Science.gov

Sample records for double-strand breaks leads

  1. Rb inactivation leads to E2F1-mediated DNA double-strand break accumulation.

    PubMed

    Pickering, M T; Kowalik, T F

    2006-02-02

    Although it is unclear which cellular factor(s) is responsible for the genetic instability associated with initiating and sustaining cell transformation, it is known that many cancers have mutations that inactivate the Rb-mediated proliferation pathway. We show here that pRb inactivation and the resultant deregulation of one E2F family member, E2F1, leads to DNA double-strand break (DSB) accumulation in normal diploid human cells. These DSBs occur independent of Atm, p53, caspases, reactive oxygen species, and apoptosis. Moreover, E2F1 does not contribute to c-Myc-associated DSBs, indicating that the DSBs associated with these oncoproteins arise through distinct pathways. We also find E2F1-associated DSBs in an Rb mutated cancer cell line in the absence of an exogenous DSB stimulus. These basal, E2F1-associated DSBs are not observed in a p16(ink4a) inactivated cancer cell line that retains functional pRb, unless pRb is depleted. Thus, Rb status is key to regulating both the proliferation promoting functions associated with E2F and for preventing DNA damage accumulation if E2F1 becomes deregulated. Taken together, these data suggest that loss of Rb creates strong selective pressure, via DSB accumulation, for inactivating p53 mutations and that E2F1 contributes to the genetic instability associated with transformation and tumorigenesis.

  2. Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting.

    PubMed

    Chung, Woo-Hyun; Zhu, Zhu; Papusha, Alma; Malkova, Anna; Ira, Grzegorz

    2010-05-13

    The formation of single-stranded DNA (ssDNA) at double-strand break (DSB) ends is essential in repair by homologous recombination and is mediated by DNA helicases and nucleases. Here we estimated the length of ssDNA generated during DSB repair and analyzed the consequences of elimination of processive resection pathways mediated by Sgs1 helicase and Exo1 nuclease on DSB repair fidelity. In wild-type cells during allelic gene conversion, an average of 2-4 kb of ssDNA accumulates at each side of the break. Longer ssDNA is formed during ectopic recombination or break-induced replication (BIR), reflecting much slower repair kinetics. This relatively extensive resection may help determine sequences involved in homology search and prevent recombination within short DNA repeats next to the break. In sgs1Delta exo1Delta mutants that form only very short ssDNA, allelic gene conversion decreases 5-fold and DSBs are repaired by BIR or de novo telomere formation resulting in loss of heterozygosity. The absence of the telomerase inhibitor, PIF1, increases de novo telomere pathway usage to about 50%. Accumulation of Cdc13, a protein recruiting telomerase, at the break site increases in sgs1Delta exo1Delta, and the requirement of the Ku complex for new telomere formation is partially bypassed. In contrast to this decreased and alternative DSB repair, the efficiency and accuracy of gene targeting increases dramatically in sgs1Delta exo1Delta cells, suggesting that transformed DNA is very stable in these mutants. Altogether these data establish a new role for processive resection in the fidelity of DSB repair.

  3. 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.

  4. Do DNA double-strand breaks induced by Alu I lead to development of novel aberrations in the second and third post-treatment mitoses?

    SciTech Connect

    Wojcik, A.; Bonk, K.; Mueller, M.U.; Streffer, C.; Obe, G.

    1996-02-01

    Several authors have reported that ionizing radiation can give rise to novel aberrations several mitotic divisions after the exposure. At our institute this phenomenon has been observed in mouse preimplantation embryos. This cell system is uniquely well suited for such investigations because the first three cell divisions show a high degree of synchrony. Thus the expression of chromosomal aberrations at the first, second and third mitosis after irradiation can be scored unambiguously. To investigate whether DNA double-strand breaks may be the lesions responsible for the delayed expression of chromosomal aberrations, we have studied the frequencies of aberrations in the first, second and third mitosis after treatment of one-cell mouse embryos with the restriction enzyme Alu I. Embryos were permeabilized with Streptolysin-O. The results indicate that the induction of double-strand breaks does not lead to novel aberrations in the third post-treatment mitosis. Several embryos scored at the second mitosis showed very high numbers of aberrations, indicating that Alu I may remain active in the cells for a period of one cell cycle. After treatment with Streptolysin-O alone, enhanced aberration frequencies were observed in the third post-treatment mitosis, suggesting that membrane damage has a delayed effect on the cellular integrity. 44 refs., 3 figs., 3 tabs.

  5. 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

  6. DNA Double-Strand Breaks, Chromosomal Rearrangements, and GenomicInstability

    SciTech Connect

    Morgan, W.F.; Corcoran, J.; Hartmann, A.; Kaplan, M.I.; Limoli,C.L.; Ponnaiya, B.

    1998-03-09

    DNA double-strand breaks can lead to chromosomalrearrangements at the first mitosis after exposure to the DNAstrand-breaking agent. The evidence suggests a number of differentpathways for DNA double-strand break rejoining in mammalian cells, but itis unclear what factors determine the fate of the induced break andwhether or not it will lead to chromosomal rearrangement. If a cell doessurvive and proliferate after DNA cleavage, delayed chromosomalinstability can be observedin the clonal descendants of the exposedcell. Most, but not all DNA double-strand breaking agents are effectiveat inducing this delayed chromosomal instability. In this paper, wereview the evidence for the role of the DNA double-strand break indirectly induced and delayed chromosomal rearrangements. Copyright 1998Elsevier Science B.V.

  7. Partners and pathwaysrepairing a double-strand break.

    PubMed

    Haber, J E

    2000-06-01

    Double-strand chromosome breaks can arise in a number of ways, by ionizing radiation, by spontaneous chromosome breaks during DNA replication, or by the programmed action of endonucleases, such as in meiosis. Broken chromosomes can be repaired either by one of several homologous recombination mechanisms, or by a number of nonhomologous repair processes. Many of these pathways compete actively for the repair of a double-strand break. Which of these repair pathways is used appears to be regulated developmentally, genetically and during the cell cycle.

  8. 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.

  9. Heavy ion induced double strand breaks in bacteria and bacteriophages

    NASA Astrophysics Data System (ADS)

    Micke, U.; Schäfer, M.; Anton, A.; Horneck, G.; Bücker, H.

    DNA damage induced by heavy ions in bacterial cells and bacteriophages such as Bacillus subtilis, E. coli and Bacteriophage Tl were investigated by analyzing the double strand breaks in the chromosomal DNA. This kind of lesion is considered as one of the main reasons for lethal events. To analyze double strand breaks in long molecules of DNA - up to some Mbp in length - the technique of pulse field agarose gel electrophoresis has been used. This allows the detection of one double strand break per genome. Cell lysis and DNA isolation were performed in small agarose blocks directly. This procedure secured minimum DNA destruction by shearing forces. After running a gel, the DNA was stained with ethidium bromide. The light intensity of ethidium bromide fluorescence for both the outcoming (running) DNA and the remaining intact DNA were measured by scanning. The mean number of double strand breaks was calculated by determining the quotient of these intensities. Strand break induction after heavy ion and X-ray irradiation was compared.

  10. Targeting DNA double-strand breaks with TAL effector nucleases.

    PubMed

    Christian, Michelle; Cermak, Tomas; Doyle, Erin L; Schmidt, Clarice; Zhang, Feng; Hummel, Aaron; Bogdanove, Adam J; Voytas, Daniel F

    2010-10-01

    Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.

  11. Double strand breaks may be a missing link between entropy and aging.

    PubMed

    Lenart, Peter; Bienertová-Vašků, Julie

    2016-07-01

    It has been previously suggested that an increase in entropy production leads to aging. However, the mechanisms linking increased entropy production in living mass to aging are currently unclear. Even though entropy cannot be easily associated with any specific molecular damage, the increase of entropy in structural mass may be connected with heat stress, which is known to generate double strand breaks. Double strand breaks, which are in turn known to play an important role in process of aging, are thus connected to both aging and an increase of entropy. In view of these associations, we propose a new model where the increase of entropy leads to the formation of double strand breaks, resulting in an aging phenotype. This not only offers a new perspective on aging research and facilitates experimental validation, but could also serve as a useful explanatory tool. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. Radiation Induced DNA Double-Strand Breaks in Radiology.

    PubMed

    Kuefner, M A; Brand, M; Engert, C; Schwab, S A; Uder, M

    2015-10-01

    Shortly after the discovery of X-rays, their damaging effect on biological tissues was observed. The determination of radiation exposure in diagnostic and interventional radiology is usually based on physical measurements or mathematical algorithms with standardized dose simulations. γ-H2AX immunofluorescence microscopy is a reliable and sensitive method for the quantification of radiation induced DNA double-strand breaks (DSB) in blood lymphocytes. The detectable amount of these DNA damages correlates well with the dose received. However, the biological radiation damage depends not only on dose but also on other individual factors like radiation sensitivity and DNA repair capacity. Iodinated contrast agents can enhance the x-ray induced DNA damage level. After their induction DSB are quickly repaired. A protective effect of antioxidants has been postulated in experimental studies. This review explains the prinicple of the γ-H2AX technique and provides an overview on studies evaluating DSB in radiologic examinations. Radiologic examinations including CT and angiography induce DNA double-strand breaks. Even after mammography a slight but significant increase is detectable in peripheral blood lymphocytes. The number of radiation induced double-strand breaks correlates well with the radiation dose. Individual factors including radiation sensitivity, DNA repair capacity and the application of iodinated contrast media has an influence on the DNA damage level. © Georg Thieme Verlag KG Stuttgart · New York.

  13. Signaling of double strand breaks and deprotected telomeres in Arabidopsis

    PubMed Central

    Amiard, Simon; Gallego, Maria E.; White, Charles I.

    2013-01-01

    Failure to repair DNA double strand breaks (DSB) can lead to chromosomal rearrangements and eventually to cancer or cell death. Radiation and environmental pollutants induce DSB and this is of particular relevance to plants due to their sessile life style. DSB also occur naturally in cells during DNA replication and programmed induction of DSB initiates the meiotic recombination essential for gametogenesis in most eukaryotes. The linear nature of most eukaryotic chromosomes means that each chromosome has two “broken” ends. Chromosome ends, or telomeres, are protected by nucleoprotein caps which avoid their recognition as DSB by the cellular DNA repair machinery. Deprotected telomeres are recognized as DSB and become substrates for recombination leading to chromosome fusions, the “bridge-breakage-fusion” cycle, genome rearrangements and cell death. The importance of repair of DSB and the severity of the consequences of their misrepair have led to the presence of multiple, robust mechanisms for their detection and repair. After a brief overview of DSB repair pathways to set the context, we present here an update of current understanding of the detection and signaling of DSB in the plant, Arabidopsis thaliana. PMID:24137170

  14. 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.

  15. 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.

  16. 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.

  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. 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.

  20. Entropy in DNA Double-Strand Break, Detection and Signaling

    NASA Astrophysics Data System (ADS)

    Zhang, Yang; Schindler, Christina; Heermann, Dieter

    2014-03-01

    In biology, the term entropy is often understood as a measure of disorder - a restrictive interpretation that can even be misleading. Recently it has become clearer and clearer that entropy, contrary to conventional wisdom, can help to order and guide biological processes in living cells. DNA double-strand breaks (DSBs) are among the most dangerous lesions and efficient damage detection and repair is essential for organism viability. However, what remains unknown is the precise mechanism of targeting the site of damage within billions of intact nucleotides and a crowded nuclear environment, a process which is often referred to as recruitment or signaling. Here we show that the change in entropy associated with inflicting a DSB facilitates the recruitment of damage sensor proteins. By means of computational modeling we found that higher mobility and local chromatin structure accelerate protein association at DSB ends. We compared the effect of different chromatin architectures on protein dynamics and concentrations in the vicinity of DSBs, and related these results to experiments on repair in heterochromatin. Our results demonstrate how entropy contributes to a more efficient damage detection. We identify entropy as the physical basis for DNA double-strand break signaling.

  1. Ku-dependent and Ku-independent end-joining pathways lead to chromosomal rearrangements during double-strand break repair in Saccharomyces cerevisiae.

    PubMed Central

    Yu, Xin; Gabriel, Abram

    2003-01-01

    Chromosomal double-strand breaks (DSBs) can be repaired by either homology-dependent or homology-independent pathways. Nonhomologous repair mechanisms have been relatively less well studied, despite their potential importance in generating chromosomal rearrangements. We have developed a Saccharomyces cerevisiae-based assay to identify and characterize homology-independent chromosomal rearrangements associated with repair of a unique DSB generated within an engineered URA3 gene. Approximately 1% of successfully repaired cells have accompanying chromosomal rearrangements consisting of large insertions, deletions, aberrant gene conversions, or other more complex changes. We have analyzed rearrangements in isogenic wild-type, rad52, yku80, and rad52 yku80 strains, to determine the types of events that occur in the presence or absence of these key repair proteins. Deletions were found in all strain backgrounds, but insertions were dependent upon the presence of Yku80p. A rare RAD52- and YKU80-independent form of deletion was present in all strains. These events were characterized by long one-sided deletions (up to 13 kb) and extensive imperfect overlapping sequences (7-22 bp) at the junctions. Our results demonstrate that the frequency and types of repair events depend on the specific genetic context. This approach can be applied to a number of problems associated with chromosome stability. PMID:12663527

  2. 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.

  3. 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.

  4. Repair of Double-Strand Breaks by End Joining

    PubMed Central

    Chiruvella, Kishore K.; Liang, Zhuobin; Wilson, Thomas E.

    2013-01-01

    Nonhomologous end joining (NHEJ) refers to a set of genome maintenance pathways in which two DNA double-strand break (DSB) ends are (re)joined by apposition, processing, and ligation without the use of extended homology to guide repair. Canonical NHEJ (c-NHEJ) is a well-defined pathway with clear roles in protecting the integrity of chromosomes when DSBs arise. Recent advances have revealed much about the identity, structure, and function of c-NHEJ proteins, but many questions exist regarding their concerted action in the context of chromatin. Alternative NHEJ (alt-NHEJ) refers to more recently described mechanism(s) that repair DSBs in less-efficient backup reactions. There is great interest in defining alt-NHEJ more precisely, including its regulation relative to c-NHEJ, in light of evidence that alt-NHEJ can execute chromosome rearrangements. Progress toward these goals is reviewed. PMID:23637284

  5. Repair of double-strand breaks by end joining.

    PubMed

    Chiruvella, Kishore K; Liang, Zhuobin; Wilson, Thomas E

    2013-05-01

    Nonhomologous end joining (NHEJ) refers to a set of genome maintenance pathways in which two DNA double-strand break (DSB) ends are (re)joined by apposition, processing, and ligation without the use of extended homology to guide repair. Canonical NHEJ (c-NHEJ) is a well-defined pathway with clear roles in protecting the integrity of chromosomes when DSBs arise. Recent advances have revealed much about the identity, structure, and function of c-NHEJ proteins, but many questions exist regarding their concerted action in the context of chromatin. Alternative NHEJ (alt-NHEJ) refers to more recently described mechanism(s) that repair DSBs in less-efficient backup reactions. There is great interest in defining alt-NHEJ more precisely, including its regulation relative to c-NHEJ, in light of evidence that alt-NHEJ can execute chromosome rearrangements. Progress toward these goals is reviewed.

  6. 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.

  7. A double-strand break can trigger immunoglobulin gene conversion

    PubMed Central

    Bastianello, Giulia; Arakawa, Hiroshi

    2017-01-01

    All three B cell-specific activities of the immunoglobulin (Ig) gene re-modeling system—gene conversion, somatic hypermutation and class switch recombination—require activation-induced deaminase (AID). AID-induced DNA lesions must be further processed and dissected into different DNA recombination pathways. In order to characterize potential intermediates for Ig gene conversion, we inserted an I-SceI recognition site into the complementarity determining region 1 (CDR1) of the Ig light chain locus of the AID knockout DT40 cell line, and conditionally expressed I-SceI endonuclease. Here, we show that a double-strand break (DSB) in CDR1 is sufficient to trigger Ig gene conversion in the absence of AID. The pattern and pseudogene usage of DSB-induced gene conversion were comparable to those of AID-induced gene conversion; surprisingly, sometimes a single DSB induced multiple gene conversion events. These constitute direct evidence that a DSB in the V region can be an intermediate for gene conversion. The fate of the DNA lesion downstream of a DSB had more flexibility than that of AID, suggesting two alternative models: (i) DSBs during the physiological gene conversion are in the minority compared to single-strand breaks (SSBs), which are frequently generated following DNA deamination, or (ii) the physiological gene conversion is mediated by a tightly regulated DSB that is locally protected from non-homologous end joining (NHEJ) or other non-homologous DNA recombination machineries. PMID:27701075

  8. 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

  9. 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.

  10. Selective mitochondrial DNA degradation following double-strand breaks.

    PubMed

    Moretton, Amandine; Morel, Frédéric; Macao, Bertil; Lachaume, Philippe; Ishak, Layal; Lefebvre, Mathilde; Garreau-Balandier, Isabelle; Vernet, Patrick; Falkenberg, Maria; Farge, Géraldine

    2017-01-01

    Mitochondrial DNA (mtDNA) can undergo double-strand breaks (DSBs), caused by defective replication, or by various endogenous or exogenous sources, such as reactive oxygen species, chemotherapeutic agents or ionizing radiations. MtDNA encodes for proteins involved in ATP production, and maintenance of genome integrity following DSBs is thus of crucial importance. However, the mechanisms involved in mtDNA maintenance after DSBs remain unknown. In this study, we investigated the consequences of the production of mtDNA DSBs using a human inducible cell system expressing the restriction enzyme PstI targeted to mitochondria. Using this system, we could not find any support for DSB repair of mtDNA. Instead we observed a loss of the damaged mtDNA molecules and a severe decrease in mtDNA content. We demonstrate that none of the known mitochondrial nucleases are involved in the mtDNA degradation and that the DNA loss is not due to autophagy, mitophagy or apoptosis. Our study suggests that a still uncharacterized pathway for the targeted degradation of damaged mtDNA in a mitophagy/autophagy-independent manner is present in mitochondria, and might provide the main mechanism used by the cells to deal with DSBs.

  11. DNA Double Strand Breaks: A Common Theme in Neurodegenerative Diseases.

    PubMed

    Merlo, Daniela; Mollinari, Cristiana; Racaniello, Mauro; Garaci, Enrico; Cardinale, Alessio

    2016-01-01

    Accumulation of DNA damage and impairment of DNA repair systems are involved in the pathogenesis of different neurodegenerative diseases. Whenever DNA damage is too extensive, the DNA damage response pathway provides for triggering cellular senescence and/or apoptosis. However, whether the increased level of DNA damage in neurodegenerative disorders is a cause rather than the consequence of neurodegenerative events remains to be established. Among possible DNA lesions, DNA double strand breaks (DSBs) are rare events, nevertheless they are the most lethal form of DNA damage. In neurons, DSBs are particularly deleterious because of their reduced DNA repair capability as compared to proliferating cells. Here, we provide a description of DSB repair systems and describe human studies showing the presence of several types of DNA lesions in three major neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Then, we analyze the role of DSB accumulation and deficiency of DSB repair systems in neurodegeneration by examining studies on animal models of neurodegenerative diseases.

  12. Chromatin modifications and DNA repair: beyond double-strand breaks

    PubMed Central

    House, Nealia C. M.; Koch, Melissa R.; Freudenreich, Catherine H.

    2014-01-01

    DNA repair must take place in the context of chromatin, and chromatin modifications and DNA repair are intimately linked. The study of double-strand break repair has revealed numerous histone modifications that occur after induction of a DSB, and modification of the repair factors themselves can also occur. In some cases the function of the modification is at least partially understood, but in many cases it is not yet clear. Although DSB repair is a crucial activity for cell survival, DSBs account for only a small percentage of the DNA lesions that occur over the lifetime of a cell. Repair of single-strand gaps, nicks, stalled forks, alternative DNA structures, and base lesions must also occur in a chromatin context. There is increasing evidence that these repair pathways are also regulated by histone modifications and chromatin remodeling. In this review, we will summarize the current state of knowledge of chromatin modifications that occur during non-DSB repair, highlighting similarities and differences to DSB repair as well as remaining questions. PMID:25250043

  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. DNA double-strand breaks caused by replication arrest.

    PubMed Central

    Michel, B; Ehrlich, S D; Uzest, M

    1997-01-01

    We report here that DNA double-strand breaks (DSBs) form in Escherichia coli upon arrest of replication forks due to a defect in, or the inhibition of, replicative DNA helicases. The formation of DSBs was assessed by the appearance of linear DNA detected by pulse-field gel electrophoresis. Processing of DSBs by recombination repair or linear DNA degradation was abolished by mutations in recBCD genes. Two E. coli replicative helicases were tested, Rep, which is essential in recBC mutants, and DnaB. The proportion of linear DNA increased up to 50% upon shift of rep recBTS recCTS cells to restrictive temperature. No increase in linear DNA was observed in the absence of replicating chromosomes, indicating that the formation of DSBs in rep strains requires replication. Inhibition of the DnaB helicase either by a strong replication terminator or by a dnaBTS mutation led to the formation of linear DNA, showing that blocked replication forks are prone to DSB formation. In wild-type E. coli, linear DNA was detected in the absence of RecBC or of both RecA and RecD. This reveals the existence of a significant amount of spontaneous DSBs. We propose that some of them may also result from the impairment of replication fork progression. PMID:9029161

  15. ATM controls meiotic double-strand-break formation.

    PubMed

    Lange, Julian; Pan, Jing; Cole, Francesca; Thelen, Michael P; Jasin, Maria; Keeney, Scott

    2011-10-16

    In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation. However, improperly repaired DSBs can cause meiotic arrest or mutation; thus, having too many DSBs is probably as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse and SPO11 and its accessory factors remain abundant long after most DSB formation ceases, implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signalling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least tenfold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency.

  16. Have a break: determinants of meiotic DNA double strand break (DSB) formation and processing in plants.

    PubMed

    Edlinger, Bernd; Schlögelhofer, Peter

    2011-03-01

    Meiosis is an essential process for sexually reproducing organisms, leading to the formation of specialized generative cells. This review intends to highlight current knowledge of early events during meiosis derived from various model organisms, including plants. It will particularly focus on cis- and trans-requirements of meiotic DNA double strand break (DSB) formation, a hallmark event during meiosis and a prerequisite for recombination of genetic traits. Proteins involved in DSB formation in different organisms, emphasizing the known factors from plants, will be introduced and their functions outlined. Recent technical advances in DSB detection and meiotic recombination analysis will be reviewed, as these new tools now allow analysis of early meiotic recombination in plants with incredible accuracy. To anticipate future directions in plant meiosis research, unpublished results will be included wherever possible.

  17. 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.

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

    PubMed Central

    Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

    2014-01-01

    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. PMID:25287622

  19. DNA Double-Strand Break Rejoining in Complex Normal Tissues

    SciTech Connect

    Ruebe, Claudia E.; Kuehne, Martin; Fricke, Andreas

    2008-11-15

    Purpose: The clinical radiation responses of different organs vary widely and likely depend on the intrinsic radiosensitivities of their different cell populations. Double-strand breaks (DSBs) are the most deleterious form of DNA damage induced by ionizing radiation, and the cells' capacity to rejoin radiation-induced DSBs is known to affect their intrinsic radiosensitivity. To date, only little is known about the induction and processing of radiation-induced DSBs in complex normal tissues. Using an in vivo model with repair-proficient mice, the highly sensitive {gamma}H2AX immunofluorescence was established to investigate whether differences in DSB rejoining could account for the substantial differences in clinical radiosensitivity observed among normal tissues. Methods and Materials: After whole body irradiation of C57BL/6 mice (0.1, 0.5, 1.0, and 2.0 Gy), the formation and rejoining of DSBs was analyzed by enumerating {gamma}H2AX foci in various organs representative of both early-responding (small intestine) and late-responding (lung, brain, heart, kidney) tissues. Results: The linear dose correlation observed in all analyzed tissues indicated that {gamma}H2AX immunofluorescence allows for the accurate quantification of DSBs in complex organs. Strikingly, the various normal tissues exhibited identical kinetics for {gamma}H2AX foci loss, despite their clearly different clinical radiation responses. Conclusion: The identical kinetics of DSB rejoining measured in different organs suggest that tissue-specific differences in radiation responses are independent of DSB rejoining. This finding emphasizes the fundamental role of DSB repair in maintaining genomic integrity, thereby contributing to cellular viability and functionality and, thus, tissue homeostasis.

  20. DNA Mismatch Repair-Induced Double-Strand Breaks

    PubMed Central

    Nowosielska, Anetta; Marinus, M. G.

    2007-01-01

    Escherichia coli dam mutants are sensitized to the cytotoxic action of base analogs, cisplatin and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), while their mismatch repair (MMR)-deficient derivatives are tolerant to these agents. We showed previously, using pulse field gel electrophoresis, that MMR-mediated double-strand breaks (DSBs) are produced by cisplatin in dam recB (Ts) cells at the non-permissive temperature. We demonstrate here that the majority of these DSBs require DNA replication for their formation, consistent with a model in which replication forks collapse at nicks or gaps formed during MMR. DSBs were also detected in dam recB(Ts) ada ogt cells exposed to MNNG in a dose- and MMR-dependent manner. In contrast to cisplatin, the formation of these DSBs was not affected by DNA replication and it is proposed that two separate mechanisms result in DSB formation. Replication-independent DSBs arise from overlapping base excision and MMR repair tracts on complementary strands and constitute the majority of detectable DSBs in dam recB(Ts) ada ogt cells exposed to MNNG. Replication-dependent DSBs result from replication fork collapse at O6-meG base pairs undergoing MMR futile cycling and are more likely to contribute to cytotoxicity. This model is consistent with the observation that fast-growing dam recB (Ts) ada ogt cells, which have more chromosome replication origins, are more sensitive to the cytotoxic effect of MNNG than the same cells growing slowly. PMID:17827074

  1. 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.

  2. 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.

  3. DNA double-strand breaks after percutaneous transluminal angioplasty.

    PubMed

    Geisel, Dominik; Heverhagen, Johannes T; Kalinowski, Marc; Wagner, Hans-Joachim

    2008-09-01

    To determine exemplarily the amount of DNA damage and the repair kinetics after interventional radiologic procedures by using visualization of foci of the phosphorylated form of the H2AX histone variant (gammaH2AX) to quantify DNA double-strand breaks (DSBs) at percutaneous transluminal angioplasty (PTA) of the lower limb arteries. After local ethics committee approval and written informed consent were obtained, five patients (two women, three men; mean age, 64.4 years; age range, 45-76 years) scheduled for computed tomography (CT) and 20 patients (six women, 14 men; mean age, 68.5 years; age range, 53-85 years) scheduled for PTA of lower limb arteries were prospectively entered into the study. Blood samples were taken before the first exposure to ionizing radiation and 5 minutes, 1 hour, 6 hours, and 24 hours after the last exposure. Additional samples were taken from the irradiated limb (femoral vein) of three patients who underwent PTA--before the first radiation exposure, 5 and 10 minutes after the first exposure, and 5 minutes after the last exposure. Lymphocytes were isolated, fixed, and stained with anti-gammaH2AX antibody, and gammaH2AX focus yields were determined with fluorescence microscopy. Data were analyzed with linear regression and two-sample F tests. Mean increase in number of gammaH2AX foci after CT (7.78 per 1 Gy x cm) depended linearly on dose-length product (r = 0.997). Number of foci reached background levels within 24 hours. Mean numbers of gammaH2AX foci per cell increased by factors of 4.08-20.67 in blood samples taken 5 minutes after PTA compared with mean numbers of foci before PTA. Mean radiation dose increase, 6.56/(10 Gy x cm(2)), depended linearly on dose-area product (r = 0.993). Maximal focus yield in cells taken directly from the irradiated limb was higher than that in cells from the systemic circulation (by mean factor of 1.46). Data showed compromised DSB repair capacity after PTA (P < .05). Mean number of foci at 24 hours (0

  4. 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.

  5. Quantitative, non-invasive imaging of radiation-induced DNA double strand breaks in vivo

    PubMed Central

    Li, Wenrong; Li, Fang; Huang, Qian; Shen, Jingping; Wolf, Frank; He, Yujun; Liu, Xinjian; Hu, Y. Angela; Bedford, Joel. S.; Li, Chuan-Yuan

    2011-01-01

    DNA double strand breaks is a major form of DNA damage and a key mechanism through which radiotherapy and some chemotherapeutic agents kill cancer cells. Despite its importance, measuring DNA double strand breaks is still a tedious task that is normally carried out by gel electrophoresis or immunofluorescence staining. Here we report a novel approach to image and quantify DNA double strand breaks in live mammalian cells through bi-fragment luciferase reconstitution. N- and C- terminal fragments of firefly luciferase gene were fused with H2AX and MDC1 genes, respectively. Our strategy was based on the established fact that at the sites of DNA double strand breaks, H2AX protein is phosphoryated and physically associates with the MDC1 protein, thus bringing together N- and C- luciferase fragments and reconstituting luciferase activity. Our strategy allowed serial, non-invasive quantification of DNA double strand breaks in cells irradiated with x-rays and 56Fe ions. Furthermore, it allowed for the evaluation of DNA double strand breaks (DSBs) non-invasively in vivo in irradiated tumors over two weeks. Surprisingly, we detected a second wave of DSB induction in irradiated tumor cells days after radiation exposure in addition to the initial rapid induction of DSBs. We conclude that our new split-luciferase based method for imaging γ-H2AX-MDC1 interaction is a powerful new tool to study DNA double strand break repair kinetics in vivo with considerable advantage for experiments requiring observations over an extended period of time. PMID:21527553

  6. Molecular characterization of DNA double strand breaks with tip-enhanced Raman scattering.

    PubMed

    Lipiec, Ewelina; Sekine, Ryo; Bielecki, Jakub; Kwiatek, Wojciech M; Wood, Bayden R

    2014-01-03

    DNA double strand breaks (DSBs) are deadly lesions that can lead to genetic defects and cell apoptosis. Techniques that directly detect DNA DSBs include scanning electron microscopy, atomic force microscopy (AFM), and fluorescence based approaches. While these techniques can be used to identify DSBs they provide no information on the molecular events occurring at the break. Tip-enhanced Raman scattering (TERS) can provide molecular information from DNA at the nanoscale and in combination with AFM provides a new way to visualize and characterize the molecular structure of DSBs. DSBs result from cleavage at the 3'- and 5'-bonds of deoxyribose upon exposure to UVC radiation based on the observation of POH and methyl/methylene deformation modes enhanced in the TERS spectra. It is hypothesized that strand fragments are hydrogen-terminated at the lesion, indicating the action of free radicals during photon exposure. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. 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

  8. 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.

  9. 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.

  10. 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)

  11. Gamma-irradiated quiescent cells repair directly induced double-strand breaks but accumulate persistent double-strand breaks during subsequent DNA replication.

    PubMed

    Minakawa, Yusuke; Atsumi, Yuko; Shinohara, Akira; Murakami, Yasufumi; Yoshioka, Ken-Ichi

    2016-07-01

    H2AX is expressed at very low levels in quiescent normal cells in vivo and in vitro. Such cells repair DNA double-strand breaks (DSBs) induced by γ-irradiation through a transient stabilization of H2AX. However, the resultant cells accumulate small numbers of irreparable (or persistent) DSBs via an unknown mechanism. We found that quiescent cells that had repaired DSBs directly induced by γ-rays were prone to accumulate DSBs during the subsequent DNA replication. Unlike directly induced DSBs, secondary DSBs were not efficiently repaired, although Rad51 and 53BP1 were recruited to these sites. H2AX was dramatically stabilized in response to DSBs directly caused by γ-rays, enabling γH2AX foci formation and DSB repair, whereas H2AX was barely stabilized in response to secondary DSBs, in which γH2AX foci were small and DSBs were not efficiently repaired. Our results show a pathway that leads to the persistent DSB formation after γ-irradiation. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  12. 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.

