; Huep, Gunnar; Appelhagen, Ingo; Viehoever, Prisca; Li, Yong; Weisshaar, Bernd

    2015-11-02

    Transformation by Agrobacterium tumefaciens, an important tool in modern plant research, involves the integration of T-DNA initially present on a plasmid in agrobacteria into the genome of plant cells. The process of attachment of the agrobacteria to plant cells and the transport of T-DNA into the cell and further to the nucleus has been well described. However, the exact mechanism of integration into the host's DNA is still unclear, although several models have been proposed. During confirmation of T-DNA insertion alleles from the GABI-Kat collection of Arabidopsis thaliana mutants, we have generated about 34,000 sequences from the junctions between inserted T-DNA and adjacent genome regions. Here, we describe the evaluation of this dataset with regard to existing models for T-DNA integration. The results suggest that integration into the plant genome is mainly mediated by the endogenous plant DNA repair machinery. The observed integration events showed characteristics highly similar to those of repair sites of double-strand breaks with respect to microhomology and deletion sizes. In addition, we describe unexpected integration events, such as large deletions and inversions at the integration site that are relevant for correct interpretation of results from T-DNA insertion mutants in reverse genetics experiments.

  10. The over-expression of the β2 catalytic subunit of the proteasome decreases homologous recombination and impairs DNA double-strand break repair in human cells.

    PubMed

    Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

    2011-01-01

    By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

  11. The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

    PubMed Central

    Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

    2011-01-01

    By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p. PMID:21660142

  12. Depletion of Histone Demethylase Jarid1A Resulting in Histone Hyperacetylation and Radiation Sensitivity Does Not Affect DNA Double-Strand Break Repair

    PubMed Central

    Penterling, Corina; Drexler, Guido A.; Böhland, Claudia; Stamp, Ramona; Wilke, Christina; Braselmann, Herbert; Caldwell, Randolph B.; Reindl, Judith; Girst, Stefanie; Greubel, Christoph; Siebenwirth, Christian; Mansour, Wael Y.; Borgmann, Kerstin; Dollinger, Günther; Unger, Kristian; Friedl, Anna A.

    2016-01-01

    Histone demethylases have recently gained interest as potential targets in cancer treatment and several histone demethylases have been implicated in the DNA damage response. We investigated the effects of siRNA-mediated depletion of histone demethylase Jarid1A (KDM5A, RBP2), which demethylates transcription activating tri- and dimethylated lysine 4 at histone H3 (H3K4me3/me2), on growth characteristics and cellular response to radiation in several cancer cell lines. In unirradiated cells Jarid1A depletion lead to histone hyperacetylation while not affecting cell growth. In irradiated cells, depletion of Jarid1A significantly increased cellular radiosensitivity. Unexpectedly, the hyperacetylation phenotype did not lead to disturbed accumulation of DNA damage response and repair factors 53BP1, BRCA1, or Rad51 at damage sites, nor did it influence resolution of radiation-induced foci or rejoining of reporter constructs. We conclude that the radiation sensitivity observed following depletion of Jarid1A is not caused by a deficiency in repair of DNA double-strand breaks. PMID:27253695

  13. ATM- and ATR-mediated phosphorylation of XRCC3 regulates DNA double-strand break-induced checkpoint activation and repair.

    PubMed

    Somyajit, Kumar; Basavaraju, Shivakumar; Scully, Ralph; Nagaraju, Ganesh

    2013-05-01

    The RAD51 paralogs XRCC3 and RAD51C have been implicated in homologous recombination (HR) and DNA damage responses. However, the molecular mechanism(s) by which these paralogs regulate HR and DNA damage signaling remains obscure. Here, we show that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We find that RAD51C but not XRCC2 is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G2 phases. XRCC3 phosphorylation is required for chromatin loading of RAD51 and HR-mediated repair of double-strand breaks (DSBs). Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G2/M checkpoint independently of its phosphorylation. Strikingly, we find that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation is required for the HR-mediated recovery of collapsed replication forks but is dispensable for the restart of stalled replication forks. Together, these findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair.