  13. Zinc chromate induces chromosome instability and DNA double strand breaks in human lung cells

    SciTech Connect

    Xie Hong; Holmes, Amie L.; Young, Jamie L.; Qin Qin; Joyce, Kellie; Pelsue, Stephen C.; Peng Cheng; Wise, Sandra S.; Jeevarajan, Antony S.; Wallace, William T.; Hammond, Dianne; Wise, John Pierce E-mail: John.Wise@usm.maine.edu

    2009-02-01

    Hexavalent chromium Cr(VI) is a respiratory toxicant and carcinogen, with solubility playing an important role in its carcinogenic potential. Zinc chromate, a water insoluble or 'particulate' Cr(VI) compound, has been shown to be carcinogenic in epidemiology studies and to induce tumors in experimental animals, but its genotoxicity is poorly understood. Our study shows that zinc chromate induced concentration-dependent increases in cytotoxicity, chromosome damage and DNA double strand breaks in human lung cells. In response to zinc chromate-induced breaks, MRE11 expression was increased and ATM and ATR were phosphorylated, indicating that the DNA double strand break repair system was initiated in the cells. In addition, our data show that zinc chromate-induced double strand breaks were only observed in the G2/M phase population, with no significant amount of double strand breaks observed in G1 and S phase cells. These data will aid in understanding the mechanisms of zinc chromate toxicity and carcinogenesis.

  14. Inactivation, DNA double strand break induction and their rejoining in bacterial cells irradiated with heavy ions

    NASA Technical Reports Server (NTRS)

    Schaefer, M.; Zimmermann, H.; Schmitz, C.

    1994-01-01

    Besides inactivation one of the major interests in our experiments is to study the primary damage in the DNA double strand breaks (DSB) after heavy ion irradiation. These damages lead not only to cell death but also under repair activities to mutations. In further experiments we have investigated the inactivation with two different strains of Deinococcus radiodurans (R1, Rec 30) and the induction of DSB as well as the rejoining of DSB in stationary cells of E. coli (strain B/r) irradiated with radiations of different quality. In the latter case irradiations were done so that the cell survival was roughly at the same level. We measured the DSB using the pulse field gelelectrophoresis which allows to separate between intact (circular) and damaged (linear) DNA. The irradiated cells were transferred to NB medium and incubated for different times to allow rejoining.

  15. DNA double-strand breaks induced by high NaCl occur predominantly in gene deserts

    PubMed Central

    Dmitrieva, Natalia I.; Cui, Kairong; Kitchaev, Daniil A.; Zhao, Keji; Burg, Maurice B.

    2011-01-01

    High concentration of NaCl increases DNA breaks both in cell culture and in vivo. The breaks remain elevated as long as NaCl concentration remains high and are rapidly repaired when the concentration is lowered. The exact nature of the breaks, and their location, has not been entirely clear, and it has not been evident how cells survive, replicate, and maintain genome integrity in environments like the renal inner medulla in which cells are constantly exposed to high NaCl concentration. Repair of the breaks after NaCl is reduced is accompanied by formation of foci containing phosphorylated H2AX (γH2AX), which occurs around DNA double-strand breaks and contributes to their repair. Here, we confirm by specific comet assay and pulsed-field electrophoresis that cells adapted to high NaCl have increased levels of double-strand breaks. Importantly, γH2AX foci that occur during repair of the breaks are nonrandomly distributed in the mouse genome. By chromatin immunoprecipitation using anti-γH2AX antibody, followed by massive parallel sequencing (ChIP-Seq), we find that during repair of double-strand breaks induced by high NaCl, γH2AX is predominantly localized to regions of the genome devoid of genes (“gene deserts”), indicating that the high NaCl-induced double-strand breaks are located there. Localization to gene deserts helps explain why the DNA breaks are less harmful than are the random breaks induced by genotoxic agents such as UV radiation, ionizing radiation, and oxidants. We propose that the universal presence of NaCl around animal cells has directly influenced the evolution of the structure of their genomes. PMID:22106305

  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. Spo11 and the Formation of DNA Double-Strand Breaks in Meiosis.

    PubMed

    Keeney, Scott

    2008-01-01

    Meiotic recombination is carried out through a specialized pathway for the formation and repair of DNA double-strand breaks made by the Spo11 protein, a relative of archaeal topoisomerase VI. This review summarizes recent studies that provide insight to the mechanism of DNA cleavage by Spo11, functional interactions of Spo11 with other proteins required for break formation, mechanisms that control the timing of recombination initiation, and evolutionary conservation and divergence of these processes.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome.

    PubMed Central

    Phillips, J W; Morgan, W F

    1994-01-01

    We examined DNA double-strand-break-induced mutations in the endogenous adenine phosphoribosyl-transferase (APRT) gene in cultured Chinese hamster ovary cells after exposure to restriction endonucleases. PvuII, EcoRV, and StuI, all of which produce blunt-end DNA double-strand breaks, were electroporated into CHO-AT3-2 cells hemizygous at the APRT locus. Colonies of viable cells containing mutations at APRT were expanded, and the mutations that occurred during break repair were analyzed at the DNA sequence level. Restriction enzyme-induced mutations consisted of small deletions of 1 to 36 bp, insertions, and combinations of insertions and deletions at the cleavage sites. Most of the small deletions involved overlaps of one to four complementary bases at the recombination junctions. Southern blot analysis revealed more complex mutations, suggesting translocation, inversion, or insertion of larger chromosomal fragments. These results indicate that blunt-end DNA double-strand breaks can induce illegitimate (nonhomologous) recombination in mammalian chromosomes and that they play an important role in mutagenesis. Images PMID:8065314

  3. 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.

  4. 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.

  5. 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.

  6. Effects of heavy ions on inactivation and DNA double strand breaks in Deinococcus radiodurans R1.

    PubMed

    Zimmermann, H; Schafer, M; Schmitz, C; Bucker, H

    1994-10-01

    Inactivation and double strand break (dsb) induction after heavy ion irradiation were studied in stationary phase cells of the highly radiation resistant bacterium Deinococcus radiodurans R1. There is evidence that the radiation sensitivity of this bacterium is nearly independent on energy in the range of up to 15 MeV/u for lighter ions (Ar). The responses to dsb induction for charged particles show direct relationship between increasing radiation dose and residual intact DNA.

  7. 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

  8. Choreographing the Double Strand Break Response: Ubiquitin and SUMO Control of Nuclear Architecture

    PubMed Central

    Harding, Shane M.; Greenberg, Roger A.

    2016-01-01

    The cellular response to DNA double strand breaks (DSBs) is a multifaceted signaling program that centers on post-translational modifications including phosphorylation, ubiquitylation and SUMOylation. In this review we discuss how ubiquitin and SUMO orchestrate the recognition of DSBs and explore how this influences chromatin organization. We discuss functional outcomes of this response including transcriptional silencing and how pre-existing chromatin states may control the DSB response and the maintenance of genomic stability. PMID:27375678

  9. Processing of 3'-phosphoglycolate-terminated DNA double strand breaks by Artemis nuclease.

    PubMed

    Povirk, Lawrence F; Zhou, Tong; Zhou, Ruizhe; Cowan, Morton J; Yannone, Steven M

    2007-02-09

    The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double strand breaks. To assess the possibility that Artemis acts on oxidatively modified double strand break termini, its activity toward model DNA substrates, bearing either 3'-hydroxyl or 3'-phosphoglycolate moieties, was examined. A 3'-phosphoglycolate had little effect on Artemis-mediated trimming of long 3' overhangs (> or =9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3'-phosphoglycolates on overhangs of 4-5 bases promoted Artemis-mediated removal of a single 3'-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3' overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was completely dependent on DNA-dependent protein kinase and ATP and was largely dependent on Ku, which markedly stimulated Artemis activity toward all 3' overhangs. Together, these data suggest that efficient Artemis-mediated cleavage of 3' overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3' to the cleavage site, as well as 2 unpaired nucleotides 5' to the cleavage site. Shorter 3'-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis but much more slowly. Consistent with a role for Artemis in repair of terminally blocked double strand breaks in vivo, human cells lacking Artemis exhibited hypersensitivity to x-rays, bleomycin, and neocarzinostatin, which all induce 3'-phosphoglycolate-terminated double strand breaks.

  10. Myonuclear breakdown in sporadic inclusion body myositis is accompanied by DNA double strand breaks.

    PubMed

    Nishii, Makoto; Nakano, Satoshi; Nakamura, Seika; Wate, Reika; Shinde, Akiyo; Kaneko, Satoshi; Kusaka, Hirofumi

    2011-05-01

    Rimmed vacuoles in sporadic inclusion body myositis (s-IBM) contain nuclear remnants. We sought to determine if the nuclear degeneration seen in s-IBM is associated with DNA damage. In muscle biopsy specimens from ten patients with s-IBM and 50 controls, we immunolocalized 1) phosphorylated histone H2AX (γ-H2AX), which is a sensitive immunocytochemical marker of DNA double-strand breaks and 2) DNA-PK, which is an enzyme involved in double-strand break repair. In s-IBM, vacuolar peripheries often showed strong immunoreactivity to γ-H2AX and the three components of DNA-PK (DNA-PKcs, Ku70, and Ku80). A triple fluorescence study of Ku70, emerin, and DNA displayed nuclear breakdown and it suggested impaired nuclear incorporation of Ku70. The percentage of positive nuclei for γ-H2AX was significantly higher in vacuolated fibers than non-vacuolated fibers in s-IBM, or fibers in polymyosits. We hypothesize that a dysfunction of nuclear envelope may cause nuclear fragility, double-strand breaks and impaired nuclear transport in s-IBM. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Simulation of the Formation of DNA Double Strand Breaks and Chromosome Aberrations in Irradiated Cells

    NASA Technical Reports Server (NTRS)

    Plante, Ianik; Ponomarev, Artem L.; Wu, Honglu; Blattnig, Steve; George, Kerry

    2014-01-01

    The formation of DNA double-strand breaks (DSBs) and chromosome aberrations is an important consequence of ionizing radiation. To simulate DNA double-strand breaks and the formation of chromosome aberrations, we have recently merged the codes RITRACKS (Relativistic Ion Tracks) and NASARTI (NASA Radiation Track Image). The program RITRACKS is a stochastic code developed to simulate detailed event-by-event radiation track structure: [1] This code is used to calculate the dose in voxels of 20 nm, in a volume containing simulated chromosomes, [2] The number of tracks in the volume is calculated for each simulation by sampling a Poisson distribution, with the distribution parameter obtained from the irradiation dose, ion type and energy. The program NASARTI generates the chromosomes present in a cell nucleus by random walks of 20 nm, corresponding to the size of the dose voxels, [3] The generated chromosomes are located within domains which may intertwine, and [4] Each segment of the random walks corresponds to approx. 2,000 DNA base pairs. NASARTI uses pre-calculated dose at each voxel to calculate the probability of DNA damage at each random walk segment. Using the location of double-strand breaks, possible rejoining between damaged segments is evaluated. This yields various types of chromosomes aberrations, including deletions, inversions, exchanges, etc. By performing the calculations using various types of radiations, it will be possible to obtain relative biological effectiveness (RBE) values for several types of chromosome aberrations.

  12. 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.

  13. Immunofluorescent Detection of DNA Double Strand Breaks induced by High-LET Radiation

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.; Wu, Honglu; Desai, Nirav

    2004-01-01

    Within cell nuclei, traversing charged heavy ion particles lead to the accumulation of proteins related to DNA lesions and repair along the ion trajectories. Irradiation using a standard geometric setup with the beam path perpendicular to the cell monolayer generates discrete foci of several proteins known to localize at sites of DNA double strand breaks (DSBs). One such molecule is the histone protein H2AX (gamma-H2AX), which gets rapidly phosphorylated in response to ionizing radiation. Here we present data obtained with a modified irradiation geometry characterized by a beam path parallel to a monolayer of human fibroblast cells. This new irradiation geometry leads to the formation of gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in the x/y plane, thus enabling the analysis of the fluorescence distributions along the particle trajectories. Qualitative analysis of these distributions presented insights into the DNA repair kinetics along the primary track structure and visualization of possible chromatin movement. We also present evidence of colocalization of gamma-H2AX with several other proteins in responses to ionizing radiation exposure. Analysis of gamma-H2AX has the potential to provide useful information on human cell responses to high LET radiation after exposure to space-like radiation.

  14. Self-inflicted DNA double-strand breaks sustain tumorigenicity and stemness of cancer cells.

    PubMed

    Liu, Xinjian; Li, Fang; Huang, Qian; Zhang, Zhengxiang; Zhou, Ling; Deng, Yu; Zhou, Min; Fleenor, Donald E; Wang, He; Kastan, Michael B; Li, Chuan-Yuan

    2017-03-24

    DNA double-strand breaks (DSBs) are traditionally associated with cancer through their abilities to cause chromosomal instabilities or gene mutations. Here we report a new class of self-inflicted DNA DSBs that can drive tumor growth irrespective of their effects on genomic stability. We discover a mechanism through which cancer cells cause DSBs in their own genome spontaneously independent of reactive oxygen species or replication stress. In this mechanism, low-level cytochrome c leakage from the mitochondria leads to sublethal activation of apoptotic caspases and nucleases, which causes DNA DSBs. In response to these spontaneous DNA DSBs, ATM, a key factor involved in DNA damage response, is constitutively activated. Activated ATM leads to activation of transcription factors NF-κB and STAT3, known drivers of tumor growth. Moreover, self-inflicted DNA DSB formation and ATM activation are important in sustaining the stemness of patient-derived glioma cells. In human tumor tissues, elevated levels of activated ATM correlate with poor patient survival. Self-inflicted DNA DSBs therefore are functionally important for maintaining the malignancy of cancer cells.Cell Research advance online publication 24 March 2017; doi: 10.1038/cr.2017.41.

  15. Immunofluorescent Detection of DNA Double Strand Breaks induced by High-LET Radiation

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.; Wu, Honglu; Desai, Nirav

    2004-01-01

    Within cell nuclei, traversing charged heavy ion particles lead to the accumulation of proteins related to DNA lesions and repair along the ion trajectories. Irradiation using a standard geometric setup with the beam path perpendicular to the cell monolayer generates discrete foci of several proteins known to localize at sites of DNA double strand breaks (DSBs). One such molecule is the histone protein H2AX (gamma-H2AX), which gets rapidly phosphorylated in response to ionizing radiation. Here we present data obtained with a modified irradiation geometry characterized by a beam path parallel to a monolayer of human fibroblast cells. This new irradiation geometry leads to the formation of gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in the x/y plane, thus enabling the analysis of the fluorescence distributions along the particle trajectories. Qualitative analysis of these distributions presented insights into the DNA repair kinetics along the primary track structure and visualization of possible chromatin movement. We also present evidence of colocalization of gamma-H2AX with several other proteins in responses to ionizing radiation exposure. Analysis of gamma-H2AX has the potential to provide useful information on human cell responses to high LET radiation after exposure to space-like radiation.

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

    PubMed

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

    2017-04-01

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

  17. 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.

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

    PubMed Central

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

    2011-01-01

    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. PMID:21689668

  19. 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.

  20. 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.

  1. 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.

  2. REV7 counteracts DNA double-strand break resection and impacts PARP inhibition

    PubMed Central

    Xu, Guotai; Yuan, Jingsong; Mistrik, Martin; Bouwman, Peter; Bartkova, Jirina; Gogola, Ewa; Warmerdam, Daniël; Barazas, Marco; Jaspers, Janneke E.; Watanabe, Kenji; Pieterse, Mark; Kersbergen, Ariena; Sol, Wendy; Celie, Patrick H. N.; Schouten, Philip C.; van den Broek, Bram; Salman, Ahmed; Nieuwland, Marja; de Rink, Iris; de Ronde, Jorma; Jalink, Kees; Boulton, Simon J.; Chen, Junjie; van Gent, Dik C.; Bartek, Jiri; Jonkers, Jos; Borst, Piet; Rottenberg, Sven

    2015-01-01

    Summary Error-free repair of DNA double-strand breaks (DSB) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway1. In the absence of BRCA1-mediated HR, administration of PARP inhibitors induces synthetic lethality of tumor cells of patients with breast or ovarian cancers2,3. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration4. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases5. In particular, little is known about BRCA1-independent restoration of HR. Here, we show that loss of REV7 (also known as MAD2L2) re-establishes CtIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and appears to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance6. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining (NHEJ) during immunoglobulin class switch recombination. Our results reveal an unexpected critical function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells. PMID:25799992

  3. 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

  4. DNA double-strand breaks disrupted the spindle assembly in porcine oocytes.

    PubMed

    Wang, HaiYang; Luo, YiBo; Zhao, Ming-Hui; Lin, ZiLi; Kwon, Jeongwoo; Cui, Xiang-Shun; Kim, Nam-Hyung

    2016-02-01

    We used etoposide (25-100 µg/mL) to induce DNA double-strand breaks (DSBs) in porcine oocytes at the germinal vesicle (GV) stage to determine how such damage affects oocyte maturation. We observed that DNA damage did not delay the rate of germinal vesicle breakdown (GVBD), but did inhibit the final stages of maturation, as indicated by the failure to extrude the first polar body. Oocytes with low levels of DSBs failed to effectively activate ataxia telangiectasia-mutated (ATM) kinase, while those with severe DNA DSBs failed to activate checkpoint kinase 1 (CHK1)--the two regulators of the DNA damage response pathway--indicating that porcine oocytes lack an efficient G2/M phase checkpoint. DSBs induced spindle defects and chromosomal misalignments, leading to the arrest of these oocytes at meiotic metaphase I. The activity of maturation-promoting factor also did not increase appropriately in oocytes with DNA DSBs, although its abundance was sufficient to promote GVBD and chromosomal condensation. Following parthenogenetic activation, embryos from etoposide-treated oocytes formed numerous micronuclei. Thus, our results indicate that DNA DSBs do not efficiently activate the ATM/CHK1-dependent DNA-damage checkpoint in porcine oocytes, allowing these DNA-impaired oocytes to enter M phase. Oocytes with DNA damage did, however, arrest at metaphase I in response to spindle defects and chromosomal misalignments, which limited the ability of these oocytes to reach meiotic metaphase II.

  5. Nucleolar Reorganization Upon Site-Specific Double-Strand Break Induction.

    PubMed

    Franek, Michal; Kovaříková, Alena; Bártová, Eva; Kozubek, Stanislav

    2016-11-01

    DNA damage response (DDR) in ribosomal genes and mechanisms of DNA repair in embryonic stem cells (ESCs) are less explored nuclear events. DDR in ESCs should be unique due to their high proliferation rate, expression of pluripotency factors, and specific chromatin signature. Given short population doubling time and fast progress through G1 phase, ESCs require a sustained production of rRNA, which leads to the formation of large and prominent nucleoli. Although transcription of rRNA in the nucleolus is relatively well understood, little is known about DDR in this nuclear compartment. Here, we directed formation of double-strand breaks in rRNA genes with I- PpoI endonuclease, and we studied nucleolar morphology, DDR, and chromatin modifications. We observed a pronounced formation of I- PpoI-induced nucleolar caps, positive on BRCA1, NBS1, MDC1, γH2AX, and UBF1 proteins. We showed interaction of nucleolar protein TCOF1 with HDAC1 and TCOF1 with CARM1 after DNA injury. Moreover, H3R17me2a modification mediated by CARM1 was found in I- PpoI-induced nucleolar caps. Finally, we report that heterochromatin protein 1 is not involved in DNA repair of nucleolar caps.

  6. 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.

  7. Generation of Gross Chromosomal Rearrangements by a Single Engineered DNA Double Strand Break

    PubMed Central

    Qiu, Zhijun; Zhang, Zhenhua; Roschke, Anna; Varga, Tamas; Aplan, Peter D.

    2017-01-01

    Gross chromosomal rearrangements (GCRs), including translocations, inversions amplifications, and deletions, can be causal events leading to malignant transformation. GCRs are thought to be triggered by DNA double strand breaks (DSBs), which in turn can be spontaneous or induced by external agents (eg. cytotoxic chemotherapy, ionizing radiation). It has been shown that induction of DNA DSBs at two defined loci can produce stable balanced chromosomal translocations, however, a single engineered DNA DSB could not. Herein, we report that although a single engineered DNA DSB in H2AX “knockdown” cells did not generate GCRs, repair of a single engineered DNA DSB in fibroblasts that had ablated H2ax did produce clonal, stable GCRs, including balanced translocations and megabase-pair inversions. Upon correction of the H2ax deficiency, cells no longer generated GCRs following a single engineered DNA DSB. These findings demonstrate that clonal, stable GCRs can be produced by a single engineered DNA DSB in H2ax knockout cells, and that the production of these GCRs is ameliorated by H2ax expression. PMID:28225067

  8. DNA Double-Strand Breaks: A Potential Causative Factor for Mammalian Aging?

    PubMed Central

    Han, Li; Mitchell, James R.; Hasty, Paul

    2008-01-01

    Aging is a pleiotropic and stochastic process influenced by both genetics and environment. As a result the fundamental underlying causes of aging are controversial and likely diverse. Genome maintenance and in particular the repair of DNA damage is critical to ensure longevity needed for reproduction and as a consequence imperfections or defects in maintaining the genome may contribute to aging. There are many forms of DNA damage with double-strand breaks (DSBs) being the most toxic. Here we discuss DNA DSBs as a potential causative factor for aging including factors that generate DNA DSBs, pathways that repair DNA DSBs, consequences of faulty or failed DSB repair and how these consequences may lead to age-dependent decline in fitness. At the end we compare mouse models of premature aging that are defective for repairing either DSBs or UV light-induced lesions. Based on these comparisons we believe the basic mechanisms responsible for their aging phenotypes are fundamentally different demonstrating the complex and pleiotropic nature of this process. PMID:18346777

  9. Double-strand breaks associated with repetitive DNA can reshape the genome

    PubMed Central

    Argueso, Juan Lucas; Westmoreland, James; Mieczkowski, Piotr A.; Gawel, Malgorzata; Petes, Thomas D.; Resnick, Michael A.

    2008-01-01

    Ionizing radiation is an established source of chromosome aberrations (CAs). Although double-strand breaks (DSBs) are implicated in radiation-induced and other CAs, the underlying mechanisms are poorly understood. Here, we show that, although the vast majority of randomly induced DSBs in G2 diploid yeast cells are repaired efficiently through homologous recombination (HR) between sister chromatids or homologous chromosomes, ≈2% of all DSBs give rise to CAs. Complete molecular analysis of the genome revealed that nearly all of the CAs resulted from HR between nonallelic repetitive elements, primarily Ty retrotransposons. Nonhomologous end-joining (NHEJ) accounted for few, if any, of the CAs. We conclude that only those DSBs that fall at the 3–5% of the genome composed of repetitive DNA elements are efficient at generating rearrangements with dispersed small repeats across the genome, whereas DSBs in unique sequences are confined to recombinational repair between the large regions of homology contained in sister chromatids or homologous chromosomes. Because repeat-associated DSBs can efficiently lead to CAs and reshape the genome, they could be a rich source of evolutionary change. PMID:18701715

  10. Mechanisms and Consequences of Double-strand DNA Break Formation in Chromatin

    PubMed Central

    Cannan, Wendy J.; Pederson, David S.

    2016-01-01

    All organisms suffer double-strand breaks (DSBs) in their DNA as a result of exposure to ionizing radiation. DSBs can also form when replication forks encounter DNA lesions or repair intermediates. The processing and repair of DSBs can lead to mutations, loss of heterozygosity, and chromosome rearrangements that result in cell death or cancer. The most common pathway used to repair DSBs in metazoans (non-homologous DNA end joining) is more commonly mutagenic than the alternative pathway (homologous recombination mediated repair). Thus, factors that influence the choice of pathways used DSB repair can affect an individual’s mutation burden and risk of cancer. This review describes radiological, chemical and biological mechanisms that generate DSBs, and discusses the impact of such variables as DSB etiology, cell type, cell cycle, and chromatin structure on the yield, distribution, and processing of DSBs. The final section focuses on nucleosome-specific mechanisms that influence DSB production, and the possible relationship between higher order chromosome coiling and chromosome shattering (chromothripsis). PMID:26040249

  11. Genome-wide redistribution of meiotic double-strand breaks in Saccharomyces cerevisiae.

    PubMed

    Robine, Nicolas; Uematsu, Norio; Amiot, Franck; Gidrol, Xavier; Barillot, Emmanuel; Nicolas, Alain; Borde, Valérie

    2007-03-01

    Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs) catalyzed by the Spo11 protein. DSBs are not randomly distributed along chromosomes. To better understand factors that control the distribution of DSBs in budding yeast, we have examined the genome-wide binding and cleavage properties of the Gal4 DNA binding domain (Gal4BD)-Spo11 fusion protein. We found that Gal4BD-Spo11 cleaves only a subset of its binding sites, indicating that the association of Spo11 with chromatin is not sufficient for DSB formation. In centromere-associated regions, the centromere itself prevents DSB cleavage by tethered Gal4BD-Spo11 since its displacement restores targeted DSB formation. In addition, we observed that new DSBs introduced by Gal4BD-Spo11 inhibit surrounding DSB formation over long distances (up to 60 kb), keeping constant the number of DSBs per chromosomal region. Together, these results demonstrate that the targeting of Spo11 to new chromosomal locations leads to both local stimulation and genome-wide redistribution of recombination initiation and that some chromosomal regions are inherently cold regardless of the presence of Spo11.

  12. 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.

  13. A critical role for topoisomerase IIb and DNA double strand breaks in transcription

    PubMed Central

    Calderwood, Stuart K.

    2016-01-01

    ABSTRACT Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb. PMID:27100743

  14. c-Myc Suppression of DNA Double-strand Break Repair12

    PubMed Central

    Li, Zhaozhong; Owonikoko, Taofeek K; Sun, Shi-Yong; Ramalingam, Suresh S; Doetsch, Paul W; Xiao, Zhi-Qiang; Khuri, Fadlo R; Curran, Walter J; Deng, Xingming

    2012-01-01

    c-Myc is a transcriptional factor that functions as a central regulator of cell growth, proliferation, and apoptosis. Overexpression of c-Myc also enhances DNA double-strand breaks (DSBs), genetic instability, and tumorigenesis. However, the mechanism(s) involved remains elusive. Here, we discovered that γ-ray ionizing radiation-induced DSBs promote c-Myc to form foci and to co-localize with γ-H2AX. Conditional expression of c-Myc in HO15.19 c-Myc null cells using the Tet-Off/Tet-On inducible system results in down-regulation of Ku DNA binding and suppressed activities of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and DNA end-joining, leading to inhibition of DSB repair and enhanced chromosomal and chromatid breaks. Expression of c-Myc reduces both signal and coding joins with decreased fidelity during V(D)J recombination. Mechanistically, c-Myc directly interacts with Ku70 protein through its Myc box II (MBII) domain. Removal of the MBII domain from c-Myc abrogates its inhibitory effects on Ku DNA binding, DNA-PKcs, and DNA end-joining activities, which results in loss of c-Myc's ability to block DSB repair and V(D)J recombination. Interestingly, c-Myc directly disrupts the Ku/DNA-PKcs complex in vitro and in vivo. Thus, c-Myc suppression of DSB repair and V(D)J recombination may occur through inhibition of the nonhomologous end-joining pathway, which provides insight into the mechanism of c-Myc in the development of tumors through promotion of genomic instability. PMID:23308051

  15. Smoking during pregnancy causes double-strand DNA break damage to the placenta.

    PubMed

    Slatter, Tania L; Park, Lydia; Anderson, Karyn; Lailai-Tasmania, Viwa; Herbison, Peter; Clow, William; Royds, Janice A; Devenish, Celia; Hung, Noelyn A

    2014-01-01

    Despite the adverse effects of smoking, many pregnancies are exposed to tobacco smoke. Recent studies have investigated whether smoking damages placental DNA by measuring DNA adducts. This study investigated whether a more severe lesion, double-strand DNA breaks, was also present in the tobacco smoking-exposed placenta. Term placentae from women who smoked during their entire pregnancies (n = 52), from those who had ceased smoking for at least 4 weeks before delivery (previous smokers, n = 34), and from nonsmoking women (n = 150) were examined using the DNA double-strand break marker phosphorylated γ H2AX. The extent of DNA damage was assessed according to cell type and additional markers were applied for cell fate (apoptosis and DNA repair), and function (human chorionic gonadotropin, human placental lactogen, and glucose transporter 1), to characterize the effect of the DNA damage on placental integrity. Marked phosphorylated γ H2AX-positive cells occurred in the villous syncytiotrophoblast and syncytial knot nuclei in placentae from smokers (P < .001). Phosphorylated γ H2AX foci did not colocalize with the DNA repair protein 53BP1, and damaged nuclei had a marked reduction in expression of human chorionic gonadotropin, human placental lactogen, and glucose transporter 1. Minimal DNA damage, similar to nonsmokers, was present in previous smokers including those that had ceased smoking for just over 4 weeks before delivery. In summary, smoking during pregnancy was associated with marked double-strand DNA break damage to the syncytiotrophoblast. We suggest that smoking cessation is important to prevent additional DNA damage and to facilitate DNA repair.

  16. Processing of 3'-Phosphoglycolate-Terminated DNA Double-StrandBreaks by Artemis Nuclease

    SciTech Connect

    Povrik, Lawrence F.; Zhou, Tong; Zhou, Ruizhe; Cowan, Morton J.; Yannone, Steven M.

    2005-10-01

    The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double-strand breaks. To assess the possibility that Artemis functions on oxidatively modified double-strand break termini, its activity toward model DNA substrates, bearing either 3{prime}-hydroxyl or 3{prime}-phosphoglycolate moieties, was examined. A 3{prime}-phosphoglycolate had little effect on Artemis-mediated trimming of long 3{prime} overhangs (>9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3{prime}-phosphoglycolates on overhangs of 4-5 bases promoted selective Artemis-mediated trimming of a single 3{prime}-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3{prime} overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was dependent upon Ku, DNA-dependent protein kinase, and ATP. Together, these data suggest that Artemis-mediated cleavage of 3{prime} overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3{prime} to the cleavage site. Shorter 3{prime}-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis, but much less efficiently. Consistent with the in vitro substrate specificity of Artemis, human cells lacking Artemis exhibited hypersensitivity to X-rays, bleomycin and neocarzinostatin, which all induce 3{prime}-phosphoglycolate-terminated double-strand breaks. Collectively, these results suggest that 3{prime}-phosphoglycolate termini and/or specific classes of DNA ends that arise from such blocked termini are relevant Artemis substrates in vivo.

  17. 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.

  18. Bleomycin-induced double-strand breaks in mitochondrial DNA of Drosophila cells are repaired.

    PubMed

    Morel, Frederic; Renoux, Monique; Lachaume, Philippe; Alziari, Serge

    2008-01-01

    Mitochondrial DNA lesions cause numerous human diseases, and it is therefore important to identify the mechanisms whereby the mitochondrion repairs the damage. We have studied in cultured Drosophila cells the repair of bleomycin-induced double-strand breaks (DSBs) in mitochondrial DNA. Our results show that DSBs are repaired as rapidly and effectively in the mitochondria as in the nucleus. DNA repair is complete within 2h following bleomycin treatment, showing that Drosophila mitochondria have an effective system of DSB repair. The mechanism and mitochondrial proteins involved remain to be identified.

  19. DNA double-strand breaks measured in individual cells subjected to gel electrophoresis

    SciTech Connect

    Olive, P.L.; Wlodek, D.; Banath, J.P. )

    1991-09-01

    Microscopic examination of individual mammalian cells embedded in agarose, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye provides a novel way of measuring DNA damage and more importantly, of assessing heterogeneity in DNA damage within a mixed population of cells. With this method, DNA double-strand breaks can be detected in populations of cells exposed to X-ray doses as low as 5 Gy. The radiation dose-response relationship for initial formation of double-strand breaks was identical for cell lines irradiated in G1, regardless of their sensitivity to killing by ionizing radiation. However, for cells irradiated in S phase, DNA migration was significantly reduced. For Chinese hamster V79 cells, Chinese hamster ovary cells, WiDr human colon carcinoma cells, and L5178Y-R mouse lymphoblastoid cells, S-phase DNA appeared to be about 3 times less sensitive to X-ray damage than DNA from other phases of the cell cycle. However, for the very radiosensitive L5178Y-S cells, the migration of replicating DNA was reduced only slightly. For Chinese hamster V79 and Chinese hamster ovary cells, damage was repaired at a similar rate in all cells of the population, and 85% of the breaks were rejoined within 2 h after irradiation. The radiosensitive L5178Y-S cells repaired damage more slowly than V79 or Chinese hamster ovary cells; 2 h after exposure to 50 Gy, approximately 50% of the damage was still present.

  20. Accumulation of DNA Double-Strand Breaks in Normal Tissues After Fractionated Irradiation

    SciTech Connect

    Ruebe, Claudia E.