  14. Double strand-breaks and DNA-to-protein cross-links induced by fast neutrons in bacteriophage DNA.

    PubMed

    Hawkins, R B

    1979-01-01

    Coliphage T7 was suspended in tryptone broth and exposed to a mixture of fast neutrons and gamma radiation. Plaque survival, double strand-breaks and DNA-to-protein cross-linkage were examined and the results compared with those found in phage exposed to gamma radiation alone. Neutral sucrose density sedimentation patterns indicate that neutron-induced double strand-breaks sometimes occur in clusters of more than 100 in the same phage and that the effeciency with which double strand-breaks form is about 50 times that of gamma-induced double strand-breaks. Neutron-induced protein-to-DNA cross-links probably also occur in clusters with enhanced efficiency relative to low LET radiation.

  15. Role of β-catenin-regulated CCN matricellular proteins in epithelial repair after inflammatory lung injury

    PubMed Central

    McClendon, Jazalle; Aschner, Yael; Briones, Natalie; Young, Scott K.; Lau, Lester F.; Kahn, Michael; Downey, Gregory P.

    2013-01-01

    Repair of the lung epithelium after injury is integral to the pathogenesis and outcomes of diverse inflammatory lung diseases. We previously reported that β-catenin signaling promotes epithelial repair after inflammatory injury, but the β-catenin target genes that mediate this effect are unknown. Herein, we examined which β-catenin transcriptional coactivators and target genes promote epithelial repair after inflammatory injury. Transmigration of human neutrophils across cultured monolayers of human lung epithelial cells resulted in a fall in transepithelial resistance and the formation of discrete areas of epithelial denudation (“microinjury”), which repaired via cell spreading by 96 h. In mice treated with intratracheal (i.t.) LPS or keratinocyte chemokine, neutrophil emigration was associated with increased permeability of the lung epithelium, as determined by increased bronchoalveolar lavage (BAL) fluid albumin concentration, which decreased over 3–6 days. Activation of β-catenin/p300-dependent gene expression using the compound ICG-001 accelerated epithelial repair in vitro and in murine models. Neutrophil transmigration induced epithelial expression of the β-catenin/p300 target genes Wnt-induced secreted protein (WISP) 1 and cysteine-rich (Cyr) 61, as determined by real-time PCR (qPCR) and immunostaining. Purified neutrophil elastase induced WISP1 upregulation in lung epithelial cells, as determined by qPCR. WISP1 expression increased in murine lungs after i.t. LPS, as determined by ELISA of the BAL fluid and qPCR of whole lung extracts. Finally, recombinant WISP1 and Cyr61 accelerated repair, and Cyr61-neutralizing antibodies delayed repair of the injured epithelium in vitro. We conclude that β-catenin/p300-dependent expression of WISP1 and Cyr61 is critical for epithelial repair and represents a potential therapeutic target to promote epithelial repair after inflammatory injury. PMID:23316072

  16. Putting a break on protein translocation: metabolic regulation of mitochondrial protein import.

    PubMed

    Herrmann, Johannes M

    2009-04-01

    Sequence-inherent targeting information directs polypeptides synthesized in the cytosol to their respective cellular compartment. Some proteins use ambiguous sorting signals or specific folding properties to be dually distributed between the cytosol and mitochondria. A study published in this issue of Molecular Microbiology shows that in the case of fumarase this distribution is controlled by the metabolic state of yeast cells. The metabolite-dependent distribution of fumarase represents an exciting example of regulated protein import into mitochondria that shows that eukaryotes can adapt the intracellular protein distribution to their physiological conditions.

  17. Protein phosphatase 5 is necessary for ATR-mediated DNA repair

    SciTech Connect

    Kang, Yoonsung; Cheong, Hyang-Min; Lee, Jung-Hee; Song, Peter I.; Lee, Kwang-Ho; Kim, Sang-Yong; Jun, Jae Yeoul; You, Ho Jin

    2011-01-07

    Research highlights: {yields} Serine/threonine protein phosphatase 5 (PP5) has been shown to participate in ataxia telangiectasia-mutated (ATM)- and ATR (ATM- and Rad3-related)-mediated checkpoint pathways, which plays an important role in the DNA damage response and maintenance of genomic stability. {yields} However, it is not clear exactly how PP5 participates in this process. {yields} Our results indicate that PP5 is more closely related with ATR-mediated pathway than ATM-mediated pathway in DNA damage repair. -- Abstract: Several recent studies have shown that protein phosphatase 5 (PP5) participates in cell cycle arrest after DNA damage, but its roles in DNA repair have not yet been fully characterized. We investigated the roles of PP5 in the repair of ultraviolet (UV)- and neocarzinostatin (NCS)-induced DNA damage. The results of comet assays revealed different repair patterns in UV- and NCS-exposed U2OS-PS cells. PP5 is only essential for Rad3-related (ATR)-mediated DNA repair. Furthermore, the phosphorylation of 53BP1 and BRCA1, important mediators of DNA damage repair, and substrates of ATR and ATM decreased in U2OS-PS cells exposed to UV radiation. In contrast, the cell cycle arrest proteins p53, CHK1, and CHK2 were normally phosphorylated in U2OS and U2OS-PS cells exposed to UV radiation or treated with NCS. In view of these results, we suggest that PP5 plays a crucial role in ATR-mediated repair of UV-induced DNA damage.

  18. PolyQ-dependent RNA–protein assemblies control symmetry breaking

    PubMed Central

    Lee, ChangHwan; Occhipinti, Patricia

    2015-01-01

    Dendritic growth in fungi and neurons requires that multiple axes of polarity are established and maintained within the same cytoplasm. We have discovered that transcripts encoding key polarity factors including a formin, Bni1, and a polarisome scaffold, Spa2, are nonrandomly clustered in the cytosol to initiate and maintain sites of polarized growth in the fungus Ashbya gossypii. This asymmetric distribution requires the mRNAs to interact with a polyQ-containing protein, Whi3, and a Pumilio protein with a low-complexity sequence, Puf2. Cells lacking Whi3 or Puf2 had severe defects in establishing new sites of polarity and failed to localize Bni1 protein. Interaction of mRNAs with Whi3 and Puf2 promotes enrichment of transcripts at established sites of polarized growth and clustering of polarity transcripts throughout the cell body. Thus, aggregation-prone proteins make functional assemblies to position polarity transcripts, and nonrandom positioning of transcripts is required for symmetry-breaking events. This reveals a physiological function for polyQ-driven assemblies in regulating cell polarity. PMID:25713414

  19. RAD50 Is Required for Efficient Initiation of Resection and Recombinational Repair at Random, γ-Induced Double-Strand Break Ends

    PubMed Central

    Westmoreland, Jim; Ma, Wenjian; Yan, Yan; Van Hulle, Kelly; Malkova, Anna; Resnick, Michael A.

    2009-01-01

    Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent. PMID:19763170

  20. Proteasome inhibition rescues clinically significant unstable variants of the mismatch repair protein Msh2

    PubMed Central

    Arlow, Tim; Scott, Kristan; Wagenseller, Aubrey; Gammie, Alison

    2013-01-01

    MSH2 is required for DNA mismatch repair recognition in eukaryotes. Deleterious mutations in human MSH2 account for approximately half of the alleles associated with a common hereditary cancer syndrome. Previously, we characterized clinically identified MSH2 missense mutations, using yeast as a model system, and found that the most common cause of defective DNA mismatch repair was low levels of the variant Msh2 proteins. Here, we show that increased protein turnover is responsible for the reduced cellular levels. Increasing gene dosage of more than half of the missense alleles fully restored function. A titration experiment revealed that raising the expression level of one variant to less than wild-type levels restored mismatch repair, suggesting that overexpression is not always required to regain function. We found that the ubiquitin-mediated proteasome degradation pathway is the major mechanism for increased turnover of the Msh2 variants and identified the primary ubiquitin ligase as San1. Deletion of San1 restored protein levels for all but one variant, but did not elevate wild-type Msh2 levels. The unstable variants interacted with San1, whereas wild-type Msh2 did not. Additionally, san1Δ suppressed the mismatch repair defect of unstable variants. Of medical significance, the clinically approved drug Bortezomib partially restored protein levels and mismatch repair function for low-level variants and reversed the resistance to cisplatin, a common chemotherapeutic. Our results provide the foundation for an innovative therapeutic regime for certain mismatch-repair-defective cancers that are refractory to conventional chemotherapies. PMID:23248292

  1. The PTEN phosphatase functions cooperatively with the Fanconi anemia proteins in DNA crosslink repair

    PubMed Central

    Vuono, Elizabeth A.; Mukherjee, Ananda; Vierra, David A.; Adroved, Morganne M.; Hodson, Charlotte; Deans, Andrew J.; Howlett, Niall G.