    2010-03-15

    Purpose: There is increasing evidence that genetic factors regulating the recognition and/or repair of DNA double-strand breaks (DSBs) are responsible for differences in radiosensitivity among patients. Genetically defined DSB repair capacities are supposed to determine patients' individual susceptibility to develop adverse normal tissue reactions after radiotherapy. In a preclinical murine model, we analyzed the impact of different DSB repair capacities on the cumulative DNA damage in normal tissues during the course of fractionated irradiation. Material and Methods: Different strains of mice with defined genetic backgrounds (SCID{sup -/-} homozygous, ATM{sup -/-} homozygous, ATM{sup +/-}heterozygous, and ATM{sup +/+}wild-type mice) were subjected to single (2 Gy) or fractionated irradiation (5 x 2 Gy). By enumerating gammaH2AX foci, the formation and rejoining of DSBs were analyzed in organs representative of both early-responding (small intestine) and late-responding tissues (lung, kidney, and heart). Results: In repair-deficient SCID{sup -/-} and ATM{sup -/-}homozygous mice, large proportions of radiation-induced DSBs remained unrepaired after each fraction, leading to the pronounced accumulation of residual DNA damage after fractionated irradiation, similarly visible in early- and late-responding tissues. The slight DSB repair impairment of ATM{sup +/-}heterozygous mice was not detectable after single-dose irradiation but resulted in a significant increase in unrepaired DSBs during the fractionated irradiation scheme. Conclusions: Radiation-induced DSBs accumulate similarly in acute- and late-responding tissues during fractionated irradiation, whereas the whole extent of residual DNA damage depends decisively on the underlying genetically defined DSB repair capacity. Moreover, our data indicate that even minor impairments in DSB repair lead to exceeding DNA damage accumulation during fractionated irradiation and thus may have a significant impact on normal

  1. 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.

  2. DNA double-strand breaks activate ATM independent of mitochondrial dysfunction in A549 cells.

    PubMed

    Kalifa, Lidza; Gewandter, Jennifer S; Staversky, Rhonda J; Sia, Elaine A; Brookes, Paul S; O'Reilly, Michael A

    2014-10-01

    Excessive nuclear or mitochondrial DNA damage can lead to mitochondrial dysfunction, decreased energy production, and increased generation of reactive oxygen species (ROS). Although numerous cell signaling pathways are activated when cells are injured, the ataxia telangiectasia mutant (ATM) protein has emerged as a major regulator of the response to both mitochondrial dysfunction and nuclear DNA double-strand breaks (DSBs). Because mitochondrial dysfunction is often a response to excessive DNA damage, it has been difficult to determine whether nuclear and/or mitochondrial DNA DSBs activate ATM independent of mitochondrial dysfunction. In this study, mitochondrial and nuclear DNA DSBs were generated in the A549 human lung adenocarcinoma cell line by infecting with retroviruses expressing the restriction endonuclease PstI fused to a mitochondrial targeting sequence (MTS) or nuclear localization sequence (NLS) and a hemagglutinin antigen epitope tag (HA). Expression of MTS-PstI-HA or NLS-PstI-HA activated the DNA damage response defined by phosphorylation of ATM, the tumor suppressor protein p53 (TP53), KRAB-associated protein (KAP)-1, and structural maintenance of chromosomes (SMC)-1. Phosphorylated ATM and SMC1 were detected in nuclear fractions, whereas phosphorylated TP53 and KAP1 were detected in both mitochondrial and nuclear fractions. PstI also enhanced expression of the cyclin-dependent kinase inhibitor p21 and inhibited cell growth. This response to DNA damage occurred in the absence of detectable mitochondrial dysfunction and excess production of ROS. These findings reveal that DNA DSBs are sufficient to activate ATM independent of mitochondrial dysfunction and suggest that the activated form of ATM and some of its substrates are restricted to the nuclear compartment, regardless of the site of DNA damage. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. NR4A2 Promotes DNA Double-strand Break Repair Upon Exposure to UVR.

    PubMed

    Yin, Kelvin; Chhabra, Yash; Tropée, Romain; Lim, Yi Chieh; Fane, Mitchell; Dray, Eloise; Sturm, Richard A; Smith, Aaron G

    2017-09-01

    Exposure of melanocytes to ultraviolet radiation (UVR) induces the formation of UV lesions that can produce deleterious effects in genomic DNA. Encounters of replication forks with unrepaired UV lesions can lead to several complex phenomena, such as the formation of DNA double-strand breaks (DSBs). The NR4A family of nuclear receptors are transcription factors that have been associated with mediating DNA repair functions downstream of the MC1R signaling pathway in melanocytes. In particular, emerging evidence shows that upon DNA damage, the NR4A2 receptor can translocate to sites of UV lesion by mechanisms requiring post-translational modifications within the N-terminal domain and at a serine residue in the DNA-binding domain at position 337. Following this, NR4A2 aids in DNA repair by facilitating chromatin relaxation, allowing accessibility for DNA repair machinery. Using A2058 and HT144 melanoma cells engineered to stably express wild-type or mutant forms of the NR4A2 proteins, we reveal that the expression of functional NR4A2 is associated with elevated cytoprotection against UVR. Conversely, knockdown of NR4A2 expression by siRNA results in a significant loss of cell viability after UV insult. By analyzing the kinetics of the ensuing 53BP1 and RAD51 foci following UV irradiation, we also reveal that the expression of mutant NR4A2 isoforms, lacking the ability to translocate, transactivate, or undergo phosphorylation, display compromised repair capacity.Implications: These data expand the understanding of the mechanism by which the NR4A2 nuclear receptor can facilitate DNA DSB repair. Mol Cancer Res; 15(9); 1184-96. ©2017 AACR. ©2017 American Association for Cancer Research.

  4. 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.

  5. 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

  6. DNA double strand breaks in the acute phase after synchrotron pencilbeam irradiation

    NASA Astrophysics Data System (ADS)

    Fernandez-Palomo, C.; Bräuer-Krisch, E.; Trippel, M.; Schroll, C.; Requardt, H.; Bartzsch, S.; Nikkhah, G.; Schültke, E.

    2013-07-01

    Introduction. At the biomedical beamline of the European Synchrotron Radiation Facility (ESRF), we have established a method to study pencilbeam irradiation in-vivoin small animal models. The pencilbeam irradiation technique is based on the principle of microbeam irradiation, a concept of spatially fractionated high-dose irradiation. Using γH2AX as marker, we followed the development of DNA double strand breaks over 48 hrs after whole brain irradiation with the pencilbeam technique. Method. Almost square pencilbeams with an individual size of 51 × 50 μm were produced with an MSC collimator using a step and shoot approach, while the animals were moved vertically through the beam. The center-to-center distance (ctc) was 400 μm, with a peak-to-valley dose ratio (PVDR) of about 400. Five groups of healthy adult mice received peak irradiation doses of either 330 Gy or 2,460 Gy and valley doses of 0.82 Gy and 6.15 Gy, respectively. Animals were sacrificed at 2, 12 and 48 hrs after irradiation. Results. DNA double strand breaks are observed in the path of the pencilbeam. The size of the damaged volume undergoes changes within the first 48 hours after irradiation. Conclusions. The extent of DNA damage caused by pencilbeam irradiation, as assessed by H2AX antibody staining, is dose- dependent.

  7. 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.

  8. 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.

  9. 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.

  10. Monitoring homology search during DNA double-strand break repair in vivo.

    PubMed

    Renkawitz, Jörg; Lademann, Claudio A; Kalocsay, Marian; Jentsch, Stefan

    2013-04-25

    Homologous recombination (HR) is crucial for genetic exchange and accurate repair of DNA double-strand breaks and is pivotal for genome integrity. HR uses homologous sequences for repair, but how homology search, the exploration of the genome for homologous DNA sequences, is conducted in the nucleus remains poorly understood. Here, we use time-resolved chromatin immunoprecipitations of repair proteins to monitor homology search in vivo. We found that homology search proceeds by a probing mechanism, which commences around the break and samples preferentially on the broken chromosome. However, elements thought to instruct chromosome loops mediate homology search shortcuts, and centromeres, which cluster within the nucleus, may facilitate homology search on other chromosomes. Our study thus reveals crucial parameters for homology search in vivo and emphasizes the importance of linear distance, chromosome architecture, and proximity for recombination efficiency. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. The cytotoxicity of (-)-lomaiviticin A arises from induction of double-strand breaks in DNA

    NASA Astrophysics Data System (ADS)

    Colis, Laureen C.; Woo, Christina M.; Hegan, Denise C.; Li, Zhenwu; Glazer, Peter M.; Herzon, Seth B.

    2014-06-01

    The metabolite (-)-lomaiviticin A, which contains two diazotetrahydrobenzo[b]fluorene (diazofluorene) functional groups, inhibits the growth of cultured human cancer cells at nanomolar-picomolar concentrations; however, the mechanism responsible for the potent cytotoxicity of this natural product is not known. Here we report that (-)-lomaiviticin A nicks and cleaves plasmid DNA by a pathway that is independent of reactive oxygen species and iron, and that the potent cytotoxicity of (-)-lomaiviticin A arises from the induction of DNA double-strand breaks (dsbs). In a plasmid cleavage assay, the ratio of single-strand breaks (ssbs) to dsbs is 5.3 ± 0.6:1. Labelling studies suggest that this cleavage occurs via a radical pathway. The structurally related isolates (-)-lomaiviticin C and (-)-kinamycin C, which contain one diazofluorene, are demonstrated to be much less effective DNA cleavage agents, thereby providing an explanation for the enhanced cytotoxicity of (-)-lomaiviticin A compared to that of other members of this family.

  12. Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks.

    PubMed

    Aymard, François; Bugler, Beatrix; Schmidt, Christine K; Guillou, Emmanuelle; Caron, Pierre; Briois, Sébastien; Iacovoni, Jason S; Daburon, Virginie; Miller, Kyle M; Jackson, Stephen P; Legube, Gaëlle

    2014-04-01

    Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.

  13. 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

  14. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing.

    PubMed

    Crosetto, Nicola; Mitra, Abhishek; Silva, Maria Joao; Bienko, Magda; Dojer, Norbert; Wang, Qi; Karaca, Elif; Chiarle, Roberto; Skrzypczak, Magdalena; Ginalski, Krzysztof; Pasero, Philippe; Rowicka, Maga; Dikic, Ivan

    2013-04-01

    We present a genome-wide approach to map DNA double-strand breaks (DSBs) at nucleotide resolution by a method we termed BLESS (direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing). We validated and tested BLESS using human and mouse cells and different DSBs-inducing agents and sequencing platforms. BLESS was able to detect telomere ends, Sce endonuclease-induced DSBs and complex genome-wide DSB landscapes. As a proof of principle, we characterized the genomic landscape of sensitivity to replication stress in human cells, and we identified >2,000 nonuniformly distributed aphidicolin-sensitive regions (ASRs) overrepresented in genes and enriched in satellite repeats. ASRs were also enriched in regions rearranged in human cancers, with many cancer-associated genes exhibiting high sensitivity to replication stress. Our method is suitable for genome-wide mapping of DSBs in various cells and experimental conditions, with a specificity and resolution unachievable by current techniques.

  15. Initiation of meiotic recombination by double-strand DNA breaks in S. pombe.

    PubMed

    Klar, A J; Miglio, L M

    1986-08-29

    Mitotic gene conversion and reciprocal recombination have recently been shown to be efficiently initiated by double-strand DNA breaks (DSBs) in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. We tested whether DSBs could also initiate meiotic recombination at the mat1 locus in S. pombe. The mat1 switching-mechanism-generated DSB found in mitotically growing cells can be repaired without mat1 switching, since strains deleted for both donor loci (mat2-P and mat3-M) have the break but do not produce inviable cells. A (mat1-P X mat1-M) cross produced a high frequency (20%) of 3:1 gene conversions of mat1 in meiotic tetrads. Gene conversion events were associated with the recombination of flanking markers. Strains lacking the DSB failed to convert. Thus, the DSB at mat1 promotes efficient meiotic recombination in fission yeast.

  16. 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

  17. Essential role for DNA-PKcs in DNA double strand break repair and apoptosis in ATM deficient lymphocytes

    PubMed Central

    Callén, Elsa; Jankovic, Mila; Wong, Nancy; Zha, Shan; Chen, Hua-Tang; Difilippantonio, Simone; Di Virgilio, Michela; Heidkamp, Gordon; Alt, Frederick W.; Nussenzweig, André; Nussenzweig, Michel

    2009-01-01

    SUMMARY The DNA double strand break (DSB) repair protein DNA-PKcs and the signal transducer ATM are both activated by DNA breaks and phosphorylate similar substrates in vitro, yet appear to have distinct functions in vivo. Here we show that ATM and DNA-PKcs have overlapping functions in lymphocytes. Ablation of both kinase activities in cells undergoing immunoglobulin class switch recombination leads to a compound defect in switching, and a synergistic increase in chromosomal fragmentation, DNA insertions and translocations due to aberrant processing of DSBs. These abnormalities are attributed to a compound deficiency in phosphorylation of key proteins required for DNA repair, class switching and cell death. Notably, both kinases are required for normal levels of p53 phosphorylation in B and T cells and p53 dependent apoptosis. Our experiments reveal a DNA-PKcs-dependent pathway that regulates DNA repair and activation of p53 in the absence of ATM. PMID:19450527

  18. RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

    PubMed Central

    Dillingham, Mark S.; Kowalczykowski, Stephen C.

    2008-01-01

    Summary: The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA, protecting the bacteria from phages and extraneous chromosomal DNA. The RecBCD enzyme is, however, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzyme's vigorous nuclease activity, switches the polarity of the attenuated nuclease activity to the 5′ strand, changes the operation of its motor subunits, and instructs the enzyme to begin loading the RecA protein onto the resultant Chi-containing single-stranded DNA. This enzyme is a prototypical example of a molecular machine: the protein architecture incorporates several autonomous functional domains that interact with each other to produce a complex, sequence-regulated, DNA-processing machine. In this review, we discuss the biochemical mechanism of the RecBCD enzyme with particular emphasis on new developments relating to the enzyme's structure and DNA translocation mechanism. PMID:19052323

  19. 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.

  20. Resection is responsible for loss of transcription around a double-strand break in Saccharomyces cerevisiae.

    PubMed

    Manfrini, Nicola; Clerici, Michela; Wery, Maxime; Colombo, Chiara Vittoria; Descrimes, Marc; Morillon, Antonin; d'Adda di Fagagna, Fabrizio; Longhese, Maria Pia

    2015-07-31

    Emerging evidence indicate that the mammalian checkpoint kinase ATM induces transcriptional silencing in cis to DNA double-strand breaks (DSBs) through a poorly understood mechanism. Here we show that in Saccharomyces cerevisiae a single DSB causes transcriptional inhibition of proximal genes independently of Tel1/ATM and Mec1/ATR. Since the DSB ends undergo nucleolytic degradation (resection) of their 5'-ending strands, we investigated the contribution of resection in this DSB-induced transcriptional inhibition. We discovered that resection-defective mutants fail to stop transcription around a DSB, and the extent of this failure correlates with the severity of the resection defect. Furthermore, Rad9 and generation of γH2A reduce this DSB-induced transcriptional inhibition by counteracting DSB resection. Therefore, the conversion of the DSB ends from double-stranded to single-stranded DNA, which is necessary to initiate DSB repair by homologous recombination, is responsible for loss of transcription around a DSB in S. cerevisiae.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Functions and regulation of the MRX complex at DNA double-strand breaks.

    PubMed

    Gobbini, Elisa; Cassani, Corinne; Villa, Matteo; Bonetti, Diego; Longhese, Maria P

    2016-07-27

    DNA double-strand breaks (DSBs) pose a serious threat to genome stability and cell survival. Cells possess mechanisms that recognize DSBs and promote their repair through either homologous recombination (HR) or non-homologous end joining (NHEJ). The evolutionarily conserved Mre11-Rad50-Xrs2 (MRX) complex plays a central role in the cellular response to DSBs, as it is implicated in controlling end resection and in maintaining the DSB ends tethered to each other. Furthermore, it is responsible for DSB signaling by activating the checkpoint kinase Tel1 that, in turn, supports MRX function in a positive feedback loop. The present review focuses mainly on recent works in the budding yeast Saccharomyces cerevisiae to highlight structure and regulation of MRX as well as its interplays with Tel1.

  6. Coupling end resection with the checkpoint response at DNA double-strand breaks.

    PubMed

    Villa, Matteo; Cassani, Corinne; Gobbini, Elisa; Bonetti, Diego; Longhese, Maria Pia

    2016-10-01

    DNA double-strand breaks (DSBs) are a nasty form of damage that needs to be repaired to ensure genome stability. The DSB ends can undergo a strand-biased nucleolytic processing (resection) to generate 3'-ended single-stranded DNA (ssDNA) that channels DSB repair into homologous recombination. Generation of ssDNA also triggers the activation of the DNA damage checkpoint, which couples cell cycle progression with DSB repair. The checkpoint response is intimately linked to DSB resection, as some checkpoint proteins regulate the resection process. The present review will highlight recent works on the mechanism and regulation of DSB resection and its interplays with checkpoint activation/inactivation in budding yeast.

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

    PubMed Central

    Pâques, Frédéric; Haber, James E.

    1999-01-01

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

  8. A computational approach to the relationship between radiation induced double strand breaks and translocations

    NASA Technical Reports Server (NTRS)

    Holley, W. R.; Chatterjee, A.

    1994-01-01

    A theoretical framework is presented which provides a quantitative analysis of radiation induced translocations between the ab1 oncogene on CH9q34 and a breakpoint cluster region, bcr, on CH 22q11. Such translocations are associated frequently with chronic myelogenous leukemia. The theory is based on the assumption that incorrect or unfaithful rejoining of initial double strand breaks produced concurrently within the 200 kbp intron region upstream of the second abl exon, and the 16.5 kbp region between bcr exon 2 and exon 6 interact with each other, resulting in a fusion gene. for an x-ray dose of 100 Gy, there is good agreement between the theoretical estimate and the one available experimental result. The theory has been extended to provide dose response curves for these types of translocations. These curves are quadratic at low doses and become linear at high doses.

  9. 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.

  10. Control of Meiotic Crossovers: From Double-Strand Break Formation to Designation

    PubMed Central

    Gray, Stephen

    2017-01-01

    Meiosis, the mechanism of creating haploid gametes, is a complex cellular process observed across sexually reproducing organisms. Fundamental to meiosis is the process of homologous recombination, whereby DNA double-strand breaks are introduced into the genome and are subsequently repaired to generate either noncrossovers or crossovers. Although homologous recombination is essential for chromosome pairing during prophase I, the resulting crossovers are critical for maintaining homolog interactions and enabling accurate segregation at the first meiotic division. Thus, the placement, timing, and frequency of crossover formation must be exquisitely controlled. In this review, we discuss the proteins involved in crossover formation, the process of their formation and designation, and the rules governing crossovers, all within the context of the important landmarks of prophase I. We draw together crossover designation data across organisms, analyze their evolutionary divergence, and propose a universal model for crossover regulation. PMID:27648641

  11. [Bacterial infections as seen from the eukaryotic genome: DNA double strand breaks, inflammation and cancer].

    PubMed

    Lemercier, Claudie

    2014-01-01

    An increasing number of studies report that infection by pathogenic bacteria alters the host genome, producing highly hazardous DNA double strand breaks for the eukaryotic cell. Even when DNA repair occurs, it often leaves "scars" on chromosomes that might generate genomic instability at the next cell division. Chronic intestinal inflammation promotes the expansion of genotoxic bacteria in the intestinal microbiote which in turn triggers tumor formation and colon carcinomas. Bacteria act at the level of the host DNA repair machinery. They also highjack the host cell cycle to allow themselves time for replication in an appropriate reservoir. However, except in the case of bacteria carrying the CDT nuclease, the molecular mechanisms responsible for DNA lesions are not well understood, even if reactive oxygen species released during infection make good candidates. © 2014 médecine/sciences – Inserm.

  12. Physiological Brain Activity Causes DNA Double Strand Breaks in Neurons — Exacerbation by Amyloid-β

    PubMed Central

    Suberbielle, Elsa; Sanchez, Pascal E.; Kravitz, Alexxai V.; Wang, Xin; Ho, Kaitlyn; Eilertson, Kirsten; Devidze, Nino; Kreitzer, Anatol C.; Mucke, Lennart

    2013-01-01

    We show that a natural behavior, exploration of a novel environment, causes DNA double-strand breaks (DSBs) in neurons of young adult wildtype mice. DSBs occurred in multiple brain regions, were most abundant in the dentate gyrus, which is involved in spatial learning and memory, and were repaired within 24 hours. Increasing neuronal activity by sensory or optogenetic stimulation increased neuronal DSBs in relevant but not irrelevant networks. Human amyloid precursor protein (hAPP) transgenic mice, which simulate key aspects of Alzheimer's disease, had increased neuronal DSBs at baseline and more severe and prolonged DSBs after exploration. Interventions that suppress aberrant neuronal activity and improve memory in hAPP mice normalized their levels of DSBs. Blocking extrasynaptic NMDA-type glutamate receptors prevented amyloid-β (Aβ)-induced DSBs in neuronal cultures. Thus, transient increases in neuronal DSBs occur as a result of physiological brain activity and Aβ exacerbates DNA damage, most likely by eliciting synaptic dysfunction. PMID:23525040

  13. DNA Replication Triggered by Double-Stranded Breaks in E. coli: Dependence on Homologous Recombination Functions

    PubMed Central

    Asai, Tsuneaki; Bates, David B.; Kogoma, Tokio

    2010-01-01

    Summary Homologous recombination-dependent DNA replication (RDR) of a λ cos site-carrying plasmid is demonstrated in E. coli cells when the cells express λ terminase that introduces a double-stranded break into the cos site. RDR occurs in normal wild-type cells if the plasmid also contains the recombination hotspot χ. χ is dispensable when cells are induced for the SOS response or contain a recD mutation. recBC sbcA mutant cells are also capable of RDR induction. A recN mutation greatly reduces RDR in normal cells, but not in SOS-induced cells. RDR proceeds by the θ mode or rolling circle mode of DNA synthesis, yielding covalently closed circular plasmid monomers or linear plasmid multimers, respectively. Previously described inducible stable DNA replication is considered to be a special type of RDR that starts exclusively from specific sites (or/Ms) on the chromosome. PMID:7923355

  14. 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.

  15. Significant correlation of species longevity with DNA double strand break recognition but not with telomere length.

    PubMed

    Lorenzini, Antonello; Johnson, F Brad; Oliver, Anthony; Tresini, Maria; Smith, Jasmine S; Hdeib, Mona; Sell, Christian; Cristofalo, Vincent J; Stamato, Thomas D

    2009-01-01

    The identification of the cellular mechanisms responsible for the wide differences in species lifespan remains one of the major unsolved problems of the biology of aging. We measured the capacity of nuclear protein to recognize DNA double strand breaks (DSBs) and telomere length of skin fibroblasts derived from mammalian species that exhibit wide differences in longevity. Our results indicate DNA DSB recognition increases exponentially with longevity. Further, an analysis of the level of Ku80 protein in human, cow, and mouse suggests that Ku levels vary dramatically between species and these levels are strongly correlated with longevity. In contrast mean telomere length appears to decrease with increasing longevity of the species, although not significantly. These findings suggest that an enhanced ability to bind to DNA ends may be important for longevity. A number of possible roles for increased levels of Ku and DNA-PKcs are discussed.

  16. Targeted molecular trait stacking in cotton through targeted double-strand break induction

    PubMed Central

    D'Halluin, Kathleen; Vanderstraeten, Chantal; Van Hulle, Jolien; Rosolowska, Joanna; Van Den Brande, Ilse; Pennewaert, Anouk; D'Hont, Kristel; Bossut, Martine; Jantz, Derek; Ruiter, Rene; Broadhvest, Jean

    2013-01-01

    Recent developments of tools for targeted genome modification have led to new concepts in how multiple traits can be combined. Targeted genome modification is based on the use of nucleases with tailor-made specificities to introduce a DNA double-strand break (DSB) at specific target loci. A re-engineered meganuclease was designed for specific cleavage of an endogenous target sequence adjacent to a transgenic insect control locus in cotton. The combination of targeted DNA cleavage and homologous recombination–mediated repair made precise targeted insertion of additional trait genes (hppd, epsps) feasible in cotton. Targeted insertion events were recovered at a frequency of about 2% of the independently transformed embryogenic callus lines. We further demonstrated that all trait genes were inherited as a single genetic unit, which will simplify future multiple-trait introgression. PMID:23777410

  17. 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.

  18. DNA double-strand breaks and ATM activation by transcription-blocking DNA lesions.

    PubMed

    Sordet, Olivier; Nakamura, Asako J; Redon, Christophe E; Pommier, Yves

    2010-01-15

    A taxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA double-strand break (DSB) response. We recently showed that transcription-blocking topoisomerase I cleavage complexes (TOP1cc) produce DSBs related to R-loop formation and activate ATM in post-mitotic neurons and lymphocytes. Here we discuss how TOP1cc can produce transcription arrest with R-loop formation and generate DSBs that activate ATM, as well as data suggesting that those transcription-dependent DSBs tend to form at the IgH locus and at specific genomic sites. We also address the potential roles of ATM in response to transcription-blocking TOP1cc.

  19. Dynamics of DNA double-strand breaks revealed by clustering of damaged chromosome domains.

    PubMed

    Aten, Jacob A; Stap, Jan; Krawczyk, Przemek M; van Oven, Carel H; Hoebe, Ron A; Essers, Jeroen; Kanaar, Roland

    2004-01-02

    Interactions between ends from different DNA double-strand breaks (DSBs) can produce tumorigenic chromosome translocations. Two theories for the juxta-position of DSBs in translocations, the static "contact-first" and the dynamic "breakage-first" theory, differ fundamentally in their requirement for DSB mobility. To determine whether or not DSB-containing chromosome domains are mobile and can interact, we introduced linear tracks of DSBs in nuclei. We observed changes in track morphology within minutes after DSB induction, indicating movement of the domains. In a subpopulation of cells, the domains clustered. Juxtaposition of different DSB-containing chromosome domains through clustering, which was most extensive in G1 phase cells, suggests an adhesion process in which we implicate the Mre11 complex. Our results support the breakage-first theory to explain the origin of chromosomal translocations.

  20. 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

  1. γ-H2AX as a biomarker for DNA double-strand breaks in ecotoxicology.

    PubMed

    Gerić, Marko; Gajski, Goran; Garaj-Vrhovac, Vera

    2014-07-01

    The visualisation of DNA damage response proteins enables the indirect measurement of DNA damage. Soon after the occurrence of a DNA double-strand break (DSB), the formation of γ-H2AX histone variants is to be expected. This review is focused on the potential use of the γ-H2AX foci assay in assessing the genotoxicity of environmental contaminants including cytostatic pharmaceuticals, since standard methods may not be sensitive enough to detect the damaging effect of low environmental concentrations of such drugs. These compounds are constantly released into the environment, potentially representing a threat to water quality, aquatic organisms, and, ultimately, human health. Our review of the literature revealed that this method could be used in the biomonitoring and risk assessment of aquatic systems affected by wastewater from the production, usage, and disposal of cytostatic pharmaceuticals. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. 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

  3. Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination

    PubMed Central

    Tillman, Robert E.; Wooley, Andrea L.; Hughes, Maureen M.; Wehrly, Tara D.; Swat, Wojciech; Sleckman, Barry P.

    2002-01-01

    Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRβ locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination. PMID:11828005

  4. DNA phosphorothioate modifications influence the global transcriptional response and protect DNA from double-stranded breaks

    PubMed Central

    Gan, Rui; Wu, Xiaolin; He, Wei; Liu, Zhenhua; Wu, Shuangju; Chen, Chao; Chen, Si; Xiang, Qianrong; Deng, Zixin; Liang, Dequan; Chen, Shi; Wang, Lianrong

    2014-01-01

    The modification of DNA by phosphorothioate (PT) occurs when the non-bridging oxygen in the sugar-phosphate backbone of DNA is replaced with sulfur. This DNA backbone modification was recently discovered and is governed by the dndABCDE genes in a diverse group of bacteria and archaea. However, the biological function of DNA PT modifications is poorly understood. In this study, we employed the RNA-seq analysis to characterize the global transcriptional changes in response to PT modifications. Our results show that DNA without PT protection is susceptible to DNA damage caused by the dndFGHI gene products. The DNA double-stranded breaks then trigger the SOS response, cell filamentation and prophage induction. Heterologous expression of dndBCDE conferring DNA PT modifications at GPSA and GPST prevented the damage in Salmonella enterica. Our data provide insights into the physiological role of the DNA PT system. PMID:25319634

  5. 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

  6. Conservative inheritance of newly synthesized DNA in double-strand break-induced gene conversion.

    PubMed

    Ira, Grzegorz; Satory, Dominik; Haber, James E

    2006-12-01

    To distinguish among possible mechanisms of repair of a double-strand break (DSB) by gene conversion in budding yeast, Saccharomyces cerevisiae, we employed isotope density transfer to analyze budding yeast mating type (MAT) gene switching in G2/M-arrested cells. Both of the newly synthesized DNA strands created during gene conversion are found at the repaired locus, leaving the donor unchanged. These results support suggestions that mitotic DSBs are primarily repaired by a synthesis-dependent strand-annealing mechanism. We also show that the proportion of crossing-over associated with DSB-induced ectopic recombination is not affected by the presence of nonhomologous sequences at one or both ends of the DSB or the presence of additional sequences that must be copied from the donor.

  7. APOBEC3 cytidine deaminases in double-strand DNA break repair and cancer promotion.

    PubMed

    Nowarski, Roni; Kotler, Moshe

    2013-06-15

    High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR. ©2013 AACR.

  8. APOBEC3 Cytidine Deaminases in Double-Strand DNA Break Repair and Cancer Promotion

    PubMed Central

    Nowarski, Roni; Kotler, Moshe

    2013-01-01

    High frequency of cytidine to thymidine conversions were identified in the genome of several types of cancer cells. In breast cancer cells these mutations are clustered in long DNA regions associated with ssDNA, double-strand DNA breaks (DSBs) and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs and clustered mutations. PMID:23598277

  9. 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.

  10. 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.

  11. 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.

  12. Interplay between the cell cycle and double-strand break response in mammalian cells.

    PubMed

    Beishline, Kate; Azizkhan-Clifford, Jane

    2014-01-01

    The cell cycle is intimately associated with the ability of cells to sense and respond to and repair DNA damage. Understanding how cell cycle progression, particularly DNA replication and cell division, are regulated and how DNA damage can affect these processes has been the subject of intense research. Recent evidence suggests that the repair of DNA damage is regulated by the cell cycle, and that cell cycle factors are closely associated with repair factors and participate in cellular decisions regarding how to respond to and repair damage. Precise regulation of cell cycle progression in the presence of DNA damage is essential to maintain genomic stability and avoid the accumulation of chromosomal aberrations that can promote tumor formation. In this review, we discuss the current understanding of how mammalian cells induce cell cycle checkpoints in response to DNA double-strand breaks. In addition, we discuss how cell cycle factors modulate DNA repair pathways to facilitate proper repair of DNA lesions.

  13. DNA helicases Sgs1 and BLM promote DNA double-strand break resection.

    PubMed

    Gravel, Serge; Chapman, J Ross; Magill, Christine; Jackson, Stephen P

    2008-10-15

    A key cellular response to DNA double-strand breaks (DSBs) is 5'-to-3' DSB resection by nucleases to generate regions of ssDNA that then trigger cell cycle checkpoint signaling and DSB repair by homologous recombination (HR). Here, we reveal that in the absence of exonuclease Exo1 activity, deletion or mutation of the Saccharomyces cerevisiae RecQ-family helicase, Sgs1, causes pronounced hypersensitivity to DSB-inducing agents. Moreover, we establish that this reflects severely compromised DSB resection, deficient DNA damage signaling, and strongly impaired HR-mediated repair. Furthermore, we show that the mammalian Sgs1 ortholog, BLM--whose deficiency causes cancer predisposition and infertility in people--also functions in parallel with Exo1 to promote DSB resection, DSB signaling and resistance to DSB-generating agents. Collectively, these data establish evolutionarily conserved roles for the BLM and Sgs1 helicases in DSB processing, signaling, and repair.

  14. Functions and regulation of the MRX complex at DNA double-strand breaks

    PubMed Central

    Gobbini, Elisa; Cassani, Corinne; Villa, Matteo; Bonetti, Diego; Longhese, Maria P.