    2016-01-01

    Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure and increased cancer risk. The FA proteins function primarily in DNA interstrand crosslink (ICL) repair. Here, we have examined the role of the PTEN phosphatase in this process. We have established that PTEN-deficient cells, like FA cells, exhibit increased cytotoxicity, chromosome structural aberrations, and error-prone mutagenic DNA repair following exposure to ICL-inducing agents. The increased ICL sensitivity of PTEN-deficient cells is caused, in part, by elevated PLK1 kinase-mediated phosphorylation of FANCM, constitutive FANCM polyubiquitination and degradation, and the consequent inefficient assembly of the FA core complex, FANCD2, and FANCI into DNA repair foci. We also establish that PTEN function in ICL repair is dependent on its protein phosphatase activity and ability to be SUMOylated, yet is independent of its lipid phosphatase activity. Finally, via epistasis analysis, we demonstrate that PTEN and FANCD2 function cooperatively in ICL repair. PMID:27819275

  2. Thrombospondin-1 might be a therapeutic target to suppress RB cells by regulating the DNA double-strand breaks repair

    PubMed Central

    Zhang, Zhang; Zhang, Ping; Yang, Ying; Wu, Nandan; Xu, Lijun; Zhang, Jing; Ge, Jian; Yu, Keming; Zhuang, Jing

    2016-01-01

    Retinoblastoma (RB) arises from the retina, and its growth usually occurs under the retina and toward the vitreous. Ideal therapy should aim to inhibit the tumor and protect neural cells, increasing the patient's life span and quality of life. Previous studies have demonstrated that Thrombospondin-1 (TSP-1) is associated with neurogenesis, neovascularization and tumorigenesis. However, at present, the bioactivity of TSP-1 in retinoblastoma has not been defined. Herein, we demonstrated that TSP-1 was silenced in RB cell lines and clinical tumor samples. HDAC inhibitor, Trichostatin A (TSA), could notably transcriptionally up-regulate TSP-1 in RB cells, WERI-Rb1 cells and Y79 cells. Moreover, we found human recombinant TSP-1 (hTSP-1) could significantly inhibit the cell viability of RB cells both in vitro and in vivo. Interestingly, hTSP-1 could significantly induce the expression of γ-H2AX, a well-characterized in situ marker of DNA double-strand breaks (DSBs) in RB cells. The DNA NHEJ pathway in WERI-Rb1 cells could be significantly inhibited by hTSP-1. A mutation in Rb1 might be involved in the hTSP-1-medicated γ-H2AX increasing in WERI-Rb1 cells. Furthermore, hTSP-1 could inhibit RB cells while promoting retinal neurocyte survival in the neuronal and retinoblastoma cell co-culture system. As such, TSP-1 may become a therapeutic target for treatment of retinoblastoma. PMID:26756218

  3. Thrombospondin-1 might be a therapeutic target to suppress RB cells by regulating the DNA double-strand breaks repair.

    PubMed

    Chen, Pei; Yu, Na; Zhang, Zhang; Zhang, Ping; Yang, Ying; Wu, Nandan; Xu, Lijun; Zhang, Jing; Ge, Jian; Yu, Keming; Zhuang, Jing

    2016-02-02

    Retinoblastoma (RB) arises from the retina, and its growth usually occurs under the retina and toward the vitreous. Ideal therapy should aim to inhibit the tumor and protect neural cells, increasing the patient's life span and quality of life. Previous studies have demonstrated that Thrombospondin-1 (TSP-1) is associated with neurogenesis, neovascularization and tumorigenesis. However, at present, the bioactivity of TSP-1 in retinoblastoma has not been defined. Herein, we demonstrated that TSP-1 was silenced in RB cell lines and clinical tumor samples. HDAC inhibitor, Trichostatin A (TSA), could notably transcriptionally up-regulate TSP-1 in RB cells, WERI-Rb1 cells and Y79 cells. Moreover, we found human recombinant TSP-1 (hTSP-1) could significantly inhibit the cell viability of RB cells both in vitro and in vivo. Interestingly, hTSP-1 could significantly induce the expression of γ-H2AX, a well-characterized in situ marker of DNA double-strand breaks (DSBs) in RB cells. The DNA NHEJ pathway in WERI-Rb1 cells could be significantly inhibited by hTSP-1. A mutation in Rb1 might be involved in the hTSP-1-medicated γ-H2AX increasing in WERI-Rb1 cells. Furthermore, hTSP-1 could inhibit RB cells while promoting retinal neurocyte survival in the neuronal and retinoblastoma cell co-culture system. As such, TSP-1 may become a therapeutic target for treatment of retinoblastoma.

  4. Bone morphogenetic proteins: Signaling periodontal bone regeneration and repair.

    PubMed

    Anusuya, G Sai; Kandasamy, M; Jacob Raja, S A; Sabarinathan, S; Ravishankar, P; Kandhasamy, Balu

    2016-10-01

    Bone morphogenetic proteins (BMPs) are a group of growth factors also known as cytokines and as metabologens. Originally discovered by their ability to induce the formation of bone and cartilage, BMPs are now considered to constitute a group of pivotal morphogenetic signals, orchestrating tissue architecture throughout the body. The important functioning of BMP signals in physiology is emphasized by the multitude of roles for dysregulated BMP signaling in pathological processes. A study done wherein it was found that protein extracts from bone implanted into the animals at nonbone sites induced the formation of new cartilage and bone tissue. This protein extract contained multiple factors that stimulated bone formation and was termed as "BMP." There are at least 15 different BMPs identified to date and are a part of the transforming growth factor-β super family. The most widely studied BMPs are BMP-2, BMP-3 (osteogenin), BMP-4, and BMP-7 (osteogenic protein-1). Now, any recombination type of morphogenic proteins have been synthesized, for example - recombinant human BMPs.

  5. Bone morphogenetic proteins: Signaling periodontal bone regeneration and repair

    PubMed Central

    Anusuya, G. Sai; Kandasamy, M.; Jacob Raja, S. A.; Sabarinathan, S.; Ravishankar, P.; Kandhasamy, Balu

    2016-01-01

    Bone morphogenetic proteins (BMPs) are a group of growth factors also known as cytokines and as metabologens. Originally discovered by their ability to induce the formation of bone and cartilage, BMPs are now considered to constitute a group of pivotal morphogenetic signals, orchestrating tissue architecture throughout the body. The important functioning of BMP signals in physiology is emphasized by the multitude of roles for dysregulated BMP signaling in pathological processes. A study done wherein it was found that protein extracts from bone implanted into the animals at nonbone sites induced the formation of new cartilage and bone tissue. This protein extract contained multiple factors that stimulated bone formation and was termed as “BMP.” There are at least 15 different BMPs identified to date and are a part of the transforming growth factor-β super family. The most widely studied BMPs are BMP-2, BMP-3 (osteogenin), BMP-4, and BMP-7 (osteogenic protein-1). Now, any recombination type of morphogenic proteins have been synthesized, for example - recombinant human BMPs. PMID:27829744

  6. DNA replication and the repair of DNA strand breaks in nuclei of Physarum polycephalum. Terminal report, August 1, 1978-March 31, 1980

    SciTech Connect

    Brewer, E.N.; Evans, T.E.

    1980-01-01

    Nuclei isolated from Physarum are able to replicate approximately 15% of the total genome in a manner which is qualitatively similar to the DNA replication process occurring in the intact organism. Such nuclei, however, are defective in the joining of Okazaki intermediates in vitro. Two DNA polymerase species, isolated from nuclei or intact plasmodia of this organism, can be separated by sucrose density gradient centrifugation. Total DNA polymerase activity is low in nuclei isolated during mitosis. A heat-stable glycoprotein material present in aqueous nuclear extracts stimulates DNA synthesis in well-washed nuclei. A sub-nuclear preparation active in DNA synthesis in vitro has been obtained from isolated nuclei of Physarum. Radiation-induced DNA double-strand breaks are rejoined in intact plasmodia and isolated nuclei of Physarum in a cell cycle-dependent manner. This phenomenon does not appear to be due to an intrinsic difference in nuclear DNA endonuclease activity at different times of the mitotic cycle. DNA strand breaks and repair induced by the carcinogen 4-nitroquinoline-1-oxide is similar in several respects to that resulting from exposure of the organism to ionizing radiation. Temperature sensitive strains of Physarum have been constructed and preliminary genetical and biochemical characterizations have been carried out. Two of the strains appear to be conditionally defective in DNA metabolism. An isogenic ploidal series of amoebae has been prepared and characterized as to uv and ionizing radiation sensitivity (in terms of cell survival). There is a direct relationship between ploidy and resistance to uv whereas ploidal change does not appear to affect the response to ionizing radiation.