    2016-01-01

    DNA double-strand breaks (DSBs) pose a serious threat to genome stability and cell survival. Cells possess mechanisms that recognize DSBs and promote their repair through either homologous recombination (HR) or non-homologous end joining (NHEJ). The evolutionarily conserved Mre11-Rad50-Xrs2 (MRX) complex plays a central role in the cellular response to DSBs, as it is implicated in controlling end resection and in maintaining the DSB ends tethered to each other. Furthermore, it is responsible for DSB signaling by activating the checkpoint kinase Tel1 that, in turn, supports MRX function in a positive feedback loop. The present review focuses mainly on recent works in the budding yeast Saccharomyces cerevisiae to highlight structure and regulation of MRX as well as its interplays with Tel1. PMID:28357369

  15. Processing of meiotic DNA double strand breaks requires cyclin-dependent kinase and multiple nucleases.

    PubMed

    Manfrini, Nicola; Guerini, Ilaria; Citterio, Andrea; Lucchini, Giovanna; Longhese, Maria Pia

    2010-04-09

    Meiotic recombination requires the formation of programmed Spo11-dependent DNA double strand breaks (DSBs). In Saccharomyces cerevisiae, the Sae2 protein and the Mre11-Rad50-Xrs2 complex are necessary to remove the covalently attached Spo11 protein from the DNA ends, which are then resected by so far unknown nucleases. Here, we demonstrate that phosphorylation of Sae2 Ser-267 by cyclin-dependent kinase 1 (Cdk1) is required to initiate meiotic DSB resection by allowing Spo11 removal from DSB ends. This finding suggests that Cdk1 activity is required for the processing of Spo11-induced DSBs, thus providing a mechanism for coordinating DSB resection with progression through meiotic prophase. Furthermore, the helicase Sgs1 and the nucleases Exo1 and Dna2 participate in lengthening the 5'-3' resection tracts during meiosis by controlling a step subsequent to Spo11 removal.

  16. 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

  17. Artemis-dependent DNA double-strand break formation at stalled replication forks.

    PubMed

    Unno, Junya; Takagi, Masatoshi; Piao, Jinhua; Sugimoto, Masataka; Honda, Fumiko; Maeda, Daisuke; Masutani, Mitsuko; Kiyono, Tohru; Watanabe, Fumiaki; Morio, Tomohiro; Teraoka, Hirobumi; Mizutani, Shuki

    2013-06-01

    Stalled replication forks undergo DNA double-strand breaks (DSBs) under certain conditions. However, the precise mechanism underlying DSB induction and the cellular response to persistent replication fork stalling are not fully understood. Here we show that, in response to hydroxyurea exposure, DSBs are generated in an Artemis nuclease-dependent manner following prolonged stalling with subsequent activation of the ataxia-telangiectasia mutated (ATM) signaling pathway. The kinase activity of the catalytic subunit of the DNA-dependent protein kinase, a prerequisite for stimulation of the endonuclease activity of Artemis, is also required for DSB generation and subsequent ATM activation. Our findings indicate a novel function of Artemis as a molecular switch that converts stalled replication forks harboring single-stranded gap DNA lesions into DSBs, thereby activating the ATM signaling pathway following prolonged replication fork stalling. © 2013 Japanese Cancer Association.

  18. Polymorphisms in double-strand breaks repair genes are associated with impaired fertility in Chinese population.

    PubMed

    Ji, Guixiang; Yan, Lifeng; Liu, Wei; Huang, Cong; Gu, Aihua; Wang, Xinru

    2013-05-01

    The DNA double-strand breaks (DSBs) repair pathway plays a critical role in repairing double-strand breaks, and genetic variants in DSBs repair pathway genes are potential risk factors for various diseases. To test the hypothesis that polymorphisms in DSBs genes are associated with susceptibility to male infertility, we examined 11 single nucleotide polymorphisms in eight key DSBs genes (XRCC3, XRCC2, BRCA2, RAG1, XRCC5, LIG4, XRCC4 and ATM) in 580 infertility cases and 580 controls from a Chinese population-based case-control study (NJMU Infertility Study). Genotypes were determined using the OpenArray platform, and sperm DNA fragmentation was detected using the TUNEL assay. The adjusted odds ratio (OR) and 95% CI were estimated using logistic regression. The results indicate that LIG4 rs1805388 (Ex2+54C>T, Thr9Ile) T allele could increase the susceptibility to male infertility (adjusted OR=2.78; 95% CI, 1.77-4.36 for TT genotype; and adjusted OR=1.58; 95% CI, 1.77-4.36 for TC genotype respectively). In addition, the homozygous variant genotype GG of RAG1 rs2227973 (A>G, K820R) was associated with a significantly increased risk of male infertility (adjusted OR, 1.44; 95% CI, 1.01-2.04). Moreover, linear regression analysis revealed that carriers of LIG4 rs1805388 or RAG1 rs2227973 variants had a significantly higher level of sperm DNA fragmentation and that T allele carriers of LIG4 rs1805388 also had a lower level of sperm concentration when compared with common homozygous genotype carriers. This study demonstrates, for the first time, to our knowledge, that functional variants of RAG1 rs2227973 and LIG4 rs1805388 are associated with susceptibility to male infertility.

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

    PubMed

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

    2017-04-01

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

  20. 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

  1. Restriction-endonuclease-induced DNA double-strand breaks and chromosomal aberrations in mammalian cells.

    PubMed

    Bryant, P E; Johnston, P J

    1993-05-01

    Restriction endonucleases (RE) can be used to mimic and model the clastogenic effects of ionising radiation. With the development of improved techniques for cell poration: electroporation and recently streptolysin O (SLO), it has become possible more confidently to study the relationships between DNA double-strand breaks (dsb) of various types (e.g. blunt or cohesive-ended) and the frequencies of induced metaphase chromosomal aberrations or micronuclei in cytokinesis-blocked cells. Although RE-induced dsb do not mimic the chemical end-structure of radiation-induced dsb (i.e. the 'dirty' ends of radiation-induced dsb), it has become clear that cohesive-ended dsb, which are thought to be the major type of dsb induced by radiation, are much less clastogenic than blunt-ended dsb. It has also been possible, with the aid of electroporation or SLO to measure the kinetics of dsb in cells as a function of time after treatment. These experiments have shown that some RE (e.g. Pvu II) are extremely stable inside CHO cells and at high concentrations persist and induce dsb over a period of many hours following treatment. Cutting of DNA by RE is thought to be at specific recognition sequences (as in free DNA) although the frequencies of sites in native chromatin available to RE is not yet known. DNA condensation and methylation are both factors limiting the numbers of available cutting sites. Relatively little is known about the kinetics of incision or repair of RE-induced dsb in cells. Direct ligation may be a method used by cells to rejoin the bulk of RE-induced dsb, since inhibitors such as araA, araC and aphidicolin appear not prevent rejoining, although these inhibitors have been found to lead to enhanced frequencies of chromosomal aberrations. 3-Aminobenzimide, the poly-ADP ribose polymerase inhibitor is the only agent that has so far been shown to inhibit rejoining of RE-induced dsb. Data from the radiosensitive xrs5 cell line, where chromosomal aberration frequencies are

  2. Double strand breaks induced by low doses of {gamma} rays or heavy ions: Quantitation in nonradioactive human DNA

    SciTech Connect

    Sutherland, B.M.; Bennett, P.V.; Sutherland, J.C.

    1996-07-15

    We have developed a method of quantitating low frequencies (0-30 sites/10{sup 9} base pairs) of double strand breaks in {approximately}1 {mu}g of nonradioactive human DNA. Unirradiated or irradiated DNA is digested with the restriction endonuclease NotI, producing cleavage fragments that include a major group centered at {approximately}1.2-1.3 Mbp. The DNA molecules are separated as a function of size by transverse alternating field electrophoresis. The frequency of double strand breaks is computed directly from the decrease in number average molecular length induced in the 1.2 to 1.3-Mbp cleavage fragment group by {sup 137}Cs {gamma} or Fe{sup 26+} (1.1 GeV/nucleon) irradiation vs the corresponding unirradiated DNA samples. The double strand break frequency can be quantitated easily in the dose range of 0-10 cGy of {gamma} rays. The frequency of breaks per unit dose calculated for {gamma} irradiation of DNA in human cells ({approximately}4.6 double strand breaks/10{sup 9} bp/Gy) who used methods requiring higher doses. 55 refs., 4 figs.

  3. Hyperactivation of DNA-PK by double-strand break mimicking molecules disorganizes DNA damage response.

    PubMed

    Quanz, Maria; Chassoux, Danielle; Berthault, Nathalie; Agrario, Céline; Sun, Jian-Sheng; Dutreix, Marie

    2009-07-21

    Cellular response to DNA damage involves the coordinated activation of cell cycle checkpoints and DNA repair. The early steps of DNA damage recognition and signaling in mammalian cells are not yet fully understood. To investigate the regulation of the DNA damage response (DDR), we designed short and stabilized double stranded DNA molecules (Dbait) mimicking double-strand breaks. We compared the response induced by these molecules to the response induced by ionizing radiation. We show that stable 32-bp long Dbait, induce pan-nuclear phosphorylation of DDR components such as H2AX, Rpa32, Chk1, Chk2, Nbs1 and p53 in various cell lines. However, individual cell analyses reveal that differences exist in the cellular responses to Dbait compared to irradiation. Responses to Dbait: (i) are dependent only on DNA-PK kinase activity and not on ATM, (ii) result in a phosphorylation signal lasting several days and (iii) are distributed in the treated population in an "all-or-none" pattern, in a Dbait-concentration threshold dependant manner. Moreover, despite extensive phosphorylation of the DNA-PK downstream targets, Dbait treated cells continue to proliferate without showing cell cycle delay or apoptosis. Dbait treatment prior to irradiation impaired foci formation of Nbs1, 53BP1 and Rad51 at DNA damage sites and inhibited non-homologous end joining as well as homologous recombination. Together, our results suggest that the hyperactivation of DNA-PK is insufficient for complete execution of the DDR but induces a "false" DNA damage signaling that disorganizes the DNA repair system.

  4. 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.

  5. 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.

  6. Rad51-mediated double-strand break repair and mismatch correction of divergent substrates.

    PubMed

    Anand, Ranjith; Beach, Annette; Li, Kevin; Haber, James

    2017-04-20

    The Rad51 (also known as RecA) family of recombinases executes the critical step in homologous recombination: the search for homologous DNA to serve as a template during the repair of DNA double-strand breaks (DSBs). Although budding yeast Rad51 has been extensively characterized in vitro, the stringency of its search and sensitivity to mismatched sequences in vivo remain poorly defined. Here, in Saccharomyces cerevisiae, we analysed Rad51-dependent break-induced replication in which the invading DSB end and its donor template share a 108-base-pair homology region and the donor carries different densities of single-base-pair mismatches. With every eighth base pair mismatched, repair was about 14% of that of completely homologous sequences. With every sixth base pair mismatched, repair was still more than 5%. Thus, completing break-induced replication in vivo overcomes the apparent requirement for at least 6-8 consecutive paired bases that has been inferred from in vitro studies. When recombination occurs without a protruding nonhomologous 3' tail, the mismatch repair protein Msh2 does not discourage homeologous recombination. However, when the DSB end contains a 3' protruding nonhomologous tail, Msh2 promotes the rejection of mismatched substrates. Mismatch correction of strand invasion heteroduplex DNA is strongly polar, favouring correction close to the DSB end. Nearly all mismatch correction depends on the proofreading activity of DNA polymerase-δ, although the repair proteins Msh2, Mlh1 and Exo1 influence the extent of correction.

  7. 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.

  8. 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.

  9. 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.

  10. Poly(ADP-ribose) polymerase (PARP-1) is not involved in DNA double-strand break recovery

    PubMed Central

    Noël, Georges; Giocanti, Nicole; Fernet, Marie; Mégnin-Chanet, Frédérique; Favaudon, Vincent

    2003-01-01

    Background The cytotoxicity and the rejoining of DNA double-strand breaks induced by γ-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose) polymerase (PARP-1) in DNA double-strand break repair. Results PARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by γ-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose) synthesis following γ-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as γ-rays and H2O2. Conclusions The results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to γ-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose) synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks. PMID:12866953

  11. Testing Predictions of the Double-Strand Break Repair Model Relating to Crossing Over in Mammalian Cells

    PubMed Central

    Birmingham, Erin C.; Lee, Shauna A.; McCulloch, Richard D.; Baker, Mark D.

    2004-01-01

    In yeast, four-stranded, biparental “joint molecules” containing a pair of Holliday junctions are demonstrated intermediates in the repair of meiotic double-strand breaks (DSBs). Genetic and physical evidence suggests that when joint molecules are resolved by the cutting of each of the two Holliday junctions, crossover products result at least most of the time. The double-strand break repair (DSBR) model is currently accepted as a paradigm for acts of DSB repair that lead to crossing over. In this study, a well-defined mammalian gene-targeting assay was used to test predictions that the DSBR model makes about the frequency and position of hDNA in recombinants generated by crossing over. The DSBR model predicts that hDNA will frequently form on opposite sides of the DSB in the two homologous sequences undergoing recombination [half conversion (HC); 5:3, 5:3 segregation]. By examining the segregation patterns of poorly repairable small palindrome genetic markers, we show that this configuration of hDNA is rare. Instead, in a large number of recombinants, full conversion (FC) events in the direction of the unbroken chromosomal sequence (6:2 segregation) were observed on one side of the DSB. A conspicuous fraction of the unidirectional FC events was associated with normal 4:4 marker segregation on the other side of the DSB. In addition, a large number of recombinants displayed evidence of hDNA formation. In several, hDNA was symmetrical on one side of the DSB, suggesting that the two homologous regions undergoing recombination swapped single strands of the same polarity. These data are considered within the context of modified versions of the DSBR model. PMID:15579705

  12. Role of Double-Strand Break End-Tethering during Gene Conversion in Saccharomyces cerevisiae

    PubMed Central

    Haber, James E.

    2016-01-01

    Correct repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Whereas gene conversion (GC)-mediated repair is mostly error-free, repair by break-induced replication (BIR) is associated with non-reciprocal translocations and loss of heterozygosity. We have previously shown that a Recombination Execution Checkpoint (REC) mediates this competition by preventing the BIR pathway from acting on DSBs that can be repaired by GC. Here, we asked if the REC can also determine whether the ends that are engaged in a GC-compatible configuration belong to the same break, since repair involving ends from different breaks will produce potentially deleterious translocations. We report that the kinetics of repair are markedly delayed when the two DSB ends that participate in GC belong to different DSBs (termed Trans) compared to the case when both DSB ends come from the same break (Cis). However, repair in Trans still occurs by GC rather than BIR, and the overall efficiency of repair is comparable. Hence, the REC is not sensitive to the “origin” of the DSB ends. When the homologous ends for GC are in Trans, the delay in repair appears to reflect their tethering to sequences on the other side of the DSB that themselves recombine with other genomic locations with which they share sequence homology. These data support previous observations that the two ends of a DSB are usually tethered to each other and that this tethering facilitates both ends encountering the same donor sequence. We also found that the presence of homeologous/repetitive sequences in the vicinity of a DSB can distract the DSB end from finding its bona fide homologous donor, and that inhibition of GC by such homeologous sequences is markedly increased upon deleting Sgs1 but not Msh6. PMID:27074148

  13. 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

  14. Relative biological effectiveness for photons: implication of complex DNA double-strand breaks as critical lesions.

    PubMed

    Liang, Ying; Fu, Qibin; Wang, Xudong; Liu, Feng; Yang, Gen; Luo, Chunxiong; Ouyang, Qi; Wang, Yugang

    2017-03-21

    Current knowledge in radiobiology ascribes the adverse biological effects of ionizing radiation primarily to the induction of DNA double-strand breaks (DSBs), which is supposed to be potentially lethal and may be converted to lethal damage due to misrepair. Soft and ultrasoft x-rays have been found to bear elevated biological effectiveness for cell killing compared with conventional x-rays or (60)Co γ-rays. This phenomenon is qualitatively interpreted as the increased level of DSB induction for low energy photons, however, a thorough quantitative reasoning is lacking. Here, we systematically compared the relative biological effectiveness (RBE) with relative DSB induction for photons from several hundreds of eV up to MeV. Although there is an approximate two-fold increase in the yields of DSB for low energy photons found in our calculation and a large number of experimental measurements, it is far from enough to account for the three- to four-fold increase in RBE. Further theoretical investigations show that DSB complexity (additional single-strand breaks and base damage within 10 base pairs) increases notably for low energy photons, which largely reconciles the discrepancy between RBE and DSB induction. Our theoretical results are in line with accumulating experimental evidence that complex DSBs are refractory to repair machinery and may contribute predominantly to the formation of lethal damage.

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

    DOE PAGES

    Janssen, Aniek; Breuer, Gregory A.; Brinkman, Eva K.; ...

    2016-07-15

    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,more » 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.« less

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

    SciTech Connect

    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.

    2016-07-15

    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.

  17. Relative biological effectiveness for photons: implication of complex DNA double-strand breaks as critical lesions

    NASA Astrophysics Data System (ADS)

    Liang, Ying; Fu, Qibin; Wang, Xudong; Liu, Feng; Yang, Gen; Luo, Chunxiong; Ouyang, Qi; Wang, Yugang

    2017-03-01

    Current knowledge in radiobiology ascribes the adverse biological effects of ionizing radiation primarily to the induction of DNA double-strand breaks (DSBs), which is supposed to be potentially lethal and may be converted to lethal damage due to misrepair. Soft and ultrasoft x-rays have been found to bear elevated biological effectiveness for cell killing compared with conventional x-rays or 60Co γ-rays. This phenomenon is qualitatively interpreted as the increased level of DSB induction for low energy photons, however, a thorough quantitative reasoning is lacking. Here, we systematically compared the relative biological effectiveness (RBE) with relative DSB induction for photons from several hundreds of eV up to MeV. Although there is an approximate two-fold increase in the yields of DSB for low energy photons found in our calculation and a large number of experimental measurements, it is far from enough to account for the three- to four-fold increase in RBE. Further theoretical investigations show that DSB complexity (additional single-strand breaks and base damage within 10 base pairs) increases notably for low energy photons, which largely reconciles the discrepancy between RBE and DSB induction. Our theoretical results are in line with accumulating experimental evidence that complex DSBs are refractory to repair machinery and may contribute predominantly to the formation of lethal damage.

  18. The Fun30 nucleosome remodeller promotes resection of DNA double-strand break ends.

    PubMed

    Chen, Xuefeng; Cui, Dandan; Papusha, Alma; Zhang, Xiaotian; Chu, Chia-Dwo; Tang, Jiangwu; Chen, Kaifu; Pan, Xuewen; Ira, Grzegorz

    2012-09-27

    Chromosomal double-strand breaks (DSBs) are resected by 5' nucleases to form 3' single-stranded DNA substrates for binding by homologous recombination and DNA damage checkpoint proteins. Two redundant pathways of extensive resection have been described both in cells and in vitro, one relying on Exo1 exonuclease and the other on Sgs1 helicase and Dna2 nuclease. However, it remains unknown how resection proceeds within the context of chromatin, where histones and histone-bound proteins represent barriers for resection enzymes. Here we identify the yeast nucleosome-remodelling enzyme Fun30 as a factor promoting DSB end resection. Fun30 is the major nucleosome remodeller promoting extensive Exo1- and Sgs1-dependent resection of DSBs. The RSC and INO80 chromatin-remodelling complexes and Fun30 have redundant roles in resection adjacent to DSB ends. ATPase and helicase domains of Fun30, which are needed for nucleosome remodelling, are also required for resection. Fun30 is robustly recruited to DNA breaks and spreads along the DSB coincident with resection. Fun30 becomes less important for resection in the absence of the histone-bound Rad9 checkpoint adaptor protein known to block 5' strand processing and in the absence of either histone H3 K79 methylation or γ-H2A, which mediate recruitment of Rad9 (refs 9, 10). Together these data suggest that Fun30 helps to overcome the inhibitory effect of Rad9 on DNA resection.

  19. Transcription and double-strand breaks induce similar mitotic recombination events in Saccharomyces cerevisiae.

    PubMed

    González-Barrera, Sergio; García-Rubio, María; Aguilera, Andrés

    2002-10-01

    We have made a comparative analysis of double-strand-break (DSB)-induced recombination and spontaneous recombination under low- and high-transcription conditions in yeast. We constructed two different recombination substrates, one for the analysis of intermolecular gene conversions and the other for intramolecular gene conversions and inversions. Such substrates were based on the same leu2-HOr allele fused to the tet promoter and containing a 21-bp HO site. Gene conversions and inversions were differently affected by rad1, rad51, rad52, and rad59 single and double mutations, consistent with the actual view that such events occur by different recombination mechanisms. However, the effect of each mutation on each type of recombination event was the same, whether associated with transcription or induced by the HO-mediated DSB. Both the highly transcribed DNA and the HO-cut sequence acted as recipients of the gene conversion events. These results are consistent with the hypothesis that transcription promotes initiation of recombination along the DNA sequence being transcribed. The similarity between transcription-associated and DSB-induced recombination suggests that transcription promotes DNA breaks.

  20. Controlled DNA double-strand break induction in mice reveals post-damage transcriptome stability.

    PubMed

    Kim, Jeongkyu; Sturgill, David; Tran, Andy D; Sinclair, David A; Oberdoerffer, Philipp

    2016-04-20

    DNA double-strand breaks (DSBs) and their repair can cause extensive epigenetic changes. As a result, DSBs have been proposed to promote transcriptional and, ultimately, physiological dysfunction via both cell-intrinsic and cell-non-autonomous pathways. Studying the consequences of DSBs in higher organisms has, however, been hindered by a scarcity of tools for controlled DSB induction. Here, we describe a mouse model that allows for both tissue-specific and temporally controlled DSB formation at ∼140 defined genomic loci. Using this model, we show that DSBs promote a DNA damage signaling-dependent decrease in gene expression in primary cells specifically at break-bearing genes, which is reversed upon DSB repair. Importantly, we demonstrate that restoration of gene expression can occur independently of cell cycle progression, underlining its relevance for normal tissue maintenance. Consistent with this, we observe no evidence for persistent transcriptional repression in response to a multi-day course of continuous DSB formation and repair in mouse lymphocytes in vivo Together, our findings reveal an unexpected capacity of primary cells to maintain transcriptome integrity in response to DSBs, pointing to a limited role for DNA damage as a mediator of cell-autonomous epigenetic dysfunction.

  1. Live cell microscopy analysis of radiation-induced DNA double-strand break motion

    PubMed Central

    Jakob, B.; Splinter, J.; Durante, M.; Taucher-Scholz, G.

    2009-01-01

    We studied the spatiotemporal organization of DNA damage processing by live cell microscopy analysis in human cells. In unirradiated U2OS osteosarcoma and HeLa cancer cells, a fast confined and Brownian-like motion of DNA repair protein foci was observed, which was not altered by radiation. By analyzing the motional activity of GFP-53BP1 foci in live cells up to 12-h after irradiation, we detected an additional slower mobility of damaged chromatin sites showing a mean square displacement of ≈0.6 μm2/h after exposure to densely- or sparsely-ionizing radiation, most likely driven by normal diffusion of chromatin. Only occasionally, larger translational motion connected to morphological changes of the whole nucleus could be observed. In addition, there was no general tendency to form repair clusters in the irradiated cells. We conclude that long-range displacements of damaged chromatin domains do not generally occur during DNA double-strand break repair after introduction of multiple damaged sites by charged particles. The occasional and in part transient appearance of cluster formation of radiation-induced foci may represent a higher mobility of chromatin along the ion trajectory. These observations support the hypothesis that spatial proximity of DNA breaks is required for the formation of radiation-induced chromosomal exchanges. PMID:19221031

  2. 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

  3. 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.

  4. Rad51-mediated double-strand break repair and mismatch correction of divergent substrates

    PubMed Central

    Anand, Ranjith; Beach, Annette; Li, Kevin; Haber, James

    2017-01-01

    The RecA/Rad51 family of recombinases execute the critical step in homologous recombination (HR): the search for homologous DNA to serve as the template during DNA double-strand break (DSB) repair1–7. Although budding yeast Rad51 has been extensively characterized in vitro3,4,6–9, the stringency of its search and sensitivity to mismatched sequences in vivo remain poorly defined. We analyzed Rad51-dependent break-induced replication (BIR) where the invading DSB end and its donor template share 108 bp homology and the donor carries different densities of single-bp mismatches (Fig. 1a). With every 8th bp mismatched, repair was ~14% compared to completely homologous sequences. With every 6th bp mismatched, repair was >5%. Thus completing BIR in vivo overcomes the apparent requirement for at least 6–8 consecutive paired bases inferred from in vitro studies6,8. When recombination occurs without a protruding nonhomologous 3′ tail, mismatch repair protein Msh2 does not discourage homeologous recombination. However, when the DSB end contains a 3′ protruding nonhomologous tail, Msh2 promotes rejection of mismatched substrates. Mismatch correction of strand invasion heteroduplex DNA is strongly polar, favoring correction close to the DSB end. Nearly all mismatch correction depends on the proofreading activity of DNA polymerase δ, although Msh2-Mlh1 and Exo1 influence the extent of correction. PMID:28405019

  5. Double-strand break repair in tandem repeats during bacteriophage T4 infection.

    PubMed Central

    Tomso, D J; Kreuzer, K N

    2000-01-01

    Recombinational repair of double-strand breaks in tandemly repeated sequences often results in the loss of one or more copies of the repeat. The single-strand annealing (SSA) model for repair has been proposed to account for this nonconservative recombination. In this study we present a plasmid-based physical assay that measures SSA during bacteriophage T4 infection and apply this assay to the genetic analysis of break repair. SSA occurs readily in broken plasmid DNA and is independent of the strand exchange protein UvsX and its accessory factor UvsY. We use the unique features of T4 DNA metabolism to examine the link between SSA repair and DNA replication and demonstrate directly that the DNA polymerase and the major replicative helicase of the phage are not required for SSA repair. We also show that the Escherichia coli RecBCD enzyme can mediate the degradation of broken DNA during early, but not late, times of infection. Finally, we consider the status of broken ends during the course of the infection and propose a model for SSA during T4 infections. PMID:10924452

  6. Sequencing Spo11 Oligonucleotides for Mapping Meiotic DNA Double-Strand Breaks in Yeast.

    PubMed

    Lam, Isabel; Mohibullah, Neeman; Keeney, Scott

    2017-01-01

    Meiosis is a specialized form of cell division resulting in reproductive cells with a reduced, usually haploid, genome complement. A key step after premeiotic DNA replication is the occurrence of homologous recombination at multiple places throughout the genome, initiated with the formation of DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like protein Spo11. DSBs are distributed non-randomly in genomes, and understanding the mechanisms that shape this distribution is important for understanding how meiotic recombination influences heredity and genome evolution. Several methods exist for mapping where Spo11 acts. Of these, sequencing of Spo11-associated oligonucleotides (Spo11 oligos) is the most precise, specifying the locations of DNA breaks to the base pair. In this chapter we detail the steps involved in Spo11-oligo mapping in the SK1 strain of budding yeast Saccharomyces cerevisiae, from harvesting cells of highly synchronous meiotic cultures, through preparation of sequencing libraries, to the mapping pipeline used for processing the data.

  7. Mitochondrial DNA double-strand breaks in oligodendrocytes cause demyelination, axonal injury and CNS inflammation.

    PubMed

    Madsen, Pernille M; Pinto, Milena; Patel, Shreyans; McCarthy, Stephanie; Gao, Han; Taherian, Mehran; Karmally, Shaffiat; Pereira, Claudia V; Dvoriantchikova, Galina; Ivanov, Dmitry; Tanaka, Kenji F; Moraes, Carlos T; Brambilla, Roberta

    2017-09-20

    Mitochondrial dysfunction has been implicated in the pathophysiology of neurodegenerative disorders, including multiple sclerosis (MS). To date, the investigation of mitochondrial dysfunction in MS has focused exclusively on neurons, with no studies exploring whether dysregulation of mitochondrial bioenergetics and/or genetics in oligodendrocytes might be associated with the etiopathogenesis of MS and other demyelinating syndromes. To address this question, we established a mouse model where mitochondrial DNA (mtDNA) double-strand breaks (DSB) were specifically induced in myelinating oligodendrocytes (PLP:mtPstI mice) by expressing a mitochondrial-targeted endonuclease, mtPstI, starting at 3 weeks of age. In both female and male mice, DSB of oligodendroglial mtDNA caused impairment of locomotor function, chronic demyelination, glial activation and axonal degeneration, which became more severe with time of induction. In addition, after short transient induction of mtDNA DSB, PLP:mtPstI mice showed an exacerbated response to experimental autoimmune encephalomyelitis. Together, our data demonstrate that mtDNA damage can cause primary oligodendropathy which in turn triggers demyelination, proving PLP:mtPstI mice to be a useful tool to study the pathological consequences of mitochondrial dysfunction in oligodendrocytes. In addition, the demyelination and axonal loss displayed by PLP:mtPstI mice recapitulate some of the key features of chronic demyelinating syndromes, including progressive MS forms, which are not accurately reproduced in the models currently available. For this reason the PLP:mtPstI mouse represents a unique and much needed platform for testing remyelinating therapies.SIGNIFICANCE STATEMENTMadsen et al. show that oligodendrocyte-specific mtDNA double strand breaks in PLP:mtPstI mice cause oligodendrocyte death and demyelination associated with axonal damage and glial activation. Hence, PLP:mtPstI mice represent a unique tool to study the pathological

  8. 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.

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

    PubMed Central

    Wood, Richard D.; Doublié, Sylvie

    2016-01-01

    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. PMID:27264557

  10. Molecular architecture of the HerA-NurA DNA double-strand break resection complex.

    PubMed

    Byrne, Robert Thomas; Schuller, Jan Michael; Unverdorben, Pia; Förster, Friedrich; Hopfner, Karl-Peter

    2014-12-20

    DNA double-strand breaks can be repaired by homologous recombination, during which the DNA ends are long-range resected by helicase-nuclease systems to generate 3' single strand tails. In archaea, this requires the Mre11-Rad50 complex and the ATP-dependent helicase-nuclease complex HerA-NurA. We report the cryo-EM structure of Sulfolobus solfataricus HerA-NurA at 7.4Å resolution and present the pseudo-atomic model of the complex. HerA forms an ASCE hexamer that tightly interacts with a NurA dimer, with each NurA protomer binding three adjacent HerA HAS domains. Entry to NurA's nuclease active sites requires dsDNA to pass through a 23Å wide channel in the HerA hexamer. The structure suggests that HerA is a dsDNA translocase that feeds DNA into the NurA nuclease sites.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  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. 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

  18. 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.

  19. Fine-tuning the ubiquitin code at DNA double-strand breaks: deubiquitinating enzymes at work

    PubMed Central

    Citterio, Elisabetta

    2015-01-01

    Ubiquitination is a reversible protein modification broadly implicated in cellular functions. Signaling processes mediated by ubiquitin (ub) are crucial for the cellular response to DNA double-strand breaks (DSBs), one of the most dangerous types of DNA lesions. In particular, the DSB response critically relies on active ubiquitination by the RNF8 and RNF168 ub ligases at the chromatin, which is essential for proper DSB signaling and repair. How this pathway is fine-tuned and what the functional consequences are of its deregulation for genome integrity and tissue homeostasis are subject of intense investigation. One important regulatory mechanism is by reversal of substrate ubiquitination through the activity of specific deubiquitinating enzymes (DUBs), as supported by the implication of a growing number of DUBs in DNA damage response processes. Here, we discuss the current knowledge of how ub-mediated signaling at DSBs is controlled by DUBs, with main focus on DUBs targeting histone H2A and on their recent implication in stem cell biology and cancer. PMID:26442100

  20. Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation

    PubMed Central

    Gomez-Godinez, Veronica; Wu, Tao; Sherman, Adria J.; Lee, Christopher S.; Liaw, Lih-Huei; Zhongsheng, You; Yokomori, Kyoko; Berns, Michael W.