  7. Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends

    NASA Technical Reports Server (NTRS)

    Lobrich, M.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

    1995-01-01

    An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

  8. The human actin-related protein hArp5: nucleo-cytoplasmic shuttling and involvement in DNA repair.

    PubMed

    Kitayama, Kumiko; Kamo, Mariko; Oma, Yukako; Matsuda, Ryo; Uchida, Takafumi; Ikura, Tsuyoshi; Tashiro, Satoshi; Ohyama, Takashi; Winsor, Barbara; Harata, Masahiko

    2009-01-15

    Certain actin-related proteins (Arps) of budding yeast are localized in the nucleus, and have essential roles as stoichiometric components of histone acetyltransferase (HAT) and chromatin remodeling complexes. On the other hand, identification of vertebrate nuclear Arps and their functional analyses are just beginning. We show that human Arp5 (hArp5) proteins are localized in the nucleus, and that arp5Delta yeast cells are partially complemented by hArp5. Thus, hArp5 is a novel member of the nuclear Arps of vertebrates, which possess evolutionarily conserved functions from yeast to humans. We show here that hArp5 shuttles between the nucleus and the cytoplasm. Furthermore, after the induction of DNA double strand breaks (DSB), cell growth and the accumulation of phosphorylated histone H2AX (gamma-H2AX) are impaired by hArp5 depletion. Association of hArp5 with the hIno80 chromatin remodeling enzyme and decrease of chromatin-bound hIno80 by hArp5-depletion indicate that hArp5 may have a role in the recruitment of the hINO80 complex to chromatin. Overexpression of hArp5 and hIno80 enhanced gamma-H2AX accumulation. These observations suggest that hArp5 is involved in the process of DSB repair through the regulation of the chromatin remodelling machinery.

  9. A small molecule inhibitor of monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA) inhibits repair of interstrand DNA cross-link, enhances DNA double strand break, and sensitizes cancer cells to cisplatin.

    PubMed

    Inoue, Akira; Kikuchi, Sotaro; Hishiki, Asami; Shao, Youming; Heath, Richard; Evison, Benjamin J; Actis, Marcelo; Canman, Christine E; Hashimoto, Hiroshi; Fujii, Naoaki

    2014-03-07

    Small molecule inhibitors of proliferating cell nuclear antigen (PCNA)/PCNA interacting protein box (PIP-Box) interactions, including T2 amino alcohol (T2AA), inhibit translesion DNA synthesis. The crystal structure of PCNA in complex with T2AA revealed that T2AA bound to the surface adjacent to the subunit interface of the homotrimer of PCNA in addition to the PIP-box binding cavity. Because this site is close to Lys-164, which is monoubiquitinated by RAD18, we postulated that T2AA would affect monoubiquitinated PCNA interactions. Binding of monoubiquitinated PCNA and a purified pol η fragment containing the UBZ and PIP-box was inhibited by T2AA in vitro. T2AA decreased PCNA/pol η and PCNA/REV1 chromatin colocalization but did not inhibit PCNA monoubiquitination, suggesting that T2AA hinders interactions of pol η and REV1 with monoubiquitinated PCNA. Interstrand DNA cross-links (ICLs) are repaired by mechanisms using translesion DNA synthesis that is regulated by monoubiquitinated PCNA. T2AA significantly delayed reactivation of a reporter plasmid containing an ICL. Neutral comet analysis of cells receiving T2AA in addition to cisplatin revealed that T2AA significantly enhanced formation of DNA double strand breaks (DSBs) by cisplatin. T2AA promoted colocalized foci formation of phospho-ATM and 53BP1 and up-regulated phospho-BRCA1 in cisplatin-treated cells, suggesting that T2AA increases DSBs. When cells were treated by cisplatin and T2AA, their clonogenic survival was significantly less than that of those treated by cisplatin only. These findings show that the inhibitors of monoubiquitinated PCNA chemosensitize cells by inhibiting repair of ICLs and DSBs.

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

    SciTech Connect

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

    1994-04-26

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

  11. Induction and repair of DNA double-strand breaks using constant-field gel electrophoresis and apoptosis as predictive markers for sensitivity of cancer cells to cisplatin.

    PubMed

    Saleh, Ekram M; El-Awady, Raafat A; Anis, Noha; El-Sharkawy, Nahla

    2012-10-01

    This study was designed to evaluate some parameters that may play a role in the prediction of cancer cells sensitivity to cisplatin (CIS). Sensitivity, induction and repair of DNA double-strand breaks (DSB), cell cycle regulation and induction of apoptosis were measured in four cancer cell lines with different sensitivities to CIS. Using a sulphorhodamine-B assay, the cervical carcinoma cells (HeLa) were found to be the most sensitive to CIS followed by breast carcinoma cells (MCF-7) and liver carcinoma cells (HepG2). Colon carcinoma HCT116 cells were the most resistant. As measured by constant-field gel electrophoresis (CFGE), DSB induction, but not residual DSB exhibited a significant correlation with the sensitivity of cells to CIS. Flow cytometric DNA ploidy analysis revealed that 67% of HeLa cells and 10% of MCF-7 cells shift to sub-G1 phase after incubation with CIS. Additionally, CIS induced the arrest of MCF-7 cells in S-phase and the arrest of HepG2 and HCT116 cells in both S phase and G2/M phase. Determination of the Fas-L level and Caspase-9 activity indicated that CIS-induced apoptosis results from the mitochondrial (intrinsic) pathway. These results, if confirmed using clinical samples, indicate that the induction of DNA DSB as measured by CFGE and the induction of apoptosis should be considered, along with other predictive markers, in future clinical trials to develop predictive assays for platinum -based therapy.

  12. Structure of the catalytic region of DNA ligase IV in complex with an Artemis fragment sheds light on double-strand break repair.

    PubMed

    Ochi, Takashi; Gu, Xiaolong; Blundell, Tom L

    2013-04-02

    Nonhomologous end joining (NHEJ) is central to the repair of double-stranded DNA breaks throughout the cell cycle and plays roles in the development of the immune system. Although three-dimensional structures of most components of NHEJ have been defined, those of the catalytic region of DNA ligase IV (LigIV), a specialized DNA ligase known to work in NHEJ, and of Artemis have remained unresolved. Here, we report the crystal structure at 2.4 Å resolution of the catalytic region of LigIV (residues 1-609) in complex with an Artemis peptide. We describe interactions of the DNA-binding domain of LigIV with the continuous epitope of Artemis, which, together, form a three-helix bundle. A kink in the first helix of LigIV introduced by a conserved VPF motif gives rise to a hydrophobic pocket, which accommodates a conserved tryptophan from Artemis. We provide structural insights into features of LigIV among human DNA ligases.

  13. FGFR2 regulates Mre11 expression and double-strand break repair via the MEK-ERK-POU1F1 pathway in breast tumorigenesis.

    PubMed

    Huang, Yuan-Ling; Chou, Wen-Cheng; Hsiung, Chia-Ni; Hu, Ling-Yueh; Chu, Hou-Wei; Shen, Chen-Yang

    2015-06-15

    The association between breast cancer risk and genetic variants of fibroblast growth factor receptor 2 (FGFR2) has been identified and repeatedly confirmed; however, the mechanism underlying FGFR2 in breast tumorigenesis remains obscure. Given that breast tumorigenesis is particularly related to DNA double-strand-break-repair (DSBR), we examined the hypothesis that FGFR2 is involved in DSBR. Our results show that expression of Mre11, a vital exonuclease in DSBR, is downregulated by FGFR2, which is further linked to decreased DSBR. Analysis of the Mre11 promoter revealed that POU1F1 mediates FGFR2-induced Mre11 downregulation. Furthermore, ERK, downstream of FGFR2, directly interacts with and phosphorylates POU1F1, increasing POU1F1 binding capacity to the Mre11 promoter and repressing Mre11 expression, which consequently affects DSBR and sensitizes breast cancer cells to chemotherapeutic treatments. The importance of the FGFR2-Mre11-DSBR link in cancer progression is su