    2010-01-01

    In this study the femtosecond near-IR and nanosecond green lasers are used to induce alterations in mitotic chromosomes. The subsequent double-strand break responses are studied. We show that both lasers are capable of creating comparable chromosomal alterations and that a phase paling observed within 1–2 s of laser exposure is associated with an alteration of chromatin as confirmed by serial section electron microscopy, DAPI, γH2AX and phospho-H3 staining. Additionally, the accumulation of dark material observed using phase contrast light microscopy (indicative of a change in refractive index of the chromatin) ∼34 s post-laser exposure corresponds spatially to the accumulation of Nbs1, Ku and ubiquitin. This study demonstrates that chromosomes selectively altered in mitosis initiate the DNA damage response within 30 s and that the accumulation of proteins are visually represented by phase-dark material at the irradiation site, allowing us to determine the fate of the damage as cells enter G1. These results occur with two widely different laser systems, making this approach to study DNA damage responses in the mitotic phase generally available to many different labs. Additionally, we present a summary of most of the published laser studies on chromosomes in order to provide a general guide of the lasers and operating parameters used by other laboratories. PMID:20923785

  1. Radiation-induced DNA double-strand break rejoining in human tumour cells.

    PubMed Central

    Núñez, M. I.; Villalobos, M.; Olea, N.; Valenzuela, M. T.; Pedraza, V.; McMillan, T. J.; Ruiz de Almodóvar, J. M.

    1995-01-01

    Five established human breast cancer cell lines and one established human bladder cancer cell line of varying radiosensitivity have been used to determine whether the rejoining of DNA double-strand breaks (dsbs) shows a correlation with radiosensitivity. The kinetics of dsb rejoining was biphasic and both components proceeded exponentially with time. The half-time (t1/2) of rejoining ranged from 18.0 +/- 1.4 to 36.4 +/- 3.2 min (fast rejoining process) and from 1.5 +/- 0.2 to 5.1 +/- 0.2 h (slow rejoining process). We found a statistically significant relationship between the survival fraction at 2 Gy (SF2) and the t1/2 of the fast rejoining component (r = 0.949, P = 0.0039). Our results suggest that cell lines which show rapid rejoining are more radioresistant. These results support the view that, as well as the level of damage induction that we have reported previously, the repair process is a major determinant of cellular radiosensitivity. It is possible that the differences found in DNA dsb rejoining and the differences in DNA dsb induction are related by a common mechanism, e.g. conformation of chromatin in the cell. PMID:7841046

  2. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases.

    PubMed

    Frock, Richard L; Hu, Jiazhi; Meyers, Robin M; Ho, Yu-Jui; Kii, Erina; Alt, Frederick W

    2015-02-01

    Although great progress has been made in the characterization of the off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification-mediated modification of a previously published high-throughput, genome-wide, translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN nucleases revealed off-target hotspot numbers for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases more than tenfold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described previously. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.

  3. DNA double-strand breaks alter the spatial arrangement of homologous loci in plant cells.

    PubMed

    Hirakawa, Takeshi; Katagiri, Yohei; Ando, Tadashi; Matsunaga, Sachihiro

    2015-06-05

    Chromatin dynamics and arrangement are involved in many biological processes in nuclei of eukaryotes including plants. Plants have to respond rapidly to various environmental stimuli to achieve growth and development because they cannot move. It is assumed that the alteration of chromatin dynamics and arrangement support the response to these stimuli; however, there is little information in plants. In this study, we investigated the chromatin dynamics and arrangement with DNA damage in Arabidopsis thaliana by live-cell imaging with the lacO/LacI-EGFP system and simulation analysis. It was revealed that homologous loci kept a constant distance in nuclei of A. thaliana roots in general growth. We also found that DNA double-strand breaks (DSBs) induce the approach of the homologous loci with γ-irradiation. Furthermore, AtRAD54, which performs an important role in the homologous recombination repair pathway, was involved in the pairing of homologous loci with γ-irradiation. These results suggest that homologous loci approach each other to repair DSBs, and AtRAD54 mediates these phenomena.

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

    PubMed

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

    2016-11-13

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

  5. DNA double-strand breaks alter the spatial arrangement of homologous loci in plant cells

    PubMed Central

    Hirakawa, Takeshi; Katagiri, Yohei; Ando, Tadashi; Matsunaga, Sachihiro

    2015-01-01

    Chromatin dynamics and arrangement are involved in many biological processes in nuclei of eukaryotes including plants. Plants have to respond rapidly to various environmental stimuli to achieve growth and development because they cannot move. It is assumed that the alteration of chromatin dynamics and arrangement support the response to these stimuli; however, there is little information in plants. In this study, we investigated the chromatin dynamics and arrangement with DNA damage in Arabidopsis thaliana by live-cell imaging with the lacO/LacI-EGFP system and simulation analysis. It was revealed that homologous loci kept a constant distance in nuclei of A. thaliana roots in general growth. We also found that DNA double-strand breaks (DSBs) induce the approach of the homologous loci with γ-irradiation. Furthermore, AtRAD54, which performs an important role in the homologous recombination repair pathway, was involved in the pairing of homologous loci with γ-irradiation. These results suggest that homologous loci approach each other to repair DSBs, and AtRAD54 mediates these phenomena. PMID:26046331

  6. Numerical constraints and feedback control of double-strand breaks in mouse meiosis.

    PubMed

    Kauppi, Liisa; Barchi, Marco; Lange, Julian; Baudat, Frédéric; Jasin, Maria; Keeney, Scott

    2013-04-15

    Different organisms display widely different numbers of the programmed double-strand breaks (DSBs) that initiate meiotic recombination (e.g., hundreds per meiocyte in mice and humans vs. dozens in nematodes), but little is known about what drives these species-specific DSB set points or the regulatory pathways that control them. Here we examine male mice with a lowered dosage of SPO11, the meiotic DSB catalyst, to gain insight into the effect of reduced DSB numbers on mammalian chromosome dynamics. An approximately twofold DSB reduction was associated with the reduced ability of homologs to synapse along their lengths, provoking prophase arrest and, ultimately, sterility. In many spermatocytes, chromosome subsets displayed a mix of synaptic failure and synapsis with both homologous and nonhomologous partners ("chromosome tangles"). The X chromosome was nearly always involved in tangles, and small autosomes were involved more often than large ones. We conclude that homolog pairing requirements dictate DSB set points during meiosis. Importantly, our results reveal that karyotype is a key factor: Smaller autosomes and heteromorphic sex chromosomes become weak links when DSBs are reduced below a critical threshold. Unexpectedly, unsynapsed chromosome segments trapped in tangles displayed an elevated density of DSB markers later in meiotic prophase. The unsynapsed portion of the X chromosome in wild-type males also showed evidence that DSB numbers increased as prophase progressed. These findings point to the existence of a feedback mechanism that links DSB number and distribution with interhomolog interactions.

  7. Homologous Pairing Preceding SPO11 Mediated Double Strand Breaks in Mice

    PubMed Central

    Boateng, Kingsley A.; Bellani, Marina A.; Gregoretti, Ivan V.; Pratto, Florencia; Camerini-Otero, R. Daniel

    2012-01-01

    SUMMARY How homologous chromosomes (homologs) find their partner, pair and recombine during meiosis constitutes the central phenomenon in eukaryotic genetics. It is widely believed that in most organisms SPO11-mediated DNA double-strand breaks (DSBs) introduced during prophase I precede and are required for efficient homolog pairing. We now show that in the mouse, a significant level of homolog pairing precedes programmed DNA cleavage. Strikingly, this early chromosome pairing still requires SPO11, but is neither dependent on its ability to make DSBs nor homologous recombination proteins. Intriguingly, SUN1, a protein required for telomere attachment to the nuclear envelope and for post-DSB synapsis, is also required for early pre-DSB homolog pairing. Furthermore, pre-DSB pairing at telomeres persists upon entry into prophase I, and is most likely important for initiation of synapsis. Our findings suggest that the DSB-triggered homology search may mainly serve to proofread and stabilize the pre-DSB pairing of homologous chromosomes. PMID:23318132

  8. Homology-based double-strand break-induced genome engineering in plants.

    PubMed

    Steinert, Jeannette; Schiml, Simon; Puchta, Holger

    2016-07-01

    This review summarises the recent progress in DSB-induced gene targeting by homologous recombination in plants. We are getting closer to efficiently inserting genes or precisely exchanging single amino acids. Although the basic features of double-strand break (DSB)-induced genome engineering were established more than 20 years ago, only in recent years has the technique come into the focus of plant biologists. Today, most scientists apply the recently discovered CRISPR/Cas system for inducing site-specific DSBs in genes of interest to obtain mutations by non-homologous end joining (NHEJ), which is the prevailing and often imprecise mechanism of DSB repair in somatic plant cells. However, predefined changes like the site-specific insertion of foreign genes or an exchange of single amino acids can be achieved by DSB-induced homologous recombination (HR). Although DSB induction drastically enhances the efficiency of HR, the efficiency is still about two orders of magnitude lower than that of NHEJ. Therefore, significant effort have been put forth to improve DSB-induced HR based technologies. This review summarises the previous studies as well as discusses the most recent developments in using the CRISPR/Cas system to improve these processes for plants.

  9. 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.

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

    PubMed Central

    Lieber, Michael R.

    2011-01-01

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

  11. 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.

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

    SciTech Connect

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

    2015-01-12

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

  13. Castration radiosensitizes prostate cancer tissue by impairing DNA double-strand break repair.

    PubMed

    Tarish, Firas L; Schultz, Niklas; Tanoglidi, Anna; Hamberg, Hans; Letocha, Henry; Karaszi, Katalin; Hamdy, Freddie C; Granfors, Torvald; Helleday, Thomas

    2015-11-04

    Chemical castration improves responses to radiotherapy in prostate cancer, but the mechanism is unknown. We hypothesized that this radiosensitization is caused by castration-mediated down-regulation of nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). To test this, we enrolled 48 patients with localized prostate cancer in two arms of the study: either radiotherapy first or radiotherapy after neoadjuvant castration treatment. We biopsied patients at diagnosis and before and after castration and radiotherapy treatments to monitor androgen receptor, NHEJ, and DSB repair in verified cancer tissue. We show that patients receiving neoadjuvant castration treatment before radiotherapy had reduced amounts of the NHEJ protein Ku70, impaired radiotherapy-induced NHEJ activity, and higher amounts of unrepaired DSBs, measured by γ-H2AX foci in cancer tissues. This study demonstrates that chemical castration impairs NHEJ activity in prostate cancer tissue, explaining the improved response of patients with prostate cancer to radiotherapy after chemical castration. Copyright © 2015, American Association for the Advancement of Science.

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

    PubMed

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

    2014-07-10

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

  15. DNA double-strand breaks induced along the trajectory of particles

    NASA Astrophysics Data System (ADS)

    Cho, I. C.; Niu, H.; Chen, C. H.; Yu, Y. C.; Hsu, C. H.

    2011-12-01

    It is well-known that the DNA damage caused by charged particles considerably differs from damage due to electromagnetic radiation. In the case of irradiation by charged particles the DNA lesions are more complex and clustered. Such clustered damage is presumed difficult to be repaired, and is potentially lethal. In this study, we utilize a 90°-scattering system and related imaging techniques to investigate the accumulation of γ-H2AX along the trajectory of charged particles. By immunostaining the γ-H2AX protein, optical images of corresponding double strand breaks were observed using a high resolution confocal microscope. We demonstrate the difference in the accumulation of γ-H2AX from irradiation by 1 MeV protons and that of 150 keV X-rays. The acquired images were arranged and reconstructed into a 3D image using ImageJ software. We discovered that the γ-H2AX foci, following irradiation by protons, have a tendency to extend in the beam direction, while those from X-ray irradiation tend to be smaller and more randomly distributed. These results can be explained by the physical model of energy deposition.

  16. Meiotic double-strand breaks at the interface of chromosome movement, chromosome remodeling, and reductional division.

    PubMed

    Storlazzi, Aurora; Tessé, Sophie; Gargano, Silvana; James, Françoise; Kleckner, Nancy; Zickler, Denise

    2003-11-01

    Chromosomal processes related to formation and function of meiotic chiasmata have been analyzed in Sordaria macrospora. Double-strand breaks (DSBs), programmed or gamma-rays-induced, are found to promote four major events beyond recombination and accompanying synaptonemal complex formation: (1) juxtaposition of homologs from long-distance interactions to close presynaptic coalignment at midleptotene; (2) structural destabilization of chromosomes at leptotene/zygotene, including sister axis separation and fracturing, as revealed in a mutant altered in the conserved, axis-associated cohesin-related protein Spo76/Pds5p; (3) exit from the bouquet stage, with accompanying global chromosome movements, at zygotene/pachytene (bouquet stage exit is further found to be a cell-wide regulatory transition and DSB transesterase Spo11p is suggested to have a new noncatalytic role in this transition); (4) normal occurrence of both meiotic divisions, including normal sister separation. Functional interactions between DSBs and the spo76-1 mutation suggest that Spo76/Pds5p opposes local destabilization of axes at developing chiasma sites and raise the possibility of a regulatory mechanism that directly monitors the presence of chiasmata at metaphase I. Local chromosome remodeling at DSB sites appears to trigger an entire cascade of chromosome movements, morphogenetic changes, and regulatory effects that are superimposed upon a foundation of DSB-independent processes.

  17. Meiotic double-strand breaks at the interface of chromosome movement, chromosome remodeling, and reductional division

    PubMed Central

    Storlazzi, Aurora; Tessé, Sophie; Gargano, Silvana; James, Françoise; Kleckner, Nancy; Zickler, Denise

    2003-01-01

    Chromosomal processes related to formation and function of meiotic chiasmata have been analyzed in Sordaria macrospora. Double-strand breaks (DSBs), programmed or γ-rays-induced, are found to promote four major events beyond recombination and accompanying synaptonemal complex formation: (1) juxtaposition of homologs from long-distance interactions to close presynaptic coalignment at midleptotene; (2) structural destabilization of chromosomes at leptotene/zygotene, including sister axis separation and fracturing, as revealed in a mutant altered in the conserved, axis-associated cohesin-related protein Spo76/Pds5p; (3) exit from the bouquet stage, with accompanying global chromosome movements, at zygotene/pachytene (bouquet stage exit is further found to be a cell-wide regulatory transition and DSB transesterase Spo11p is suggested to have a new noncatalytic role in this transition); (4) normal occurrence of both meiotic divisions, including normal sister separation. Functional interactions between DSBs and the spo76-1 mutation suggest that Spo76/Pds5p opposes local destabilization of axes at developing chiasma sites and raise the possibility of a regulatory mechanism that directly monitors the presence of chiasmata at metaphase I. Local chromosome remodeling at DSB sites appears to trigger an entire cascade of chromosome movements, morphogenetic changes, and regulatory effects that are superimposed upon a foundation of DSB-independent processes. PMID:14563680

  18. 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

  19. DNA double strand breaks and Hsp70 expression in proton irradiated living cells

    NASA Astrophysics Data System (ADS)

    Fiedler, Anja; Reinert, Tilo; Tanner, Judith; Butz, Tilman

    2007-07-01

    DNA double strand breaks (DSBs) in living cells can be directly provoked by ionising radiation. DSBs can be visualized by immunostaining the phosphorylated histone γH2AX. Our concern was to test the feasibility of γH2AX staining for a direct visualization of single proton hits. If single protons produce detectable foci, DNA DSBs could be used as "biological track detectors" for protons. Ionising radiation can also damage proteins indirectly by inducing free radicals. Heat shock proteins (Hsp) help to refold or even degrade the damaged proteins. The level of the most famous heat shock protein Hsp70 is increased by ionising radiation. We investigated the expression of γH2AX and Hsp70 after cross and line patterned irradiation with counted numbers of 2.25 MeV protons on primary human skin fibroblasts. The proton induced DSBs appear more delocalised than it was expected by the ion hit accuracy. Cooling the cells before the irradiation reduces the delocalisation of DNA DSBs, which is probably caused by the reduced diffusion of DNA damaging agents. Proton irradiation seems to provoke protein damages mainly in the cytoplasm indicated by cytoplasmic Hsp70 aggregates. On the contrary, in control heat shocked cells the Hsp70 was predominantly localized in the cell nucleus. However, the irradiated area could not be recognized, all cells on the Si 3N 4 window showed a homogenous Hsp70 expression pattern.

  20. 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

  1. Homing endonuclease I-TevIII: dimerization as a means to a double-strand break

    PubMed Central

    Robbins, Justin B.; Stapleton, Michelle; Stanger, Matthew J.; Smith, Dorie; Dansereau, John T.; Derbyshire, Victoria; Belfort, Marlene

    2007-01-01

    Homing endonucleases are unusual enzymes, capable of recognizing lengthy DNA sequences and cleaving site-specifically within genomes. Many homing endonucleases are encoded within group I introns, and such enzymes promote the mobility reactions of these introns. Phage T4 has three group I introns, within the td, nrdB and nrdD genes. The td and nrdD introns are mobile, whereas the nrdB intron is not. Phage RB3 is a close relative of T4 and has a lengthier nrdB intron. Here, we describe I-TevIII, the H–N–H endonuclease encoded by the RB3 nrdB intron. In contrast to previous reports, we demonstrate that this intron is mobile, and that this mobility is dependent on I-TevIII, which generates 2-nt 3′ extensions. The enzyme has a distinct catalytic domain, which contains the H–N–H motif, and DNA-binding domain, which contains two zinc fingers required for interaction with the DNA substrate. Most importantly, I-TevIII, unlike the H–N–H endonucleases described so far, makes a double-strand break on the DNA homing site by acting as a dimer. Through deletion analysis, the dimerization interface was mapped to the DNA-binding domain. The unusual propensity of I-TevIII to dimerize to achieve cleavage of both DNA strands underscores the versatility of the H–N–H enzyme family. PMID:17289754

  2. 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

  3. Double-strand break-induced mitotic intrachromosomal recombination in the fission yeast Schizosaccharomyces pombe

    SciTech Connect

    Osman, F.; Fortunato, E.A.; Subramani, S.

    1996-02-01

    The Saccharomyces cerevisiae HO gene and MATa cutting site were used to introduce site-specific double-strand breaks (DSBs) within intrachromosomal recombination substrates in Schizosaccharomyces pombe. The recombination substrates consisted of nontandem direct repeats of ade6 heteroalleles. DSB induction stimulated the frequency of recombinants 2000-fold. The spectrum of DSB-induced recombinants depended on whether the DSB was introduced within one of the ade6 repeats or in intervening unique DNA. When the DSB was introduced within unique DNA, over 99.8% of the recombinants lacked the intervening DNA but retained one copy of ade6 that was wild type or either one of the heteroalleles. When the DSB was located in duplicated DNA, 77% of the recombinants were similar to the deletion types described above, but the single ade6 copy was either wild type or exclusively that of the uncut repeat. The remaining 23% of the induced recombinants were gene convertants with two copies of ade6 and the intervening sequences; the ade6 heteroallele in which the DSB was induced was the recipient of genetic information. Half-sectored colonies were isolated, analyzed and interpreted as evidence of heteroduplex DNA formation. The results are discussed in terms of current models for recombination. 81 refs., 9 figs., 3 tabs.

  4. Accumulation of Ku70 at DNA double-strand breaks in living epithelial cells

    SciTech Connect

    Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

    2011-10-15

    Ku70 and Ku80 play an essential role in the DNA double-strand break (DSB) repair pathway, i.e., nonhomologous DNA-end-joining (NHEJ). No accumulation mechanisms of Ku70 at DSBs have been clarified in detail, although the accumulation mechanism of Ku70 at DSBs plays key roles in regulating the NHEJ activity. Here, we show the essential domains for the accumulation and function of Ku70 at DSBs in living lung epithelial cells. Our results showed that EGFP-Ku70 accumulation at DSBs began immediately after irradiation. Our findings demonstrate that three domains of Ku70, i.e., the {alpha}/{beta}, DNA-binding, and Ku80-binding domains, but not the SAP domain, are necessary for the accumulation at or recognition of DSBs in the early stage after irradiation. Moreover, our findings demonstrate that the leucine at amino acid 385 of Ku70 in the Ku80-binding domain, but not the three target amino acids for acetylation in the DNA-binding domain, is involved in the localization and accumulation of Ku70 at DSBs. Furthermore, accumulations of XRCC4 and XLF, but not that of Artemis, at DSBs are dependent on the presence of Ku70. These findings suggest that Artemis can work in not only the Ku-dependent repair process, but also the Ku-independent process at DSBs in living epithelial cells.

  5. 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

  6. 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.

  7. DNA Double-Strand Breaks and Telomeres Play Important Roles in Trypanosoma brucei Antigenic Variation

    PubMed Central

    2015-01-01

    Human-infecting microbial pathogens all face a serious problem of elimination by the host immune response. Antigenic variation is an effective immune evasion mechanism where the pathogen regularly switches its major surface antigen. In many cases, the major surface antigen is encoded by genes from the same gene family, and its expression is strictly monoallelic. Among pathogens that undergo antigenic variation, Trypanosoma brucei (a kinetoplastid), which causes human African trypanosomiasis, Plasmodium falciparum (an apicomplexan), which causes malaria, Pneumocystis jirovecii (a fungus), which causes pneumonia, and Borrelia burgdorferi (a bacterium), which causes Lyme disease, also express their major surface antigens from loci next to the telomere. Except for Plasmodium, DNA recombination-mediated gene conversion is a major pathway for surface antigen switching in these pathogens. In the last decade, more sophisticated molecular and genetic tools have been developed in T. brucei, and our knowledge of functions of DNA recombination in antigenic variation has been greatly advanced. VSG is the major surface antigen in T. brucei. In subtelomeric VSG expression sites (ESs), VSG genes invariably are flanked by a long stretch of upstream 70-bp repeats. Recent studies have shown that DNA double-strand breaks (DSBs), particularly those in 70-bp repeats in the active ES, are a natural potent trigger for antigenic variation in T. brucei. In addition, telomere proteins can influence VSG switching by reducing the DSB amount at subtelomeric regions. These findings will be summarized and their implications will be discussed in this review. PMID:25576484

  8. DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

    PubMed Central

    Li, Lei; Germain, Devon R.; Poon, Ho-Yin; Hildebrandt, Matthew R.; Monckton, Elizabeth A.; McDonald, Darin; Hendzel, Michael J.

    2016-01-01

    Although RNA and RNA-binding proteins have been linked to double-strand breaks (DSBs), little is known regarding their roles in the cellular response to DSBs and, if any, in the repair process. Here, we provide direct evidence for the presence of RNA-DNA hybrids at DSBs and suggest that binding of RNA to DNA at DSBs may impact repair efficiency. Our data indicate that the RNA-unwinding protein DEAD box 1 (DDX1) is required for efficient DSB repair and cell survival after ionizing radiation (IR), with depletion of DDX1 resulting in reduced DSB repair by homologous recombination (HR). While DDX1 is not essential for end resection, a key step in homology-directed DSB repair, DDX1 is required for maintenance of the single-stranded DNA once generated by end resection. We show that transcription deregulation has a significant effect on DSB repair by HR in DDX1-depleted cells and that RNA-DNA duplexes are elevated at DSBs in DDX1-depleted cells. Based on our combined data, we propose a role for DDX1 in resolving RNA-DNA structures that accumulate at DSBs located at sites of active transcription. Our findings point to a previously uncharacterized requirement for clearing RNA at DSBs for efficient repair by HR. PMID:27550810

  9. Transcriptional effects on double-strand break-induced gene conversion tracts.

    PubMed

    Weng, Y S; Xing, D; Clikeman, J A; Nickoloff, J A

    2000-10-16

    Transcription stimulates spontaneous homologous recombination, but prior studies have not investigated the effects of transcription on double-strand break (DSB)-induced recombination in yeast. We examined products of five ura3 direct repeat substrates in yeast using alleles that were transcribed at low or high levels. In each strain, recombination was stimulated by DSBs created in vivo at an HO site in one copy of ura3. Increasing transcription levels in donor or recipient alleles did not further stimulate DSB-induced recombination, nor did it alter the relative frequencies of conversion and deletion (pop-out) events. This result is consistent with the idea that transcription enhances spontaneous recombination by increasing initiation. Gene conversion tracts were measured using silent restriction fragment length polymorphisms (RFLPs) at approximately 100bp intervals. Transcription did not alter average tract lengths, but increased transcription in donor alleles increased both the frequency of promoter-proximal (5') unidirectional tracts and conversion of 5' markers. Increased transcription in recipient alleles increased the frequency of bidirectional tracts. We demonstrate that these effects are due to transcription per se, and not just transcription factor binding. These results suggest that transcription influences aspects of gene conversion after initiation, such as strand invasion and/or mismatch repair (MMR).

  10. 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.

  11. 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.

  12. Transcription-induced DNA double strand breaks: both oncogenic force and potential therapeutic target?

    PubMed

    Haffner, Michael C; De Marzo, Angelo M; Meeker, Alan K; Nelson, William G; Yegnasubramanian, Srinivasan

    2011-06-15

    An emerging model of transcriptional activation suggests that induction of transcriptional programs, for instance by stimulating prostate or breast cells with androgens or estrogens, respectively, involves the formation of DNA damage, including DNA double strand breaks (DSB), recruitment of DSB repair proteins, and movement of newly activated genes to transcription hubs. The DSB can be mediated by the class II topoisomerase TOP2B, which is recruited with the androgen receptor and estrogen receptor to regulatory sites on target genes and is apparently required for efficient transcriptional activation of these genes. These DSBs are recognized by the DNA repair machinery triggering the recruitment of repair proteins such as poly(ADP-ribose) polymerase 1 (PARP1), ATM, and DNA-dependent protein kinase (DNA-PK). If illegitimately repaired, such DSBs can seed the formation of genomic rearrangements like the TMPRSS2-ERG fusion oncogene in prostate cancer. Here, we hypothesize that these transcription-induced, TOP2B-mediated DSBs can also be exploited therapeutically and propose that, in hormone-dependent tumors like breast and prostate cancers, a hormone-cycling therapy, in combination with topoisomerase II poisons or inhibitors of the DNA repair components PARP1 and DNA-PK, could overwhelm cancer cells with transcription-associated DSBs. Such strategies may find particular utility in cancers, like prostate cancer, which show low proliferation rates, in which other chemotherapeutic strategies that target rapidly proliferating cells have had limited success.

  13. Ataxia telangiectasia mutated activation by transcription- and topoisomerase I-induced DNA double-strand breaks.

    PubMed

    Sordet, Olivier; Redon, Christophe E; Guirouilh-Barbat, Josée; Smith, Susan; Solier, Stéphanie; Douarre, Céline; Conti, Chiara; Nakamura, Asako J; Das, Benu B; Nicolas, Estelle; Kohn, Kurt W; Bonner, William M; Pommier, Yves

    2009-08-01

    Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA damage response (DDR). As neurons have high rates of transcription that require topoisomerase I (TOP1), we investigated whether TOP1 cleavage complexes (TOP1cc)-which are potent transcription-blocking lesions-also produce transcription-dependent DNA double-strand breaks (DSBs) with ATM activation. We show the induction of DSBs and DDR activation in post-mitotic primary neurons and lymphocytes treated with camptothecin, with the induction of nuclear DDR foci containing activated ATM, gamma-H2AX (phosphorylated histone H2AX), activated CHK2 (checkpoint kinase 2), MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1). The DSB-ATM-DDR pathway was suppressed by inhibiting transcription and gamma-H2AX signals were reduced by RNase H1 transfection, which removes transcription-mediated R-loops. Thus, we propose that Top1cc produce transcription arrests with R-loop formation and generate DSBs that activate ATM in post-mitotic cells.

  14. DNA damage signaling in response to double-strand breaks during mitosis

    PubMed Central

    Giunta, Simona

    2010-01-01

    The signaling cascade initiated in response to DNA double-strand breaks (DSBs) has been extensively investigated in interphase cells. Here, we show that mitotic cells treated with DSB-inducing agents activate a “primary” DNA damage response (DDR) comprised of early signaling events, including activation of the protein kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), histone H2AX phosphorylation together with recruitment of mediator of DNA damage checkpoint 1 (MDC1), and the Mre11–Rad50–Nbs1 (MRN) complex to damage sites. However, mitotic cells display no detectable recruitment of the E3 ubiquitin ligases RNF8 and RNF168, or accumulation of 53BP1 and BRCA1, at DSB sites. Accordingly, we found that DNA-damage signaling is attenuated in mitotic cells, with full DDR activation only ensuing when a DSB-containing mitotic cell enters G1. Finally, we present data suggesting that induction of a primary DDR in mitosis is important because transient inactivation of ATM and DNA-PK renders mitotic cells hypersensitive to DSB-inducing agents. PMID:20660628

  15. Cell cycle-dependent resolution of DNA double-strand breaks

    PubMed Central

    Ambrosio, Susanna; Di Palo, Giacomo; Napolitano, Giuliana; Amente, Stefano; Dellino, Gaetano Ivan; Faretta, Mario; Pelicci, Pier Giuseppe; Lania, Luigi; Majello, Barbara

    2016-01-01

    DNA double strand breaks (DSBs) elicit prompt activation of DNA damage response (DDR), which arrests cell-cycle either in G1/S or G2/M in order to avoid entering S and M phase with damaged DNAs. Since mammalian tissues contain both proliferating and quiescent cells, there might be fundamental difference in DDR between proliferating and quiescent cells (or G0-arrested). To investigate these differences, we studied recruitment of DSB repair factors and resolution of DNA lesions induced at site-specific DSBs in asynchronously proliferating, G0-, or G1-arrested cells. Strikingly, DSBs occurring in G0 quiescent cells are not repaired and maintain a sustained activation of the p53-pathway. Conversely, re-entry into cell cycle of damaged G0-arrested cells, occurs with a delayed clearance of DNA repair factors initially recruited to DSBs, indicating an inefficient repair when compared to DSBs induced in asynchronously proliferating or G1-synchronized cells. Moreover, we found that initial recognition of DSBs and assembly of DSB factors is largely similar in asynchronously proliferating, G0-, or G1-synchronized cells. Our study thereby demonstrates that repair and resolution of DSBs is strongly dependent on the cell-cycle state. PMID:26700820

  16. Quantification and genome-wide mapping of DNA double-strand breaks.

    PubMed

    Grégoire, Marie-Chantal; Massonneau, Julien; Leduc, Frédéric; Arguin, Mélina; Brazeau, Marc-André; Boissonneault, Guylain

    2016-12-01

    DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase. We used DNA extracted from HeLa cells harboring an I-SceI cassette to induce a targeted nick or DSB and demonstrated by immunocapture of 3'-OH that a prior step of NGR allows specific determination of loci-specific or genome wide DSBs. This method can be applied to the global determination of DSBs using radioactive end labeling and can find several applications aimed at understanding the distribution and kinetics of DSBs formation and repair. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. 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.

  18. 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.

  19. 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

  20. Functional overlaps between XLF and the ATM-dependent DNA double strand break response.

    PubMed

    Kumar, Vipul; Alt, Frederick W; Oksenych, Valentyn

    2014-04-01

    Developing B and T lymphocytes generate programmed DNA double strand breaks (DSBs) during the V(D)J recombination process that assembles exons that encode the antigen-binding variable regions of antibodies. In addition, mature B lymphocytes generate programmed DSBs during the immunoglobulin heavy chain (IgH) class switch recombination (CSR) process that allows expression of different antibody heavy chain constant regions that provide different effector functions. During both V(D)J recombination and CSR, DSB intermediates are sensed by the ATM-dependent DSB response (DSBR) pathway, which also contributes to their joining via classical non-homologous end-joining (C-NHEJ). The precise nature of the interplay between the DSBR and C-NHEJ pathways in the context of DSB repair via C-NHEJ remains under investigation. Recent studies have shown that the XLF C-NHEJ factor has functional redundancy with several members of the ATM-dependent DSBR pathway in C-NHEJ, highlighting unappreciated major roles for both XLF as well as the DSBR in V(D)J recombination, CSR and C-NHEJ in general. In this review, we discuss current knowledge of the mechanisms that contribute to the repair of DSBs generated during B lymphocyte development and activation with a focus on potential functionally redundant roles of XLF and ATM-dependent DSBR factors. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. 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

  2. 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

  3. Homozygous mutation of MTPAP causes cellular radiosensitivity and persistent DNA double-strand breaks

    PubMed Central

    Martin, N T; Nakamura, K; Paila, U; Woo, J; Brown, C; Wright, J A; Teraoka, S N; Haghayegh, S; McCurdy, D; Schneider, M; Hu, H; Quinlan, A R; Gatti, R A; Concannon, P

    2014-01-01

    The study of rare human syndromes characterized by radiosensitivity has been instrumental in identifying novel proteins and pathways involved in DNA damage responses to ionizing radiation. In the present study, a mutation in mitochondrial poly-A-polymerase (MTPAP), not previously recognized for its role in the DNA damage response, was identified by exome sequencing and subsequently associated with cellular radiosensitivity. Cell lines derived from two patients with the homozygous MTPAP missense mutation were radiosensitive, and this radiosensitivity could be abrogated by transfection of wild-type mtPAP cDNA into mtPAP-deficient cell lines. Further analysis of the cellular phenotype revealed delayed DNA repair, increased levels of DNA double-strand breaks, increased reactive oxygen species (ROS), and increased cell death after irradiation (IR). Pre-IR treatment of cells with the potent anti-oxidants, α-lipoic acid and n-acetylcysteine, was sufficient to abrogate the DNA repair and clonogenic survival defects. Our results firmly establish that mutation of the MTPAP gene results in a cellular phenotype of increased DNA damage, reduced repair kinetics, increased cell death by apoptosis, and reduced clonogenic survival after exposure to ionizing radiation, suggesting a pathogenesis that involves the disruption of ROS homeostasis. PMID:24651433

  4. Functional Overlaps Between XLF and The ATM-dependent DNA Double Strand Break Response

    PubMed Central

    Kumar, Vipul; Alt, Frederick W.; Oksenych, Valentyn

    2014-01-01

    Developing B and T lymphocytes generate programmed DNA Double Strand Breaks (DSBs) during the V(D)J recombination process that assembles exons that encode the antigen-binding variable regions of antibodies. In addition, mature B lymphocytes generate programmed DSBs during the Immunoglobulin Heavy chain (IgH) Class Switch Recombination (CSR) process that allows expression of different antibody heavy chain constant regions that provide different effector functions. During both V(D)J recombination and CSR, DSB intermediates are sensed by the ATM-dependent DSB response (DSBR) pathway, which also contributes to their joining via Classical Non-Homologous End-Joining (C-NHEJ). The precise nature of the interplay between the DSBR and C-NHEJ pathways in the context of DSB repair via C-NHEJ remains under investigation. Recent studies have shown that the XLF C-NHEJ factor has functional redundancy with several members of the ATM-dependent DSBR pathway in C-NHEJ, highlighting unappreciated major roles for both XLF as well as the DSBR in V(D)J recombination, CSR and C-NHEJ in general. In this review, we discuss current knowledge of the mechanisms that contribute to the repair of DSBs generated during B lymphocyte development and activation with a focus on potential functionally redundant roles of XLF and ATM-dependent DSBR factors. PMID:24674624

  5. And yet, it moves: nuclear and chromatin dynamics of a heterochromatic double-strand break.

    PubMed

    Caridi, P Christopher; Delabaere, Laetitia; Zapotoczny, Grzegorz; Chiolo, Irene

    2017-10-05

    Heterochromatin is mostly composed of repeated DNA sequences prone to aberrant recombination. How cells maintain the stability of these sequences during double-strand break (DSB) repair has been a long-standing mystery. Studies in Drosophila cells revealed that faithful homologous recombination repair of heterochromatic DSBs relies on the striking relocalization of repair sites to the nuclear periphery before Rad51 recruitment and repair progression. Here, we summarize our current understanding of this response, including the molecular mechanisms involved, and conserved pathways in mammalian cells. We will highlight important similarities with pathways identified in budding yeast for repair of other types of repeated sequences, including rDNA and short telomeres. We will also discuss the emerging role of chromatin composition and regulation in heterochromatin repair progression. Together, these discoveries challenged previous assumptions that repair sites are substantially static in multicellular eukaryotes, that heterochromatin is largely inert in the presence of DSBs, and that silencing and compaction in this domain are obstacles to repair.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Authors.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-08-01

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

  8. 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.

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

    DOE PAGES

    Andres, Sara N.; Appel, C. Denise; Westmoreland, James W.; ...

    2015-01-12

    Ctp1 (also known as CtIP or Sae2) collaborates with Mre11-Rad50-Nbs1 to initiate repair of DNA double-strand breaks (DSBs), but its functions remain enigmatic. In this paper, we report that tetrameric Schizosaccharomyces pombe Ctp1 contains multivalent DNA-binding and DNA-bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer, we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal 'RHR' DNA-interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA-bridging activity in vitro, and both the THDD and RHR are required for efficientmore » DSB repair in S. pombe. Finally, our results establish non-nucleolytic roles of Ctp1 in binding and coordination of DSB-repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CtIP-linked Seckel and Jawad syndromes.« less

  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. Induction of DNA double-strand breaks and cellular senescence by human respiratory syncytial virus

    PubMed Central

    Martínez, Isidoro; García-Carpizo, Verónica; Guijarro, Trinidad; García-Gomez, Ana; Navarro, Diego; Aranda, Ana; Zambrano, Alberto

    2016-01-01

    ABSTRACT Human respiratory syncytial virus (HRSV) accounts for the majority of lower respiratory tract infections during infancy and childhood and is associated with significant morbidity and mortality. HRSV provokes a proliferation arrest and characteristic syncytia in cellular systems such as immortalized epithelial cells. We show here that HRSV induces the expression of DNA damage markers and proliferation arrest such as P-TP53, P-ATM, CDKN1A and γH2AFX in cultured cells secondary to the production of mitochondrial reactive oxygen species (ROS). The DNA damage foci contained γH2AFX and TP53BP1, indicative of double-strand breaks (DSBs) and could be reversed by antioxidant treatments such as N-Acetylcysteine (NAC) or reduced glutathione ethyl ester (GSHee). The damage observed is associated with the accumulation of senescent cells, displaying a canonical senescent phenotype in both mononuclear cells and syncytia. In addition, we show signs of DNA damage and aging such as γH2AFX and CDKN2A expression in the respiratory epithelia of infected mice long after viral clearance. Altogether, these results show that HRSV triggers a DNA damage-mediated cellular senescence program probably mediated by oxidative stress. The results also suggest that this program might contribute to the physiopathology of the infection, tissue remodeling and aging, and might be associated to long-term consequences of HRSV infections. PMID:26809688

  12. 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

  13. Using carbon nanotubes to induce micronuclei and double strand breaks of the DNA in human cells

    NASA Astrophysics Data System (ADS)

    Cveticanin, Jelena; Joksic, Gordana; Leskovac, Andreja; Petrovic, Sandra; Valenta Sobot, Ana; Neskovic, Olivera

    2010-01-01

    Carbon nanotubes are unique one-dimensional macromolecules with promising applications in biology and medicine. Since their toxicity is still under debate, here we present a study investigating the genotoxic properties of purified single wall carbon nanotubes (SWCNTs), multiwall carbon nanotubes (MWCNTs), and amide functionalized purified SWCNTs on cultured human lymphocytes employing cytokinesis block micronucleus assay and enumeration of γH2AX foci as a measure of double strand breaks (DSBs) of the DNA in normal human fibroblasts. SWCNTs induce micronuclei (MN) formation in lymphocytes and decrease the proliferation potential (CBPI) of cells. In a fibroblast cell line the same dose of SWCNTs induces γH2AX foci 2.7-fold higher than in a control. Amide functionalized purified SWCNTs behave differently: they do not disturb the cell proliferation potential of harvested lymphocytes, but induce micronuclei to a higher extent than SWCNTs. When applied on fibroblasts, amide functionalized SWCNTs also induce γH2AX foci, 3.18-fold higher than the control. The cellular effects of MWCNTs display the broad spectrum of clastogenic properties seen as the highest incidence of induced lymphocyte micronuclei and anaphase bridges among nuclei in binucleated cells. Surprisingly, the incidence of induced γH2AX foci was not as high as was expected by the micronucleus test, which indicates that MWCNTs act as clastogen and aneugen agents simultaneously. Biological endpoints investigated in this study indicate a close relationship between the electrochemical properties of carbon nanotubes and observed genotoxicity.

  14. 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

  15. 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

  16. Programmed induction of endoreduplication by DNA double-strand breaks in Arabidopsis.

    PubMed

    Adachi, Sumiko; Minamisawa, Kazunori; Okushima, Yoko; Inagaki, Soichi; Yoshiyama, Kaoru; Kondou, Youichi; Kaminuma, Eli; Kawashima, Mika; Toyoda, Tetsuro; Matsui, Minami; Kurihara, Daisuke; Matsunaga, Sachihiro; Umeda, Masaaki

    2011-06-14

    Genome integrity is continuously threatened by external stresses and endogenous hazards such as DNA replication errors and reactive oxygen species. The DNA damage checkpoint in metazoans ensures genome integrity by delaying cell-cycle progression to repair damaged DNA or by inducing apoptosis. ATM and ATR (ataxia-telangiectasia-mutated and -Rad3-related) are sensor kinases that relay the damage signal to transducer kinases Chk1 and Chk2 and to downstream cell-cycle regulators. Plants also possess ATM and ATR orthologs but lack obvious counterparts of downstream regulators. Instead, the plant-specific transcription factor SOG1 (suppressor of gamma response 1) plays a central role in the transmission of signals from both ATM and ATR kinases. Here we show that in Arabidopsis, endoreduplication is induced by DNA double-strand breaks (DSBs), but not directly by DNA replication stress. When root or sepal cells, or undifferentiated suspension cells, were treated with DSB inducers, they displayed increased cell size and DNA ploidy. We found that the ATM-SOG1 and ATR-SOG1 pathways both transmit DSB-derived signals and that either one suffices for endocycle induction. These signaling pathways govern the expression of distinct sets of cell-cycle regulators, such as cyclin-dependent kinases and their suppressors. Our results demonstrate that Arabidopsis undergoes a programmed endoreduplicative response to DSBs, suggesting that plants have evolved a distinct strategy to sustain growth under genotoxic stress.

  17. Programmed induction of endoreduplication by DNA double-strand breaks in Arabidopsis

    PubMed Central

    Adachi, Sumiko; Minamisawa, Kazunori; Okushima, Yoko; Inagaki, Soichi; Yoshiyama, Kaoru; Kondou, Youichi; Kaminuma, Eli; Kawashima, Mika; Toyoda, Tetsuro; Matsui, Minami; Kurihara, Daisuke; Matsunaga, Sachihiro; Umeda, Masaaki

    2011-01-01

    Genome integrity is continuously threatened by external stresses and endogenous hazards such as DNA replication errors and reactive oxygen species. The DNA damage checkpoint in metazoans ensures genome integrity by delaying cell-cycle progression to repair damaged DNA or by inducing apoptosis. ATM and ATR (ataxia-telangiectasia-mutated and -Rad3-related) are sensor kinases that relay the damage signal to transducer kinases Chk1 and Chk2 and to downstream cell-cycle regulators. Plants also possess ATM and ATR orthologs but lack obvious counterparts of downstream regulators. Instead, the plant-specific transcription factor SOG1 (suppressor of gamma response 1) plays a central role in the transmission of signals from both ATM and ATR kinases. Here we show that in Arabidopsis, endoreduplication is induced by DNA double-strand breaks (DSBs), but not directly by DNA replication stress. When root or sepal cells, or undifferentiated suspension cells, were treated with DSB inducers, they displayed increased cell size and DNA ploidy. We found that the ATM–SOG1 and ATR–SOG1 pathways both transmit DSB-derived signals and that either one suffices for endocycle induction. These signaling pathways govern the expression of distinct sets of cell-cycle regulators, such as cyclin-dependent kinases and their suppressors. Our results demonstrate that Arabidopsis undergoes a programmed endoreduplicative response to DSBs, suggesting that plants have evolved a distinct strategy to sustain growth under genotoxic stress. PMID:21613568

  18. 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.

  19. 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.

  20. 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.

  1. The MRE11 GAR motif regulates DNA double-strand break processing and ATR activation

    PubMed Central

    Yu, Zhenbao; Vogel, Gillian; Coulombe, Yan; Dubeau, Danielle; Spehalski, Elizabeth; Hébert, Josée; Ferguson, David O; Masson, Jean Yves; Richard, Stéphane

    2012-01-01

    The MRE11/RAD50/NBS1 complex is the primary sensor rapidly recruited to DNA double-strand breaks (DSBs). MRE11 is known to be arginine methylated by PRMT1 within its glycine-arginine-rich (GAR) motif. In this study, we report a mouse knock-in allele of Mre11 that substitutes the arginines with lysines in the GAR motif and generates the MRE11RK protein devoid of methylated arginines. The Mre11RK/RK mice were hypersensitive to γ-irradiation (IR) and the cells from these mice displayed cell cycle checkpoint defects and chromosome instability. Moreover, the Mre11RK/RK MEFs exhibited ATR/CHK1 signaling defects and impairment in the recruitment of RPA and RAD51 to the damaged sites. The MRKRN complex formed and localized to the sites of DNA damage and normally activated the ATM pathway in response to IR. The MRKRN complex exhibited exonuclease and DNA-binding defects in vitro responsible for the impaired DNA end resection and ATR activation observed in vivo in response to IR. Our findings provide genetic evidence for the critical role of the MRE11 GAR motif in DSB repair, and demonstrate a mechanistic link between post-translational modifications at the MRE11 GAR motif and DSB processing, as well as the ATR/CHK1 checkpoint signaling. PMID:21826105

  2. Triplex structures induce DNA double strand breaks via replication fork collapse in NER deficient cells

    PubMed Central

    Kaushik Tiwari, Meetu; Adaku, Nneoma; Peart, Natoya; Rogers, Faye A.

    2016-01-01

    Structural alterations in DNA can serve as natural impediments to replication fork stability and progression, resulting in DNA damage and genomic instability. Naturally occurring polypurine mirror repeat sequences in the human genome can create endogenous triplex structures evoking a robust DNA damage response. Failures to recognize or adequately process these genomic lesions can result in loss of genomic integrity. Nucleotide excision repair (NER) proteins have been found to play a prominent role in the recognition and repair of triplex structures. We demonstrate using triplex-forming oligonucleotides that chromosomal triplexes perturb DNA replication fork progression, eventually resulting in fork collapse and the induction of double strand breaks (DSBs). We find that cells deficient in the NER damage recognition proteins, XPA and XPC, accumulate more DSBs in response to chromosomal triplex formation than NER-proficient cells. Furthermore, we demonstrate that XPC-deficient cells are particularly prone to replication-associated DSBs in the presence of triplexes. In the absence of XPA or XPC, deleterious consequences of triplex-induced genomic instability may be averted by activating apoptosis via dual phosphorylation of the H2AX protein. Our results reveal that damage recognition by XPC and XPA is critical to maintaining replication fork integrity and preventing replication fork collapse in the presence of triplex structures. PMID:27298253

  3. Induction of DNA double-strand breaks and cellular senescence by human respiratory syncytial virus.

    PubMed

    Martínez, Isidoro; García-Carpizo, Verónica; Guijarro, Trinidad; García-Gomez, Ana; Navarro, Diego; Aranda, Ana; Zambrano, Alberto

    2016-05-18

    Human respiratory syncytial virus (HRSV) accounts for the majority of lower respiratory tract infections during infancy and childhood and is associated with significant morbidity and mortality. HRSV provokes a proliferation arrest and characteristic syncytia in cellular systems such as immortalized epithelial cells. We show here that HRSV induces the expression of DNA damage markers and proliferation arrest such as P-TP53, P-ATM, CDKN1A and γH2AFX in cultured cells secondary to the production of mitochondrial reactive oxygen species (ROS). The DNA damage foci contained γH2AFX and TP53BP1, indicative of double-strand breaks (DSBs) and could be reversed by antioxidant treatments such as N-Acetylcysteine (NAC) or reduced glutathione ethyl ester (GSHee). The damage observed is associated with the accumulation of senescent cells, displaying a canonical senescent phenotype in both mononuclear cells and syncytia. In addition, we show signs of DNA damage and aging such as γH2AFX and CDKN2A expression in the respiratory epithelia of infected mice long after viral clearance. Altogether, these results show that HRSV triggers a DNA damage-mediated cellular senescence program probably mediated by oxidative stress. The results also suggest that this program might contribute to the physiopathology of the infection, tissue remodeling and aging, and might be associated to long-term consequences of HRSV infections.

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

    PubMed

    Suzuki, Tetsuya; Yasui, Manabu; Honma, Masamitsu

    2016-12-01

    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. Copyright © 2016 Suzuki et al.

  5. 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.

  6. Chromosome position determines the success of double-strand break repair.

    PubMed

    Lee, Cheng-Sheng; Wang, Ruoxi W; Chang, Hsiao-Han; Capurso, Daniel; Segal, Mark R; Haber, James E

    2016-01-12

    Repair of a chromosomal double-strand break (DSB) by gene conversion depends on the ability of the broken ends to encounter a donor sequence. To understand how chromosomal location of a target sequence affects DSB repair, we took advantage of genome-wide Hi-C analysis of yeast chromosomes to create a series of strains in which an induced site-specific DSB in budding yeast is repaired by a 2-kb donor sequence inserted at different locations. The efficiency of repair, measured by cell viability or competition between each donor and a reference site, showed a strong correlation (r = 0.85 and 0.79) with the contact frequencies of each donor with the DSB repair site. Repair efficiency depends on the distance between donor and recipient rather than any intrinsic limitation of a particular donor site. These results further demonstrate that the search for homology is the rate-limiting step in DSB repair and suggest that cells often fail to repair a DSB because they cannot locate a donor before other, apparently lethal, processes arise. The repair efficiency of a donor locus can be improved by four factors: slower 5' to 3' resection of the DSB ends, increased abundance of replication protein factor A (RPA), longer shared homology, or presence of a recombination enhancer element adjacent to a donor.

  7. Position effects influencing intrachromosomal repair of a double-strand break in budding yeast.

    PubMed

    Wang, Ruoxi W; Lee, Cheng-Sheng; Haber, James E

    2017-01-01

    Repair of a double-strand break (DSB) by an ectopic homologous donor sequence is subject to the three-dimensional arrangement of chromosomes in the nucleus of haploid budding yeast. The data for interchromosomal recombination suggest that searching for homology is accomplished by a random collision process, strongly influenced by the contact probability of the donor and recipient sequences. Here we explore how recombination occurs on the same chromosome and whether there are additional constraints imposed on repair. Specifically, we examined how intrachromosomal repair is affected by the location of the donor sequence along the 813-kb chromosome 2 (Chr2), with a site-specific DSB created on the right arm (position 625 kb). Repair correlates well with contact frequencies determined by chromosome conformation capture-based studies (r = 0.85). Moreover, there is a profound constraint imposed by the anchoring of the centromere (CEN2, position 238 kb) to the spindle pole body. Sequences at the same distance on either side of CEN2 are equivalently constrained in recombining with a DSB located more distally on one arm, suggesting that sequences on the opposite arm from the DSB are not otherwise constrained in their interaction with the DSB. The centromere constraint can be partially relieved by inducing transcription through the centromere to inactivate CEN2 tethering. In diploid cells, repair of a DSB via its allelic donor is strongly influenced by the presence and the position of an ectopic intrachromosomal donor.

  8. DNA Double-Strand Break Repair at −15°C

    PubMed Central

    Dieser, Markus; Battista, John R.

    2013-01-01

    The survival of microorganisms in ancient glacial ice and permafrost has been ascribed to their ability to persist in a dormant, metabolically inert state. An alternative possibility, supported by experimental data, is that microorganisms in frozen matrices are able to sustain a level of metabolic function that is sufficient for cellular repair and maintenance. To examine this experimentally, frozen populations of Psychrobacter arcticus 273-4 were exposed to ionizing radiation (IR) to simulate the damage incurred from natural background IR sources in the permafrost environment from over ∼225 kiloyears (ky). High-molecular-weight DNA was fragmented by exposure to 450 Gy of IR, which introduced an average of 16 double-strand breaks (DSBs) per chromosome. During incubation at −15°C for 505 days, P. arcticus repaired DNA DSBs in the absence of net growth. Based on the time frame for the assembly of genomic fragments by P. arcticus, the rate of DNA DSB repair was estimated at 7 to 10 DSBs year−1 under the conditions tested. Our results provide direct evidence for the repair of DNA lesions, extending the range of complex biochemical reactions known to occur in bacteria at frozen temperatures. Provided that sufficient energy and nutrient sources are available, a functional DNA repair mechanism would allow cells to maintain genome integrity and augment microbial survival in icy terrestrial or extraterrestrial environments. PMID:24077718

  9. 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

  10. Telmisartan Induces Growth Inhibition, DNA Double-Strand Breaks and Apoptosis in Human Endometrial Cancer Cells

    PubMed Central

    Koyama, Naoko; Nishida, Yoshihiro; Ishii, Terukazu; Yoshida, Toshie; Furukawa, Yuichi; Narahara, Hisashi

    2014-01-01

    Telmisartan, an angiotensin II receptor type 1 blocker, is often used as an antihypertension drug, and it has also been characterized as a peroxisome proliferator-activated receptor-gamma (PPARγ) ligand. The purpose of this study was to elucidate the antitumor effects of telmisartan on endometrial cancer cells. We treated three endometrial cancer cell lines with various concentrations of telmisartan, and we investigated the effects of the telmisartan on the cell proliferation, apoptosis, and their related measurements in vitro. We also administered telmisartan to nude mice with experimental tumors to determine its in vivo effects and toxicity. All three endometrial cancer cell lines were sensitive to the growth-inhibitory effect of telmisartan. The induction of apoptosis was confirmed in concert with the altered expression of genes and proteins related to the apoptosis. We also observed that DNA double-strand breaks (DSBs) were induced in HHUA (human endometrial cancer) cells by telmisartan treatment. In addition, experiments in nude mice showed that telmisartan significantly inhibited human endometrial tumor growth, without toxic side effects. Our results suggest that telmisartan might be a new therapeutic option for the treatment of endometrial cancers. PMID:24667764

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

    PubMed Central

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

    2011-01-01

    The ability to induce synchronously a single site-specific double-strand break (DSB) in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during DSB repair. Special attention has been paid to the switching of mating-type genes in Saccharomyces cerevisiae, a process initiated by the HO endonuclease by cleaving the MAT locus. A DSB in MATa is repaired by homologous recombination—specifically, by gene conversion—using a heterochromatic donor, HMLα. Repair results in the replacement of the a-specific sequences (Ya) by Yα and switching from MATa to MATα. We report that MAT switching requires the DNA replication factor Dpb11, although it does not require the Cdc7-Dbf4 kinase or the Mcm and Cdc45 helicase components. Using Southern blot, PCR, and ChIP analysis of samples collected every 10 min, we extend previous studies of this process to identify the times for the loading of Rad51 recombinase protein onto the DSB ends at MAT, the subsequent strand invasion by the Rad51 nucleoprotein filament into the donor sequences, the initiation of new DNA synthesis, and the removal of the nonhomologous Y sequences. In addition we report evidence for the transient displacement of well-positioned nucleosomes in the HML donor locus during strand invasion. PMID:21292986

  12. No evidence for DNA double-strand breaks caused by endodontic sealers.

    PubMed

    Van Landuyt, Kirsten L; Geebelen, Benjamin; Shehata, Mostafa; Furche, Silja L; Durner, Jürgen; Van Meerbeek, Bart; Hickel, Reinhard; Reichl, Franz X

    2012-05-01

    On extrusion, endodontic sealers might come into close contact with the periapical tissues for long periods. The objective of this study was to test possible mutagenicity of resin-based endodontic sealers by evaluating their potential to induce DNA double-strand breaks (DSBs). Human gingival fibroblasts were exposed to subtoxic concentrations of eluates from 1 epoxy resin-based endodontic sealer (AH Plus Jet) and 2 methacrylate-based endodontic sealers (EndoRez and Real Seal). As control, Calcicur, a Ca(OH)(2)-based sealer, was used. The γ-H2AX immunofluorescence assay was used to microscopically detect DNA DSBs, and a custom algorithm was developed to quantify them. The cytotoxicity of the 24-hour eluates could be ranked in the following order: AH Plus Jet > Real Seal > EndoRez > Calcicur. The γ-H2AX assay revealed that 1.3%-4.3% of the cell nucleus was occupied by foci when the cells were exposed to the eluates of the endodontic sealers. This was not significantly different from the negative control group in which the cells had been exposed to medium (2.1%). No indications for increased risk of genotoxicity of resin-based root canal sealers caused by the induction of DNA DSBs were found in this study. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  13. LINE-1 methylation status of endogenous DNA double-strand breaks.

    PubMed

    Pornthanakasem, Wichai; Kongruttanachok, Narisorn; Phuangphairoj, Chutipa; Suyarnsestakorn, Chotika; Sanghangthum, Taweap; Oonsiri, Sornjarod; Ponyeam, Wanpen; Thanasupawat, Thatchawan; Matangkasombut, Oranart; Mutirangura, Apiwat

    2008-06-01

    DNA methylation and the repair of DNA double-strand breaks (DSBs) are important processes for maintaining genomic integrity. Although DSBs can be produced by numerous agents, they also occur spontaneously as endogenous DSBs (EDSBs). In this study, we evaluated the methylation status of EDSBs to determine if there is a connection between DNA methylation and EDSBs. We utilized interspersed repetitive sequence polymerase chain reaction (PCR), ligation-mediated PCR and combined bisulfite restriction analysis to examine the extent of EDSBs and methylation at long interspersed nuclear element-1 (LINE-1) sequences nearby EDSBs. We tested normal white blood cells and several cell lines derived from epithelial cancers and leukemias. Significant levels of EDSBs were detectable in all cell types. EDSBs were also found in both replicating and non-replicating cells. We found that EDSBs contain higher levels of methylation than the cellular genome. This hypermethylation is replication independent and the methylation was present in the genome at the location prior to the DNA DSB. The differences in methylation levels between EDSBs and the rest of the genome suggests that EDSBs are differentially processed, by production, end-modification, or repair, depending on the DNA methylation status.

  14. Radiation dose determines the method for quantification of DNA double strand breaks.

    PubMed

    Bulat, Tanja; Keta, Otilija; Korićanac, Lela; Žakula, Jelena; Petrović, Ivan; Ristić-Fira, Aleksandra; Todorović, Danijela

    2016-03-01

    Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci.

  15. Meiotic chromosome synapsis in yeast can occur without spo11-induced DNA double-strand breaks.

    PubMed

    Bhuiyan, Hasanuzzaman; Schmekel, Karin

    2004-10-01

    Proper chromosome segregation and formation of viable gametes depend on synapsis and recombination between homologous chromosomes during meiosis. Previous reports have shown that the synaptic structures, the synaptonemal complexes (SCs), do not occur in yeast cells with the SPO11 gene removed. The Spo11 enzyme makes double-strand breaks (DSBs) in the DNA and thereby initiates recombination. The view has thus developed that synapsis in yeast strictly depends on the initiation of recombination. Synapsis in some other species (Drosophila melanogaster and Caenorhabditis elegans) is independent of recombination events, and SCs are found in spo11 mutants. This difference between species led us to reexamine spo11 deletion mutants of yeast. Using antibodies against Zip1, a SC component, we found that a small fraction (1%) of the spo11 null mutant cells can indeed form wild-type-like SCs. We further looked for synapsis in a spo11 mutant strain that accumulates pachytene cells (spo11Delta ndt80Delta), and found that the frequency of cells with apparently complete SC formation was 10%. Other phenotypic criteria, such as spore viability and homologous chromosome juxtaposition measured by FISH labeling of chromosomal markers, agree with several previous reports of the spo11 mutant. Our results demonstrate that although the Spo11-induced DSBs obviously promote synapsis in yeast, the presence of Spo11 is not an absolute requirement for synapsis.

  16. Homologous pairing preceding SPO11-mediated double-strand breaks in mice.

    PubMed

    Boateng, Kingsley A; Bellani, Marina A; Gregoretti, Ivan V; Pratto, Florencia; Camerini-Otero, R Daniel

    2013-01-28

    How homologous chromosomes (homologs) find their partner, pair, and recombine during meiosis constitutes the central phenomenon in eukaryotic genetics. It is widely believed that, in most organisms, SPO11-mediated DNA double-strand breaks (DSBs) introduced during prophase I precede and are required for efficient homolog pairing. We now show that, in the mouse, a significant level of homolog pairing precedes programmed DNA cleavage. Strikingly, this early chromosome pairing still requires SPO11 but is not dependent on its ability to make DSBs or homologous recombination proteins. Intriguingly, SUN1, a protein required for telomere attachment to the nuclear envelope and for post-DSB synapsis, is also required for early pre-DSB homolog pairing. Furthermore, pre-DSB pairing at telomeres persists upon entry into prophase I and is most likely important for initiation of synapsis. Our findings suggest that the DSB-triggered homology search may mainly serve to proofread and stabilize the pre-DSB pairing of homologous chromosomes.

  17. 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.

  18. Coordinated nuclease activities counteract Ku at single-ended DNA double-strand breaks

    PubMed Central

    Chanut, Pauline; Britton, Sébastien; Coates, Julia; Jackson, Stephen P.; Calsou, Patrick

    2016-01-01

    Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 3′ single-strand DNA overhang by exonuclease activities in a process called DNA resection. However, it is anticipated that the highly abundant DNA end-binding protein Ku sequesters seDSBs and shields them from exonuclease activities. Despite pioneering works in yeast, it is unclear how mammalian cells counteract Ku at seDSBs to allow HR to proceed. Here we show that in human cells, ATM-dependent phosphorylation of CtIP and the epistatic and coordinated actions of MRE11 and CtIP nuclease activities are required to limit the stable loading of Ku on seDSBs. We also provide evidence for a hitherto unsuspected additional mechanism that contributes to prevent Ku accumulation at seDSBs, acting downstream of MRE11 endonuclease activity and in parallel with MRE11 exonuclease activity. Finally, we show that Ku persistence at seDSBs compromises Rad51 focus assembly but not DNA resection. PMID:27641979

  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. 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

  1. Nanoneedle insertion into the cell nucleus does not induce double-strand breaks in chromosomal DNA.

    PubMed

    Ryu, Seunghwan; Kawamura, Ryuzo; Naka, Ryohei; Silberberg, Yaron R; Nakamura, Noriyuki; Nakamura, Chikashi

    2013-09-01

    An atomic force microscope probe can be formed into an ultra-sharp cylindrical shape (a nanoneedle) using micro-fabrication techniques such as focused ion beam etching. This nanoneedle can be effectively inserted through the plasma membrane of a living cell to not only access the cytosol, but also to penetrate through the nuclear membrane. This technique shows great potential as a tool for performing intranuclear measurements and manipulations. Repeated insertions of a nanoneedle into a live cell were previously shown not to affect cell viability. However, the effect of nanoneedle insertion on the nucleus and nuclear components is still unknown. DNA is the most crucial component of the nucleus for proper cell function and may be physically damaged by a nanoneedle. To investigate the integrity of DNA following nanoneedle insertion, the occurrence of DNA double-strand breaks (DSBs) was assessed. The results showed that there was no chromosomal DNA damage due to nanoneedle insertion into the nucleus, as indicated by the expression level of γ-H2AX, a molecular marker of DSBs.

  2. Functions and regulation of human artemis in double strand break repair.

    PubMed

    Dahm, Kirsten

    2007-04-15

    Cells, which lacked the activity of the nuclease Artemis, retained approximately 10% of unrepaired double strand breaks (DSBs) at later timepoints after ionizing radiation. Ionizing radiation induced hyperphosphorylation of Artemis mainly by ATM and in ATM deficient cells to a minor extent by DNA PK. After induction of DSBs with modified ends by a high dose of calicheamicin gamma1, Artemis was phosphorylated by DNA PK. The type of calicheamicin gamma1-induced DSBs is likely to represent a subclass of DSBs induced by ionizing radiation. DNA PK-dependent phosphorylation of Artemis after treatment with DSB inducing agents increased the cellular retention of Artemis, maintained its interaction with DNA ends and activated its endonucleolytic activity. The following model is suggested: ATM-dependent phosphorylation of Artemis after ionizing radiation could prevent DNA PK-dependent phosphorylation and activation of undesired endonucleolytic activity at DSBs, which do not require endonucleolytic processing by Artemis. The Artemis:DNA PK complex could be involved in the repair of DSBs, which carry modified ends and are refractory to repair by otherwise lesion specific enzymes because of the presence of an inhibitory lesion in the opposite strand.

  3. 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

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

    PubMed

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

    2017-03-01

    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. 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. 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. 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.

  5. E2F1 promotes the recruitment of DNA repair factors to sites of DNA double-strand breaks

    PubMed Central

    Chen, Jie; Zhu, Feng; Weaks, Regina L; Biswas, Anup K; Guo, Ruifeng; Li, Yanjie

    2011-01-01

    The E2F1 transcription factor is post-translationally modified and stabilized in response to various forms of DNA damage to regulate the expression of cell cycle and pro-apoptotic genes. E2F1 also forms foci at DNA double-strand breaks (DSBs) but the function of E2F1 at sites of damage is unknown. Here we demonstrate that the absence of E2F1 leads to spontaneous DNA breaks and impaired recovery following exposure to ionizing radiation. E2F1 deficiency results in defective NBS1 phosphorylation and foci formation in response to DSBs but does not affect NBS1 expression levels. Moreover, an increased association between NBS1 and E2F1 is observed in response to DNA damage, suggesting that E2F1 may promote NBS1 foci formation through a direct or indirect interaction at sites of DNA breaks. E2F1 deficiency also impairs RPA and Rad51 foci formation indicating that E2F1 is important for DNA end resection and the formation of single-stranded DNA at DSBs. These findings establish new roles for E2F1 in the DNA damage response, which may directly contribute to DNA repair and genome maintenance. PMID:21512314

  6. Effects of Breast Shielding during Heart Imaging on DNA Double-Strand-Break Levels: A Prospective Randomized Controlled Trial.

    PubMed

    Cheezum, Michael K; Redon, Christophe E; Burrell, Allison S; Kaviratne, Anthony S; Bindeman, Jody; Maeda, Daisuke; Balmakhtar, Houria; Pezel, Ashly; Wisniewski, Piotr; Delacruz, Panfilo; Nguyen, Binh; Bonner, William M; Villines, Todd C

    2016-10-01

    Purpose To examine the effect of breast shielding on blood lymphocyte deoxyribonucleic acid (DNA) double-strand-break levels resulting from in vivo radiation and ex vivo radiation at breast-tissue level, and the effect of breast shielding on image quality. Materials and Methods The study was approved by institutional review and commpliant with HIPAA guidelines. Adult women who underwent 64-section coronary computed tomographic (CT) angiography and who provided informed consent were prospectively randomized to the use (n = 50) or absence (n = 51) of bismuth breast shields. Peripheral blood samples were obtained before and 30 minutes after in vivo radiation during CT angiography to compare DNA double-strand-break levels by γ-H2AX immunofluorescence in blood lymphocytes. To estimate DNA double-strand-break induction at breast-tissue level, a blood sample was taped to the sternum for ex vivo radiation with or without shielding. Data were analyzed by linear regression and independent sample t tests. Results Breast shielding had no effect on DNA double-strand-break levels from ex vivo radiation of blood samples under shields at breast-tissue level (unadjusted regression: β = .08; P = .43 versus no shielding), or in vivo radiation of circulating lymphocytes (β = -.07; P = .50). Predictors of increased DNA double-strand-break levels included total radiation dose, increasing tube potential, and tube current (P < .05). With current radiation exposures (median, 3.4 mSv), breast shielding yielded a 33% increase in image noise and 19% decrease in the rate of excellent quality ratings. Conclusion Among women who underwent coronary CT angiography, breast shielding had no effect on DNA double-strand-break levels in blood lymphocytes exposed to in vivo radiation, or ex vivo radiation at breast-tissue level. At present relatively low radiation exposures, breast shielding contributed to an increase in image noise and a decline in image quality. The findings support efforts to

  7. Probing Enhanced Double-Strand Break Formation at Abasic Sites within Clustered Lesions in Nucleosome Core Particles.

    PubMed

    Banerjee, Samya; Chakraborty, Supratim; Jacinto, Marco Paolo; Paul, Michael D; Balster, Morgan V; Greenberg, Marc M

    2017-01-10

    DNA is rapidly cleaved under mild alkaline conditions at apyrimidinic/apurinic sites, but the half-life is several weeks in phosphate buffer (pH 7.5). However, abasic sites are ∼100-fold more reactive within nucleosome core particles (NCPs). Histone proteins catalyze the strand scission, and at superhelical location 1.5, the histone H4 tail is largely responsible for the accelerated cleavage. The rate constant for strand scission at an abasic site is enhanced further in a nucleosome core particle when it is part of a bistranded lesion containing a proximal strand break. Cleavage of this form results in a highly deleterious double-strand break. This acceleration is dependent upon the position of the abasic lesion in the NCP and its structure. The enhancement in cleavage rate at an apurinic/apyrimidinic site rapidly drops off as the distance between the strand break and abasic site increases and is negligible once the two forms of damage are separated by 7 bp. However, the enhancement of the rate of double-strand break formation increases when the size of the gap is increased from one to two nucleotides. In contrast, the cleavage rate enhancement at 2-deoxyribonolactone within bistranded lesions is more modest, and it is similar in free DNA and nucleosome core particles. We postulate that the enhanced rate of double-strand break formation at bistranded lesions containing apurinic/apyrimidinic sites within nucleosome core particles is a general phenomenon and is due to increased DNA flexibility.

  8. Radiation-induced heat-labile sites that convert into DNA double-strand breaks

    NASA Technical Reports Server (NTRS)

    Rydberg, B.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    The yield of DNA double-strand breaks (DSBs) in SV40 DNA irradiated in aqueous solution was found to increase by more than a factor of two as a result of postirradiation incubation of the DNA at 50 degrees C and pH 8.0 for 24 h. This is in agreement with data from studies performed at 37 degrees C that were published previously. Importantly, similar results were also obtained from irradiation of mammalian DNA in agarose plugs. These results suggest that heat-labile sites within locally multiply damaged sites are produced by radiation and are subsequently transformed into DSBs. Since incubation at 50 degrees C is typically employed for lysis of cells in commonly used pulsed-field gel assays for detection of DSBs in mammalian cells, the possibility that heat-labile sites are present in irradiated cells was also studied. An increase in the apparent number of DSBs as a function of lysis time at 50 degrees C was found with kinetics that was similar to that for irradiated DNA, although the magnitude of the increase was smaller. This suggests that heat-labile sites are also formed in the cell. If this is the case, a proportion of DSBs measured by the pulsed-field gel assays may occur during the lysis step and may not be present in the cell as breaks but as heat-labile sites. It is suggested that such sites consist mainly of heat-labile sugar lesions within locally multiply damaged sites. Comparing rejoining of DSBs measured with short and long lysis procedure indicates that the heat-labile sites are repaired with fast kinetics in comparison with repair of the bulk of DSBs.

  9. Radiation-induced heat-labile sites that convert into DNA double-strand breaks

    NASA Technical Reports Server (NTRS)

    Rydberg, B.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    The yield of DNA double-strand breaks (DSBs) in SV40 DNA irradiated in aqueous solution was found to increase by more than a factor of two as a result of postirradiation incubation of the DNA at 50 degrees C and pH 8.0 for 24 h. This is in agreement with data from studies performed at 37 degrees C that were published previously. Importantly, similar results were also obtained from irradiation of mammalian DNA in agarose plugs. These results suggest that heat-labile sites within locally multiply damaged sites are produced by radiation and are subsequently transformed into DSBs. Since incubation at 50 degrees C is typically employed for lysis of cells in commonly used pulsed-field gel assays for detection of DSBs in mammalian cells, the possibility that heat-labile sites are present in irradiated cells was also studied. An increase in the apparent number of DSBs as a function of lysis time at 50 degrees C was found with kinetics that was similar to that for irradiated DNA, although the magnitude of the increase was smaller. This suggests that heat-labile sites are also formed in the cell. If this is the case, a proportion of DSBs measured by the pulsed-field gel assays may occur during the lysis step and may not be present in the cell as breaks but as heat-labile sites. It is suggested that such sites consist mainly of heat-labile sugar lesions within locally multiply damaged sites. Comparing rejoining of DSBs measured with short and long lysis procedure indicates that the heat-labile sites are repaired with fast kinetics in comparison with repair of the bulk of DSBs.

  10. Fine resolution mapping of double-strand break sites for human ribosomal DNA units.

    PubMed

    Pope, Bernard J; Mahmood, Khalid; Jung, Chol-Hee; Park, Daniel J

    2016-12-01

    DNA breakage arises during a variety of biological processes, including transcription, replication and genome rearrangements. In the context of disease, extensive fragmentation of DNA has been described in cancer cells and during early stages of neurodegeneration (Stephens et al., 2011 Stephens et al. (2011) [5]; Blondet et al., 2001 Blondet et al. (2001) [1]). Stults et al. (2009) Stults et al. (2009) [6] reported that human rDNA gene clusters are hotspots for recombination and that rDNA restructuring is among the most common chromosomal alterations in adult solid tumours. As such, analysis of rDNA regions is likely to have significant prognostic and predictive value, clinically. Tchurikov et al. (2015a, 2016) Tchurikov et al. (2015a, 2016) [7], [9] have made major advances in this direction, reporting that sites of human genome double-strand breaks (DSBs) occur frequently at sites in rDNA that are tightly linked with active transcription - the authors used a RAFT (rapid amplification of forum termini) protocol that selects for blunt-ended sites. They reported the relative frequency of these rDNA DSBs within defined co-ordinate 'windows' of varying size and made these data (as well as the relevant 'raw' sequencing information) available to the public (Tchurikov et al., 2015b). Assay designs targeting rDNA DSB hotspots will benefit greatly from the publication of break sites at greater resolution. Here, we re-analyse public RAFT data and make available rDNA DSB co-ordinates to the single-nucleotide level.

  11. 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.

  12. 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.

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

    SciTech Connect

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

    1991-05-15

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

  14. 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.

  15. Homology Requirements for Double-Strand Break-Mediated Recombination in a Phage λ-Td Intron Model System

    PubMed Central

    Parker, M. M.; Court, D. A.; Preiter, K.; Belfort, M.

    1996-01-01

    Many group I introns encode endonucleases that promote intron homing by initiating a double-strand break-mediated homologous recombination event. A td intron-phage λ model system was developed to analyze exon homology effects on intron homing and determine the role of the λ 5'-3' exonuclease complex (Redαβ) in the repair event. Efficient intron homing depended on exon lengths in the 35- to 50-bp range, although homing levels remained significantly elevated above nonbreak-mediated recombination with as little as 10 bp of flanking homology. Although precise intron insertion was demonstrated with extremely limiting exon homology, the complete absence of one exon produced illegitimate events on the side of heterology. Interestingly, intron inheritance was unaffected by the presence of extensive heterology at the double-strand break in wild-type λ, provided that sufficient homology between donor and recipient was present distal to the heterologous sequences. However, these events involving heterologous ends were absolutely dependent on an intact Red exonuclease system. Together these results indicate that heterologous sequences can participate in double-strand break-mediated repair and imply that intron transposition to heteroallelic sites might occur at break sites within regions of limited or no homology. PMID:8807281

  16. 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.

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

    PubMed Central

    Jette, Nicholas; Lees-Miller, Susan P.

    2015-01-01

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

  18. Low-Energy Ion-Species-Dependent Induction of DNA Double-Strand Breaks: Ion Energy and Fluence Thresholds.

    PubMed

    Thopan, Prutchayawoot; Yu, Liangdeng; Brown, Ian G; Tippawan, Udomrat

    2017-10-01

    The goal of this study was to determine the critical ion-radiation conditions under which heavy ion beams can induce DNA double-strand breaks. Helium, nitrogen and argon-ion beams in the energy range of 20 eV to 2 keV were used to irradiate naked DNA plasmid pGFP to fluences of 1, 2 and 4 × 10(15) ions/cm(2). The topological forms of DNA were subsequently analyzed using gel electrophoresis. The DNA forms were changed from the original supercoiled to damaged relaxed and linear forms, depending on the ion mass, energy, fluence and inertia. We found ion energy and fluence thresholds above which direct double-strand breaks can occur. The threshold is discussed in terms of the areal ion-energy density and the cross-section.

  19. Radiation-induced double-strand breaks require ATM but not Artemis for homologous recombination during S-phase

    PubMed Central

    Köcher, Sabrina; Rieckmann, Thorsten; Rohaly, Gabor; Mansour, Wael Y.; Dikomey, Ekkehard; Dornreiter, Irena; Dahm-Daphi, Jochen

    2012-01-01

    Double-strand breaks (DSBs) are repaired by two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The endonuclease Artemis and the PIK kinase Ataxia-Telangiectasia Mutated (ATM), mutated in prominent human radiosensitivity syndromes, are essential for repairing a subset of DSBs via NHEJ in G1 and HR in G2. Both proteins have been implicated in DNA end resection, a mandatory step preceding homology search and strand pairing in HR. Here, we show that during S-phase Artemis but not ATM is dispensable for HR of radiation-induced DSBs. In replicating AT cells, numerous Rad51 foci form gradually, indicating a Rad51 recruitment process that is independent of ATM-mediated end resection. Those DSBs decorated with Rad51 persisted through S- and G2-phase indicating incomplete HR resulting in unrepaired DSBs and a pronounced G2 arrest. We demonstrate that in AT cells loading of Rad51 depends on functional ATR/Chk1. The ATR-dependent checkpoint response is most likely activated when the replication fork encounters radiation-induced single-strand breaks leading to generation of long stretches of single-stranded DNA. Together, these results provide new insight into the role of ATM for initiation and completion of HR during S- and G2-phase. The DSB repair defect during S-phase significantly contributes to the radiosensitivity of AT cells. PMID:22730303

  20. DNA double-strand breaks caused by new and contemporary endodontic sealers.

    PubMed

    Eldeniz, A U; Shehata, M; Högg, C; Reichl, F X

    2016-12-01

    To investigate the cytotoxicity and genotoxicity of a new silicate-based BioRoot RCS(®) sealer in comparison with contemporary sealers. A periodontal ligament cell line using lentiviral gene transfer of human telomerase reverse transcriptase (hTERT) was used and exposed to subtoxic concentrations of 24-h eluates from two epoxy resin-based (AH Plus Jet(®) and Acroseal(®) ), four various methacrylate-based endodontic sealers (EndoREZ(®) , RealSeal(®) , RealSeal SE(®) and Hybrid Root SEAL(®) ) and three silicate-based sealers (BioRoot RCS(®) , iRootSP(®) and MTA Fillapex(®) ). The XTT-based cell viability assay was used for cytotoxicity screening of materials. The γ-H2AX assay was used for genotoxicity screening. In the γ-H2AX immunofluorescence assay, PDL-hTERT cells were exposed to eluates of the substances for 6 h and DNA double-strand breaks (DSB) were detected microscopically. Induced foci represented DSBs, which can induce ATM-dependent phosphorylation of the histone H2AX. The statistical significance of the differences between the experimental groups was compared using the Student's t-test (P < 0.05). The cytotoxicity of the 24-h eluates could be ranked in the following order: Hybrid Root SEAL(®) >RealSeal(®) >Acroseal(®) >RealSeal SE(®) ≥ AH Plus Jet(®) > EndoREZ(®) >MTA Fillapex(®) > iRoot SP(®) >BioRoot RCS(®) . In negative controls (cells which received medium only) 4.08 ± 0.53 DSB foci (mean ± SEM) whilst in positive controls 10.76 ± 4.05 DSB foci/cell were found. BioRoot RCS(®) and RealSeal SE(®) exhibited significant differences in foci formation at 1/3 EC50 compared with their 1/10 EC50 concentration (P < 0.05). Both concentrations (1/10 and 1/3 of EC50) of AH Plus Jet(®) , Acroseal(®) , RealSeal(®) and MTA Fillapex(®) sealers were not significantly different when compared with the medium control (P < 0.05). New BioRoot RCS(®) was not toxic whilst Hybrid Root SEAL(®) demonstrated more toxicity and DNA

  1. 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

  2. AAV-Mediated Gene Editing via Double-Strand Break Repair

    PubMed Central

    Hirsch, Matthew L.; Samulski, R. Jude

    2016-01-01

    Traditionally, the ability to edit the mammalian genome was inhibited by the inherent low efficiency of homologous recombination (HR; approximately <1 in a million events) and the inability to deliver DNA efficiently to dividing and non-dividing cells/tissue. Despite these limitations, creative selections designed over 20 years ago, clearly demonstrated the powerful implications of gene knock-in and knockout technology for the genetic engineering of mice (Doetschman et al. Nat 330(6148): 576–578, 1987; Thomas and Capecchi. Cell 51(3): 503–512, 1987). The development and application of recombinant vectors based on adeno-associated virus (rAAV) have helped to overcome both of the initial limitations regarding DNA delivery and the frequency of HR. Considering DNA delivery, rAAV infects non-dividing and dividing cultured cells as well as most tissues in mouse and larger animal models (including humans). At the DNA editing level, rAAV genomes have been reported to increase the frequency of HR several orders of magnitude by serving as the repair substrate (Russell and Hirata. Nat Genet 18(4): 325–330, 1998). However, reports on the ability of rAAV genomes to stimulate HR, compared to plasmid DNA and oligonucleotides, are variable, and many labs have found it necessary to augment the frequency of rAAV-induced HR using site-specific endonucleases (Ellis et al. Gene Ther, 2012; Hirsch et al. Gene Ther 17(9): 1175–1180, 2010; Porteus et al. Mol Cell Biol 23(10): 3558–3565, 2003; Radecke et al. Mol Ther 14(6): 798–808, 2006). In this protocol, we describe a method to perform rAAV-mediated double-strand break (DSB) repair for precise genetic engineering in human cells. PMID:24557911

  3. Different fates of oocytes with DNA double-strand breaks in vitro and in vivo.

    PubMed

    Lin, Fei; Ma, Xue-Shan; Wang, Zhen-Bo; Wang, Zhong-Wei; Luo, Yi-Bo; Huang, Lin; Jiang, Zong-Zhe; Hu, Meng-Wen; Schatten, Heide; Sun, Qing-Yuan

    2014-01-01

    In female mice, despite the presence of slight DNA double-strand breaks (DSBs), fully grown oocytes are able to undergo meiosis resumption as indicated by germinal vesicle breakdown (GVBD); however, severe DNA DSBs do reduce and delay entry into M phase through activation of the DNA damage checkpoint. But little is known about the effect of severe DNA DSBs on the spindle assembly checkpoint (SAC) during oocyte maturation. We showed that nearly no first polar body (PB1) was extruded at 12 h of in vitro maturation (IVM) in severe DNA DSBs oocytes, and the limited number of oocytes with PB1 were actually at telophase. However, about 60% of the severe DNA DSBs oocytes which underwent GVBD at 2 h of IVM released a PB1 at 18 h of IVM and these oocytes did reach the second metaphase (MII) stage. Chromosome spread at MI and MII stages showed that chromosomes fragmented after GVBD in severe DNA DSBs oocytes. The delayed PB1 extrusion was due to the disrupted attachment of microtubules to kinetochores and activation of the SAC. At the same time, misaligned chromosome fragments became obvious at the first metaphase (MI) in severe DNA DSBs oocytes. These data implied that the inactivation of SAC during the metaphase-anaphase transition of first meiosis was independent of chromosome integrity. Next, we induced DNA DSBs in vivo, and found that the number of superovulated oocytes per mouse was significantly reduced; moreover, this treatment increased the percentage of apoptotic oocytes. These results suggest that DNA DSBs oocytes undergo apoptosis in vivo.

  4. 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.

  5. Deficiency in the response to DNA double-strand breaks in mouse early preimplantation embryos

    SciTech Connect

    Yukawa, Masashi; Oda, Shoji; Mitani, Hiroshi; Nagata, Masao; Aoki, Fugaku . E-mail: aokif@k.u-tokyo.ac.jp

    2007-06-29

    DNA double-strand breaks (DSBs) are caused by various environmental stresses, such as ionizing radiation and DNA-damaging agents. When DSBs occur, cell cycle checkpoint mechanisms function to stop the cell cycle until all DSBs are repaired; the phosphorylation of H2AX plays an important role in this process. Mouse preimplantation-stage embryos are hypersensitive to ionizing radiation, and X-irradiated mouse zygotes are arrested at the G2 phase of the first cell cycle. To investigate the mechanisms responding to DNA damage at G2 in mouse preimplantation embryos, we examined G2/M checkpoint and DNA repair mechanisms in these embryos. Most of the one- and two-cell embryos in which DSBs had been induced by {gamma}-irradiation underwent a delay in cleavage and ceased development before the blastocyst stage. In these embryos, phosphorylated H2AX ({gamma}-H2AX) was not detected in the one- or two-cell stages by immunocytochemistry, although it was detected after the two-cell stage during preimplantation development. These results suggest that the G2/M checkpoint and DNA repair mechanisms have insufficient function in one- and two-cell embryos, causing hypersensitivity to {gamma}-irradiation. In addition, phosphorylated ataxia telangiectasia mutated protein and DNA protein kinase catalytic subunits, which phosphorylate H2AX, were detected in the embryos at one- and two-cell stages, as well as at other preimplantation stages, suggesting that the absence of {gamma}-H2AX in one- and two-cell embryos depends on some factor(s) other than these kinases.

  6. Boric Acid Reduces the Formation of DNA Double Strand Breaks and Accelerates Wound Healing Process.

    PubMed

    Tepedelen, Burcu Erbaykent; Soya, Elif; Korkmaz, Mehmet

    2016-12-01

    Boron is absorbed by the digestive and respiratory system, and it was considered that it is converted to boric acid (BA), which was distributed to all tissues above 90 %. The biochemical essentiality of boron element is caused by boric acid because it affects the activity of several enzymes involved in the metabolism. DNA damage repair mechanisms and oxidative stress regulation is quite important in the transition stage from normal to cancerous cells; thus, this study was conducted to investigate the protective effect of boric acid on DNA damage and wound healing in human epithelial cell line. For this purpose, the amount of DNA damage occurred with irinotecan (CPT-11), etoposide (ETP), doxorubicin (Doxo), and H2O2 was determined by immunofluorescence through phosphorylation of H2AX((Ser139)) and pATM((Ser1981)) in the absence and presence of BA. Moreover, the effect of BA on wound healing has been investigated in epithelial cells treated with these agents. Our results demonstrated that H2AX((Ser139)) foci numbers were significantly decreased in the presence of BA while wound healing was accelerated by BA compared to that in the control and only drug-treated cells. Eventually, the results indicate that BA reduced the formation of DNA double strand breaks caused by agents as well as improving the wound healing process. Therefore, we suggest that boric acid has important therapeutical effectiveness and may be used in the treatment of inflammatory diseases where oxidative stress and wound healing process plays an important role.

  7. 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

  8. Estimated yield of double-strand breaks from internal exposure to tritium.

    PubMed

    Chen, Jing

    2012-08-01

    Internal exposure to tritium may result in DNA lesions. Of those, DNA double-strand breaks (DSBs) are believed to be important. However, experimental and computational data of DSBs induction by tritium are very limited. In this study, microdosimetric characteristics of uniformly distributed tritium were determined in dimensions of critical significance in DNA DSBs. Those characteristics were used to identify other particles comparable to tritium in terms of microscopic energy deposition. The yield of DSBs could be strongly dependent on biological systems and cellular environments. After reviewing theoretically predicted and experimentally determined DSB yields available in the literature for low-energy electrons and high-energy protons of comparable microdosimetric characteristics to tritium in the dimensions relevant to DSBs, it is estimated that the average DSB yields of 2.7 × 10(-11), 0.93 × 10(-11), 2.4 × 10(-11) and 1.6 × 10(-11) DSBs Gy(-1) Da(-1) could be reasonable estimates for tritium in plasmid DNAs, yeast cells, Chinese hamster V79 cells and human fibroblasts, respectively. If a biological system is not specified, the DSB yield from tritium exposure can be estimated as (2.3 ± 0.7) × 10(-11) DSBs Gy(-1) Da(-1), which is a simple average over experimentally determined yields of DSBs for low-energy electrons in various biological systems without considerations of variations caused by different techniques used and obvious differences among different biological systems where the DSB yield was measured.

  9. 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.

  10. 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.

  11. A DNA double-strand break kinetic rejoining model based on the local effect model.

    PubMed

    Tommasino, F; Friedrich, T; Scholz, U; Taucher-Scholz, G; Durante, M; Scholz, M

    2013-11-01

    We report here on a DNA double-strand break (DSB) kinetic rejoining model applicable to a wide range of radiation qualities based on the DNA damage pattern predicted by the local effect model (LEM). In the LEM this pattern is derived from the SSB and DSB yields after photon irradiation in combination with an amorphous track structure approach. Together with the assumption of a giant-loop organization to describe the higher order chromatin structure this allows the definition of two different classes of DSB. These classes are defined by the level of clustering on a micrometer scale, i.e., "isolated DSB" (iDSB) are characterized by a single DSB in a giant loop and "clustered DSB" (cDSB) by two or more DSB in a loop. Clustered DSB are assumed to represent a more difficult challenge for the cell repair machinery compared to isolated DSB, and we thus hypothesize here that the fraction of isolated DSB can be identified with the fast component of rejoining, whereas clustered DSB are identified with the slow component of rejoining. The resulting predicted bi-exponential decay functions nicely reproduce the experimental curves of DSB rejoining over time obtained by means of gel electrophoresis elution techniques as reported by different labs, involving different cell types and a wide spectrum of radiation qualities. New experimental data are also presented aimed at investigating the effects of the same ion species accelerated at different energies. The results presented here further support the relevance of the proposed two classes of DSB as a basis for understanding cell response to ion irradiation. Importantly the density of DSB within DNA giant loops of around 2 Mbp size, i.e., on a micrometer scale, is identified as a key parameter for the description of radiation effectiveness.

  12. BRUCE regulates DNA double-strand break response by promoting USP8 deubiquitination of BRIT1.

    PubMed

    Ge, Chunmin; Che, Lixiao; Ren, Jinyu; Pandita, Raj K; Lu, Jing; Li, Kaiyi; Pandita, Tej K; Du, Chunying

    2015-03-17

    The DNA damage response (DDR) is crucial for genomic integrity. BRIT1 (breast cancer susceptibility gene C terminus-repeat inhibitor of human telomerase repeat transcriptase expression), a tumor suppressor and early DDR factor, is recruited to DNA double-strand breaks (DSBs) by phosphorylated H2A histone family, member X (γ-H2AX), where it promotes chromatin relaxation by recruiting the switch/sucrose nonfermentable (SWI-SNF) chromatin remodeler to facilitate DDR. However, regulation of BRIT1 recruitment is not fully understood. The baculovirus IAP repeat (BIR)-containing ubiquitin-conjugating enzyme (BRUCE) is an inhibitor of apoptosis protein (IAP). Here, we report a non-IAP function of BRUCE in the regulation of the BRIT1-SWI-SNF DSB-response pathway and genomic stability. We demonstrate that BRIT1 is K63 ubiquitinated in unstimulated cells and that deubiquitination of BRIT1 is a prerequisite for its recruitment to DSB sites by γ-H2AX. We show mechanistically that BRUCE acts as a scaffold, bridging the ubiquitin-specific peptidase 8 (USP8) and BRIT1 in a complex to coordinate USP8-catalyzed deubiquitination of BRIT1. Loss of BRUCE or USP8 impairs BRIT1 deubiquitination, BRIT1 binding with γ-H2AX, the formation of BRIT1 DNA damage foci, and chromatin relaxation. Moreover, BRUCE-depleted cells display reduced homologous recombination repair, and BRUCE-mutant mice exhibit repair defects and genomic instability. These findings identify BRUCE and USP8 as two hitherto uncharacterized critical DDR regulators and uncover a deubiquitination regulation of BRIT1 assembly at damaged chromatin for efficient DDR and genomic stability.

  13. BRUCE regulates DNA double-strand break response by promoting USP8 deubiquitination of BRIT1

    PubMed Central

    Ge, Chunmin; Che, Lixiao; Ren, Jinyu; Pandita, Raj K.; Lu, Jing; Li, Kaiyi; Pandita, Tej K.; Du, Chunying

    2015-01-01

    The DNA damage response (DDR) is crucial for genomic integrity. BRIT1 (breast cancer susceptibility gene C terminus-repeat inhibitor of human telomerase repeat transcriptase expression), a tumor suppressor and early DDR factor, is recruited to DNA double-strand breaks (DSBs) by phosphorylated H2A histone family, member X (γ-H2AX), where it promotes chromatin relaxation by recruiting the switch/sucrose nonfermentable (SWI–SNF) chromatin remodeler to facilitate DDR. However, regulation of BRIT1 recruitment is not fully understood. The baculovirus IAP repeat (BIR)-containing ubiquitin-conjugating enzyme (BRUCE) is an inhibitor of apoptosis protein (IAP). Here, we report a non-IAP function of BRUCE in the regulation of the BRIT1–SWI–SNF DSB-response pathway and genomic stability. We demonstrate that BRIT1 is K63 ubiquitinated in unstimulated cells and that deubiquitination of BRIT1 is a prerequisite for its recruitment to DSB sites by γ-H2AX. We show mechanistically that BRUCE acts as a scaffold, bridging the ubiquitin-specific peptidase 8 (USP8) and BRIT1 in a complex to coordinate USP8-catalyzed deubiquitination of BRIT1. Loss of BRUCE or USP8 impairs BRIT1 deubiquitination, BRIT1 binding with γ-H2AX, the formation of BRIT1 DNA damage foci, and chromatin relaxation. Moreover, BRUCE-depleted cells display reduced homologous recombination repair, and BRUCE-mutant mice exhibit repair defects and genomic instability. These findings identify BRUCE and USP8 as two hitherto uncharacterized critical DDR regulators and uncover a deubiquitination regulation of BRIT1 assembly at damaged chromatin for efficient DDR and genomic stability. PMID:25733871

  14. The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens

    PubMed Central

    Kamisugi, Yasuko; Whitaker, John W.

    2016-01-01

    The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency. PMID:27537368

  15. Retrospective analysis of double-strand break rejoining data collected using warm-lysis PFGE protocols.

    PubMed

    Ratnayake, R K; Semenenko, V A; Stewart, R D

    2005-06-01

    Sample preparation procedures for the pulsed-field gel electrophoresis (PFGE) assay usually involve a lysis step at temperatures as high as 50 degrees C. During this warm-lysis procedure, multiply damaged sites containing heat-labile sites (HLS) can be converted into double-strand breaks (DSB). Once formed, these DSB cannot be distinguished from the DSB formed directly by ionizing radiation. This paper develops a method to correct DSB estimates for the effects of HLS in warm-lysis protocols. A first-order repair model is used to predict the number of HLS available for conversion into DSB as a function of the time available for repair before initiating warm-lysis. A mathematical expression is derived to separate prompt DSB from those formed through the artefactual conversion of HLS into DSB. The proposed formalism only requires the specification of two adjustable parameters, both of which can be estimated from measured data. Estimates of prompt DSB yields obtained by correcting warm-lysis data are in good agreement with estimates obtained using cold-lysis protocols, which do not include the effect of HLS. The retrospective analyses of two published datasets suggest that corrections for HLS have a substantial impact on DSB yields within the first 20-30 min after irradiation. Bi-exponential fits to the DSB data for Chinese hamster ovary cells suggest that corrections for HLS reduce the half-time for fast DSB rejoining by about 15%, whereas the half-time for the slow DSB rejoining only decreases by 4%. The total DSB yield and the fraction of fast-rejoining DSB decrease by 24 and 38%, respectively, when the correction is applied. The proposed formalism can be used to characterize trends and uncertainties in DSB rejoining kinetics associated with the artefactual conversion of HLS into DSB. The retrospective application of the methodology to warm-lysis data enhances their relevance and usefulness for studies of DSB rejoining kinetics.

  16. 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.

  17. Effects of terminal nonhomology and homeology on double-strand-break-induced gene conversion tract directionality.

    PubMed Central

    Nelson, H H; Sweetser, D B; Nickoloff, J A

    1996-01-01

    Double-strand breaks (DSBs) greatly enhance gene conversion in the yeast Saccharomyces cerevisiae. In prior plasmid x chromosome crosses, conversion tracts were often short ( < 53 bp) and usually extended in only one direction from a DSB in an HO recognition sequence inserted into ura3. To allow fine-structure analysis of short and unidirectional tracts, phenotypically silent markers were introduced at 3- and 6-bp intervals flanking the HO site. These markers, which created a 70-bp homeologous region (71% homology), greatly increased the proportion of bidirectional tracts. Among products with short or unidirectional tracts, 85% were highly directional, converting markers on only one side (the nearest marker being 6 bp from the HO site). A DSB in an HO site insertion creates terminal nonhomologies. The high degree of directionality is a likely consequence of the precise cleavage at homology/nonhomology borders in hybrid DNA by Rad1/10 endonuclease. In contrast, terminal homeology alone yielded mostly unidirectional tracts. Thus, nonhomology flanked by homeology yields primarily bidirectional tracts, but terminal homeology or nonhomology alone yields primarily unidirectional tracts. These results are inconsistent with uni- and bidirectional tracts arising from one- and two-ended invasion mechanisms, respectively, as reduced homology would be expected to favor one-ended events. Tract spectra with terminal homeology alone with similar in RAD1 and rad1 cells, indicating that the high proportion of bidirectional tracts seen with homeology flanking nonhomology is not a consequence of Rad1/10 cleavage at homology/homeology boundaries. Instead, tract directionality appears to reflect the influence of the degree of broken-end homology on mismatch repair. PMID:8649406

  18. Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells.

    PubMed Central

    Taghian, D G; Nickoloff, J A

    1997-01-01

    Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion. PMID:9343400

  19. Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

    PubMed Central

    Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

    2014-01-01

    The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies. PMID:25460012

  20. 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

  1. 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.

  2. Involvement of the nuclear proteasome activator PA28γ in the cellular response to DNA double-strand breaks.

    PubMed

    Levy-Barda, Adva; Lerenthal, Yaniv; Davis, Anthony J; Chung, Young Min; Essers, Jeroen; Shao, Zhengping; van Vliet, Nicole; Chen, David J; Hu, Mickey C-T; Kanaar, Roland; Ziv, Yael; Shiloh, Yosef

    2011-12-15

    The DNA damage response (DDR) is a complex signaling network that leads to damage repair while modulating numerous cellular processes. DNA double-strand breaks (DSBs), a highly cytotoxic DNA lesion, activate this system most vigorously. The DSB response network is orchestrated by the ATM protein kinase, which phosphorylates key players in its various branches. Proteasome-mediated protein degradation plays an important role in the proteome dynamics following DNA damage induction. Here, we identify the nuclear proteasome activator PA28γ (REGγ; PSME3) as a novel DDR player. PA28γ depletion leads to cellular radiomimetic sensitivity and a marked delay in DSB repair. Specifically, PA28γ deficiency abrogates the balance between the two major DSB repair pathways--nonhomologous end-joining and homologous recombination repair. Furthermore, PA28γ is found to be an ATM target, being recruited to the DNA damage sites and required for rapid accumulation of proteasomes at these sites. Our data reveal a novel ATM-PA28γ-proteasome axis of the DDR that is required for timely coordination of DSB repair.

  3. Involvement of the nuclear proteasome activator PA28γ in the cellular response to DNA double-strand breaks

    PubMed Central

    Levy-Barda, Adva; Lerenthal, Yaniv; Davis, Anthony J; Chung, Young Min; Essers, Jeroen; Shao, Zhengping; van Vliet, Nicole; Chen, David J; Hu, Mickey C-T; Kanaar, Roland

    2011-01-01

    The DNA damage response (DDR) is a complex signaling network that leads to damage repair while modulating numerous cellular processes. DNA double-strand breaks (DSBs), a highly cytotoxic DNA lesion, activate this system most vigorously. The DSB response network is orchestrated by the ATM protein kinase, which phosphorylates key players in its various branches. Proteasome-mediated protein degradation plays an important role in the proteome dynamics following DNA damage induction. Here, we identify the nuclear proteasome activator PA28γ (REGγ; PSME3) as a novel DDR player. PA28γ depletion leads to cellular radiomimetic sensitivity and a marked delay in DSB repair. Specifically, PA28γ deficiency abrogates the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair. Furthermore, PA28γ is found to be an ATM target, being recruited to the DNA damage sites and required for rapid accumulation of proteasomes at these sites. Our data reveal a novel ATM-PA28γ-proteasome axis of the DDR that is required for timely coordination of DSB repair. PMID:22134242

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

    PubMed

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

    2014-10-17

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

  5. 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

  6. Non-random distribution of DNA double-strand breaks induced by particle irradiation

    NASA Technical Reports Server (NTRS)

    Lobrich, M.; Cooper, P. K.; Rydberg, B.; Chatterjee, A. (Principal Investigator)

    1996-01-01

    Induction of DNA double-strand breaks (dsbs) in mammalian cells is dependent on the spatial distribution of energy deposition from the ionizing radiation. For high LET particle radiations the primary ionization sites occur in a correlated manner along the track of the particles, while for X-rays these sites are much more randomly distributed throughout the volume of the cell. It can therefore be expected that the distribution of dsbs linearly along the DNA molecule also varies with the type of radiation and the ionization density. Using pulsed-field gel and conventional gel techniques, we measured the size distribution of DNA molecules from irradiated human fibroblasts in the total range of 0.1 kbp-10 Mbp for X-rays and high LET particles (N ions, 97 keV/microns and Fe ions, 150 keV/microns). On a mega base pair scale we applied conventional pulsed-field gel electrophoresis techniques such as measurement of the fraction of DNA released from the well (FAR) and measurement of breakage within a specific NotI restriction fragment (hybridization assay). The induction rate for widely spaced breaks was found to decrease with LET. However, when the entire distribution of radiation-induced fragments was analysed, we detected an excess of fragments with sizes below about 200 kbp for the particles compared with X-irradiation. X-rays are thus more effective than high LET radiations in producing large DNA fragments but less effective in the production of smaller fragments. We determined the total induction rate of dsbs for the three radiations based on a quantitative analysis of all the measured radiation-induced fragments and found that the high LET particles were more efficient than X-rays at inducing dsbs, indicating an increasing total efficiency with LET. Conventional assays that are based only on the measurement of large fragments are therefore misleading when determining total dsb induction rates of high LET particles. The possible biological significance of this non

  7. Biased Gene Conversion in Rhizobium etli Is Caused by Preferential Double-Strand Breaks on One of the Recombining Homologs

    PubMed Central

    Yáñez-Cuna, Fares Osam; Castellanos, Mildred

    2015-01-01

    ABSTRACT Gene conversion, the nonreciprocal transfer of information during homologous recombination, is the main process that maintains identity between members of multigene families. Gene conversion in the nitrogenase (nifH) multigene family of Rhizobium etli was analyzed by using a two-plasmid system, where each plasmid carried a copy of nifH. One of the nifH copies was modified, creating restriction fragment length polymorphisms (RFLPs) spaced along the gene. Once the modified plasmid was introduced into R. etli, selection was done for cointegration with a resident plasmid lacking the RFLPs. Most of the cointegrate molecules harbor gene conversion events, biased toward a gain of RFLPs. This bias may be explained under the double-strand break repair model by proposing that the nifH gene lacking the RFLPs suffers a DNA double-strand break, so the incoming plasmid functions as a template for repairing the homolog on the resident plasmid. To support this proposal, we cloned an SceI site into the nifH homolog that had the RFLPs used for scoring gene conversion. In vivo expression of the meganuclease I-SceI allowed the generation of a double-strand break on this homolog. Upon introduction of this modified plasmid into an R. etli strain lacking I-SceI, biased gene conversion still favored the retention of markers on the incoming plasmid. In contrast, when the recipient strain ectopically expressed I-SceI, a dramatic reversal in gene conversion bias was seen, favoring the preservation of resident sequences. These results show that biased gene conversion is caused by preferential double-strand breaks on one of the recombining homologs. IMPORTANCE In this work, we analyzed gene conversion by using a system that entails horizontal gene transfer followed by homologous recombination in the recipient cell. Most gene conversion events are biased toward the acquisition of the incoming sequences, ranging in size from 120 bp to 800 bp. This bias is due to preferential cutting of

  8. 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.

  9. Formation and rejoining of deoxyribonucleic acid double-strand breaks induced in isolated cell nuclei by antineoplastic intercalating agents.

    PubMed

    Pommier, Y; Schwartz, R E; Kohn, K W; Zwelling, L A

    1984-07-03

    The biochemical characteristics of the formation and disappearance of intercalator-induced DNA double-strand breaks (DSB) were studied in nuclei from mouse leukemia L1210 cells by using filter elution methodology [Bradley, M. O., & Kohn, K.W. (1979) Nucleic Acids Res. 7, 793-804]. The three intercalators used were 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), 5-iminodaunorubicin (5-ID), and ellipticine. These compounds differ in that they produced predominantly DNA single-strand breaks (SSB) (m-AMSA) or predominantly DNA double-strand breaks (ellipticine) or a mixture of both SSB and DSB (5-ID) in whole cells. In isolated nuclei, each intercalator produced DSB at a frequency comparable to that which is produced in whole cells. Moreover, these DNA breaks reversed within 30 min after drug removal. It thus appeared that neither ATP nor other nucleotides were necessary for intercalator-dependent DNA nicking-closing reactions. The formation of the intercalator-induced DSB was reduced at ice temperature. Break formation was also reduced in the absence of magnesium, at a pH above 6.4 and at NaCl concentrations above 200 mM. In the presence of ATP and ATP analogues, the intercalator-induced cleavage was enhanced. These results suggest that the intercalator-induced DSB are enzymatically mediated and that the enzymes involved in these reactions can catalyze DNA double-strand cleavage and rejoining in the absence of ATP, although the occupancy of an ATP binding site might convert the enzyme to a form more reactive to intercalators. Three inhibitors of DNA topoisomerase II--novobiocin, nalidixic acid, and norfloxacin--reduced the formation of DNA strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)

  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. Do Exogenous DNA Double-Strand Breaks Change Incomplete Synapsis and Chiasma Localization in the Grasshopper Stethophyma grossum?

    PubMed Central

    2016-01-01

    Meiotic recombination occurs as a programmed event that initiates by the formation of DNA double-strand breaks (DSBs) that give rise to the formation of crossovers that are observed as chiasmata. Chiasmata are essential for the accurate chromosome segregation and the generation of new combinations of parental alleles. Some treatments that provoke exogenous DSBs also lead to alterations in the recombination pattern of some species in which full homologous synapsis is achieved at pachytene. We have carried out a similar approach in males of the grasshopper Stethophyma grossum, whose homologues show incomplete synapsis and proximal chiasma localization. After irradiating males with γ rays we have studied the distribution of both the histone variant γ-H2AX and the recombinase RAD51. These proteins are cytological markers of DSBs at early prophase I. We have inferred synaptonemal complex (SC) formation via identification of SMC3 and RAD 21 cohesin subunits. Whereas thick and thin SMC3 filaments would correspond to synapsed and unsynapsed regions, the presence of RAD21 is only restricted to synapsed regions. Results show that irradiated spermatocytes maintain restricted synapsis between homologues. However, the frequency and distribution of chiasmata in metaphase I bivalents is slightly changed and quadrivalents were also observed. These results could be related to the singular nuclear polarization displayed by the spermatocytes of this species. PMID:28005992

  12. Do Exogenous DNA Double-Strand Breaks Change Incomplete Synapsis and Chiasma Localization in the Grasshopper Stethophyma grossum?

    PubMed

    Calvente, Adela; Santos, Juan Luis; Rufas, Julio S

    2016-01-01

    Meiotic recombination occurs as a programmed event that initiates by the formation of DNA double-strand breaks (DSBs) that give rise to the formation of crossovers that are observed as chiasmata. Chiasmata are essential for the accurate chromosome segregation and the generation of new combinations of parental alleles. Some treatments that provoke exogenous DSBs also lead to alterations in the recombination pattern of some species in which full homologous synapsis is achieved at pachytene. We have carried out a similar approach in males of the grasshopper Stethophyma grossum, whose homologues show incomplete synapsis and proximal chiasma localization. After irradiating males with γ rays we have studied the distribution of both the histone variant γ-H2AX and the recombinase RAD51. These proteins are cytological markers of DSBs at early prophase I. We have inferred synaptonemal complex (SC) formation via identification of SMC3 and RAD 21 cohesin subunits. Whereas thick and thin SMC3 filaments would correspond to synapsed and unsynapsed regions, the presence of RAD21 is only restricted to synapsed regions. Results show that irradiated spermatocytes maintain restricted synapsis between homologues. However, the frequency and distribution of chiasmata in metaphase I bivalents is slightly changed and quadrivalents were also observed. These results could be related to the singular nuclear polarization displayed by the spermatocytes of this species.

  13. 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

  14. Post-meiotic DNA double-strand breaks occur in Tetrahymena, and require Topoisomerase II and Spo11

    PubMed Central

    Akematsu, Takahiko; Fukuda, Yasuhiro; Garg, Jyoti; Fillingham, Jeffrey S; Pearlman, Ronald E; Loidl, Josef

    2017-01-01

    Based on observations of markers for DNA lesions, such as phosphorylated histone H2AX (γH2AX) and open DNA ends, it has been suggested that post-meiotic DNA double-strand breaks (PM-DSBs) enable chromatin remodeling during animal spermiogenesis. However, the existence of PM-DSBs is unconfirmed, and the mechanism responsible for their formation is unclear. Here, we report the first direct observation of programmed PM-DSBs via the electrophoretic separation of DSB-generated DNA fragments in the ciliate Tetrahymena thermophila. These PM-DSBs are accompanied by switching from a heterochromatic to euchromatic chromatin structure in the haploid pronucleus. Both a topoisomerase II paralog with exclusive pronuclear expression and Spo11 are prerequisites for PM-DSB induction. Reduced PM-DSB induction blocks euchromatin formation, characterized by histone H3K56 acetylation, leading to a failure in gametic nuclei production. We propose that PM-DSBs are responsible for histone replacement during the reprogramming of generative to undifferentiated progeny nuclei. DOI: http://dx.doi.org/10.7554/eLife.26176.001 PMID:28621664

  15. The Democratization of Gene Editing: Insights from site-specific cleavage and double-strand break repair

    PubMed Central

    Jasin, Maria; Haber, James E.

    2017-01-01

    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 occur by homologous recombination that 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. PMID:27261202

  16. 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.

  17. 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.

  18. 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

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

    PubMed Central

    Pike, Brietta L.; Heierhorst, Jörg

    2007-01-01

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

  20. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    SciTech Connect

    Fukumoto, Yasunori Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto

    2014-09-26

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.

  1. 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

  2. Myricetin induces apoptosis via endoplasmic reticulum stress and DNA double-strand breaks in human ovarian cancer cells

    PubMed Central

    XU, YE; XIE, QI; WU, SHAOHUA; YI, DAN; YU, YANG; LIU, SHIBING; LI, SONGYAN; LI, ZHIXIN

    2016-01-01

    The mechanisms underlying myricetin-induced cancer cell apoptosis remain to be elucidated. Certain previous studies have shown that myricetin induces apoptosis through the mitochondrial pathway. Apoptosis, however, can also be induced by other classical pathways, including endoplasmic reticulum (ER) stress and DNA double-strand breaks (DSBs). The aim of the present study was to assess whether these two apoptotic pathways are involved in myricetin-induced cell death in SKOV3 ovarian cancer cells. The results revealed that treatment with myricetin inhibited viability of SKOV3 cells in a dose-dependent manner. Myricetin induced nuclear chromatin condensation and fragmentation, and also upregulated the protein levels of active caspase 3 in a time-dependent manner. In addition, myricetin upregulated ER stress-associated proteins, glucose-regulated protein-78 and C/EBP homologous protein in SKOV3 cells. Phosphorylation of H2AX, a marker of DNA DSBs, was revealed to be upregulated in myricetin-treated cells. The data indicated that myricetin induces DNA DSBs and ER stress, which leads to apoptosis in SKOV3 cells. PMID:26782830

  3. Repair of ionizing radiation-induced DNA double strand breaks by non-homologous end-joining

    PubMed Central

    Mahaney, Brandi L.; Meek, Katheryn; Lees-Miller, Susan P.

    2010-01-01

    DNA double strand breaks (DSBs) are considered the most cytotoxic type of DNA lesion. They can be introduced by external sources such as ionizing radiation (IR), by chemotherapeutic drugs such as topoisomerase poisons and by normal biological processes such as V(D)J recombination. If left unrepaired, DSBs can cause cell death. If misrepaired, DSBs may lead to chromosomal translocations and genomic instability. One of the major pathways for the repair of IR-induced DSBs in mammalian cells is non-homologous end-joining (NHEJ). The main proteins required for NHEJ in mammalian cells are the Ku heterodimer, the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Artemis, XRCC4, DNA ligase IV and XLF (XRCC4-like factor, also called Cernunnos). Additional proteins including DNA polymerases μ and λ, polynucleotide kinase (PNK) and the Werner’s Syndrome helicase (WRN) may also play a role. Here, we will review our current understanding of the mechanism of NHEJ in mammalian cells and discuss the roles of DNA-PKcs and DNA-PK-mediated phosphorylation in NHEJ. PMID:19133841

  4. Irreparable complex DNA double-strand breaks induce chromosome breakage in organotypic three-dimensional human lung epithelial cell culture

    PubMed Central

    Asaithamby, Aroumougame; Hu, Burong; Delgado, Oliver; Ding, Liang-Hao; Story, Michael D.; Minna, John D.; Shay, Jerry W.; Chen, David J.

    2011-01-01

    DNA damage and consequent mutations initiate the multistep carcinogenic process. Differentiated cells have a reduced capacity to repair DNA lesions, but the biological impact of unrepaired DNA lesions in differentiated lung epithelial cells is unclear. Here, we used a novel organotypic human lung three-dimensional (3D) model to investigate the biological significance of unrepaired DNA lesions in differentiated lung epithelial cells. We showed, consistent with existing notions that the kinetics of loss of simple double-strand breaks (DSBs) were significantly reduced in organotypic 3D culture compared to kinetics of repair in two-dimensional (2D) culture. Strikingly, we found that, unlike simple DSBs, a majority of complex DNA lesions were irreparable in organotypic 3D culture. Levels of expression of multiple DNA damage repair pathway genes were significantly reduced in the organotypic 3D culture compared with those in 2D culture providing molecular evidence for the defective DNA damage repair in organotypic culture. Further, when differentiated cells with unrepaired DNA lesions re-entered the cell cycle, they manifested a spectrum of gross-chromosomal aberrations in mitosis. Our data suggest that downregulation of multiple DNA repair pathway genes in differentiated cells renders them vulnerable to DSBs, promoting genome instability that may lead to carcinogenesis. PMID:21421565

  5. Simultaneous labeling of single- and double-strand DNA breaks by DNA breakage detection-FISH (DBD-FISH).

    PubMed

    Fernández, José Luis; Cajigal, Dioleyda; Gosálvez, Jaime

    2011-01-01

    DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) permits simultaneous and selective labeling of single- and double-strand DNA breaks in individual cells, either in the whole genome or within specific DNA sequences. In this technique, cells are embedded into agarose microgels, lysed and subjected to electrophoresis under nondenaturing conditions. Subsequently, the produced "comets" are exposed to a controlled denaturation step which transforms DNA breaks into single-stranded DNA regions, detected by hybridization with whole genome fluorescent probes or the probes to specific DNA sequences. This makes possible a targeted analysis of various chromatin areas for the presence of DNA breaks. The migration length of the DBD-FISH signal is proportional to the number of double strand breaks, whereas its fluorescence intensity depends on numbers of single-strand breaks.The detailed protocol for detection of two types of DNA breaks produced by ionizing radiation is presented. The technique can be used to determine intragenomic and intercellular heterogeneity in the induction and repair of DNA damage.

  6. DNA double strand break (DSB) induction and cell survival in iodine-enhanced computed tomography (CT)

    NASA Astrophysics Data System (ADS)

    Streitmatter, Seth W.; Stewart, Robert D.; Jenkins, Peter A.; Jevremovic, Tatjana

    2017-08-01

    A multi-scale Monte Carlo model is proposed to assess the dosimetric and biological impact of iodine-based contrast agents commonly used in computed tomography. As presented, the model integrates the general purpose MCNP6 code system for larger-scale radiation transport and dose assessment with the Monte Carlo damage simulation to determine the sub-cellular characteristics and spatial distribution of initial DNA damage. The repair-misrepair-fixation model is then used to relate DNA double strand break (DSB) induction to reproductive cell death. Comparisons of measured and modeled changes in reproductive cell survival for ultrasoft characteristic k-shell x-rays (0.25-4.55 keV) up to orthovoltage (200-500 kVp) x-rays indicate that the relative biological effectiveness (RBE) for DSB induction is within a few percent of the RBE for cell survival. Because of the very short range of secondary electrons produced by low energy x-ray interactions with contrast agents, the concentration and subcellular distribution of iodine within and near cellular targets have a significant impact on the estimated absorbed dose and number of DSB produced in the cell nucleus. For some plausible models of the cell-level distribution of contrast agent, the model predicts an increase in RBE-weighted dose (RWD) for the endpoint of DSB induction of 1.22-1.40 for a 5-10 mg ml-1 iodine concentration in blood compared to an RWD increase of 1.07  ±  0.19 from a recent clinical trial. The modeled RWD of 2.58  ±  0.03 is also in good agreement with the measured RWD of 2.3  ±  0.5 for an iodine concentration of 50 mg ml-1 relative to no iodine. The good agreement between modeled and measured DSB and cell survival estimates provides some confidence that the presented model can be used to accurately assess biological dose for other concentrations of the same or different contrast agents.

  7. A new method to efficiently induce a site-specific double-strand break in the fission yeast Schizosaccharomyces pombe.

    PubMed

    Sunder, Sham; Greeson-Lott, Nikole T; Runge, Kurt W; Sanders, Steven L

    2012-07-01

    Double-strand DNA breaks are a serious threat to cellular viability and yeast systems have proved invaluable in helping to understand how these potentially toxic lesions are sensed and repaired. An important method to study the processing of DNA breaks in the budding yeast Saccharomyces cerevisiae is to introduce a unique double-strand break into the genome by regulating the expression of the site-specific HO endonuclease with a galactose inducible promoter. Variations of the HO site-specific DSB assay have been adapted to many organisms, but the methodology has seen only limited use in the fission yeast Schizosaccharomyces pombe because of the lack of a promoter capable of inducing endonuclease expression on a relatively short time scale (~1 h). We have overcome this limitation by developing a new assay in which expression of the homing endonuclease I-PpoI is tightly regulated with a tetracycline-inducible promoter. We show that induction of the I-PpoI endonuclease produces rapid cutting of a defined cleavage site (> 80% after 1 h), efficient cell cycle arrest and significant accumulation of the checkpoint protein Crb2 at break-adjacent regions in a manner that is analogous to published findings with DSBs produced by an acute exposure to ionizing irradiation. This assay provides an important new tool for the fission yeast community and, because many aspects of mammalian chromatin organization have been well-conserved in Sz. pombe but not in S. cerevisiae, also offers an attractive system to decipher the role of chromatin structure in modulating the repair of double-stranded DNA breaks. Copyright © 2012 John Wiley & Sons, Ltd.

  8. Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR.

    PubMed

    Singh, Sheetal; Shih, Shyh-Jen; Vaughan, Andrew T M

    2014-01-01

    Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter- and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks and rearrangements introduced into any identifiable genomic location. We have also applied parallel sequencing for the high-throughput analysis of inverse PCR products to facilitate the unbiased recording of all rearrangements located at a specific genomic location.

  9. Hypermutability of Damaged Single-Strand DNA Formed at Double-Strand Breaks and Uncapped Telomeres in Yeast Saccharomyces cerevisiae

    PubMed Central

    Yang, Yong; Sterling, Joan; Storici, Francesca; Resnick, Michael A.; Gordenin, Dmitry A.

    2008-01-01

    The major DNA repair pathways operate on damage in double-strand DNA because they use the intact strand as a template after damage removal. Therefore, lesions in transient single-strand stretches of chromosomal DNA are expected to be especially threatening to genome stability. To test this hypothesis, we designed systems in budding yeast that could generate many kilobases of persistent single-strand DNA next to double-strand breaks or uncapped telomeres. The systems allowed controlled restoration to the double-strand state after applying DNA damage. We found that lesions induced by UV-light and methyl methanesulfonate can be tolerated in long single-strand regions and are hypermutagenic. The hypermutability required PCNA monoubiquitination and was largely attributable to translesion synthesis by the error-prone DNA polymerase ζ. In support of multiple lesions in single-strand DNA being a source of hypermutability, analysis of the UV-induced mutants revealed strong strand-specific bias and unexpectedly high frequency of alleles with widely separated multiple mutations scattered over several kilobases. Hypermutability and multiple mutations associated with lesions in transient stretches of long single-strand DNA may be a source of carcinogenesis and provide selective advantage in adaptive evolution. PMID:19023402

  10. Cohesin promotes the repair of ionizing radiation-induced DNA double-strand breaks in replicated chromatin

    PubMed Central

    Bauerschmidt, Christina; Arrichiello, Cecilia; Burdak-Rothkamm, Susanne; Woodcock, Michael; Hill, Mark A.; Stevens, David L.; Rothkamm, Kai

    2010-01-01

    The cohesin protein complex holds sister chromatids together after synthesis until mitosis. It also contributes to post-replicative DNA repair in yeast and higher eukaryotes and accumulates at sites of laser-induced damage in human cells. Our goal was to determine whether the cohesin subunits SMC1 and Rad21 contribute to DNA double-strand break repair in X-irradiated human cells in the G2 phase of the cell cycle. RNA interference-mediated depletion of SMC1 sensitized HeLa cells to X-rays. Repair of radiation-induced DNA double-strand breaks, measured by γH2AX/53BP1 foci analysis, was slower in SMC1- or Rad21-depleted cells than in controls in G2 but not in G1. Inhibition of the DNA damage kinase DNA-PK, but not ATM, further inhibited foci loss in cohesin-depleted cells in G2. SMC1 depletion had no effect on DNA single-strand break repair in either G1 or late S/G2. Rad21 and SMC1 were recruited to sites of X-ray-induced DNA damage in G2-phase cells, but not in G1, and only when DNA damage was concentrated in subnuclear stripes, generated by partially shielded ultrasoft X-rays. Our results suggest that the cohesin complex contributes to cell survival by promoting the repair of radiation-induced DNA double-strand breaks in G2-phase cells in an ATM-dependent pathway. PMID:19906707

  11. scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair

    SciTech Connect

    Biedermann, K.A.; Sun, J.R.; Giaccia, A.J.; Tosto, L.M.; Brown, J.M. )

    1991-02-15

    C.B-17 severe combined immunodeficient (scid) mice carry the scid mutation and are severely deficient in both T cell- and B cell-mediated immunity, apparently as a result of defective V(D)J joining of the immunoglobulin and T-cell receptor gene elements. In the present studies, we have defined the tissue, cellular, and molecular basis of another characteristic of these mice: their hypersensitivity to ionizing radiation. Bone marrow stem cells, intestinal crypt cells, and epithelial skin cells from scid mice are 2- to 3-fold more sensitive when irradiated in situ than are congenic BALB/c or C.B-17 controls. Two independently isolated embryo fibroblastic scid mouse cell lines display similar hypersensitivities to gamma-rays. In addition, these cell lines are sensitive to cell killing by bleomycin, which also produces DNA strand breaks, but not by the DNA crosslinking agent mitomycin C or UV irradiation. Measurement of the rejoining of gamma-ray-induced DNA double-strand breaks by pulsed-field gel electrophoresis indicates that these animals are defective in this repair system. This suggests that the gamma-ray sensitivity of the scid mouse fibroblasts could be the result of reduced repair of DNA double-strand breaks. Therefore, a common factor may participate in both the repair of DNA double-strand breaks as well as V(D)J rejoining during lymphocyte development. This murine autosomal recessive mutation should prove extremely useful in fundamental studies of radiation-induced DNA damage and repair.

  12. Chiral symmetry breaking of a double-stranded helical chain through bend-writhe coupling

    NASA Astrophysics Data System (ADS)

    Yanao, Tomohiro; Yoshikawa, Kenichi

    2014-06-01

    This paper explores asymmetric elasticity of a double-stranded helical chain, which serves as a minimal model of biopolymers. The model consists of two elastic chains that mutually intertwine in a right-handed manner, forming a double-stranded helix. A simple numerical experiment for structural relaxation, which reduces the total elastic energy of the model monotonically without thermal fluctuations, reveals possible asymmetric elasticity inherent in the helical chain. It is first shown that a short segment of the double-stranded helical chain has a tendency to unwind when it is bent. It is also shown that a short segment of the helical chain has a tendency to writhe in the left direction upon bending. This tendency gives rise to a propensity for a longer segment of the chain to form a left-handed superhelix spontaneously upon bending. Finally, this propensity of the helical chain to form a left-handed superhelix is proposed to be a possible origin of the uniform left-handed wrapping of DNA around nucleosome core particles in nature. The results presented here could provide deeper insights into the roles and significance of helical chirality of biopolymers.

  13. What fraction of DNA double-strand breaks produced by the direct effect is accounted for by radical pairs?

    PubMed

    Peoples, Anita R; Mercer, Kermit R; Bernhard, William A

    2010-07-22

    The purpose of this investigation was to determine what fraction of double strand breaks (dsb's), generated by the direct effect of ionizing radiation on DNA, can be accounted for by radical pairs. A radical pair is defined as two radicals trapped within a separation distance of <3 nm. Q-band EPR was used to measure the yield of radical pairs in calf thymus DNA films X-irradiated at 4 K. The EPR spectrum of DNA showed no evidence of radical pairs. To determine the relative sensitivity for radical pair detection via Q-band EPR, we measured the yield of radical pairs in single crystals of thymine, G(rp-Thy). Under the same conditions employed for DNA, G(rp-Thy) was approximately 8 nmol/J. The value of G(rp-Thy), in conjunction with the measured signal-to-noise, was used to calculate an upper limit for the yield of radical pairs in DNA, G(max)(rp-DNA) < 0.7-1.4 nmol/J. The upper limit, G(max)(rp-DNA), was compared with the yield of dsb's, G(total)(dsb) = 10 nmol/J, previously measured in pUC18 DNA films by Purkayastha, S.; Milligan, J. R.; Bernhard, W. A. Radiat. Res. 2007, 168, 357. We found that G(total)(dsb) > 2 x G(max)(rp-DNA), implying that a significant fraction of dsb's were not derived from a pair of trappable radicals. At least one of the two precursors needed to form a dsb was a diamagnetic (molecular) product. The hypothesis is that EPR silent lesions are formed through a molecular pathway. For example, a two-electron oxidation of deoxyribose would result in a deoxyribose carbocation intermediate that ultimately leads to a strand break.

  14. Norfloxacin-induced DNA gyrase cleavage complexes block Escherichia coli replication forks, causing double-stranded breaks in vivo

    PubMed Central

    Pohlhaus, Jennifer Reineke; Kreuzer, Kenneth N.

    2005-01-01

    Summary Antibacterial quinolones inhibit type II DNA topoisomerases by stabilizing covalent topoisomerase-DNA cleavage complexes, which are apparently transformed into double-stranded breaks by cellular processes such as replication. We used plasmid pBR322 and two-dimensional agarose gel electrophoresis to examine the collision of replication forks with quinolone-induced gyrase-DNA cleavage complexes in Escherichia coli. Restriction endonuclease-digested DNA exhibited a bubble arc with discrete spots, indicating that replication forks had been stalled. The most prominent spot depended upon the strong gyrase binding site of pBR322, providing direct evidence that quinolone-induced cleavage complexes block bacterial replication forks in vivo. We differentiated between stalled forks that do or do not contain bound cleavage complex by extracting DNA under different conditions. Resealing conditions allow gyrase to efficiently reseal the transient breaks within cleavage complexes, while cleavage conditions cause the latent breaks to be revealed. These experiments showed that some stalled forks did not contain a cleavage complex, implying that gyrase had dissociated in vivo and yet the fork had not restarted at the time of DNA isolation. Additionally, some branched plasmid DNA isolated under resealing conditions nonetheless contained broken DNA ends. We discuss a model for the creation of double-stranded breaks by an indirect mechanism after quinolone treatment. PMID:15916595

  15. Analysis of DNA Double-Strand Breaks and Cytotoxicity after 7 Tesla Magnetic Resonance Imaging of Isolated Human Lymphocytes

    PubMed Central

    Guttek, Karina; Hartig, Roland; Godenschweger, Frank; Roggenbuck, Dirk; Ricke, Jens; Reinhold, Dirk; Speck, Oliver

    2015-01-01

    The global use of magnetic resonance imaging (MRI) is constantly growing and the field strengths increasing. Yet, only little data about harmful biological effects caused by MRI exposure are available and published research analyzing the impact of MRI on DNA integrity reported controversial results. This in vitro study aimed to investigate the genotoxic and cytotoxic potential of 7 T ultra-high-field MRI on isolated human peripheral blood mononuclear cells. Hence, unstimulated mononuclear blood cells were exposed to 7 T static magnetic field alone or in combination with maximum permissible imaging gradients and radiofrequency pulses as well as to ionizing radiation during computed tomography and γ-ray exposure. DNA double-strand breaks were quantified by flow cytometry and automated microscopy analysis of immunofluorescence stained γH2AX. Cytotoxicity was studied by CellTiter-Blue viability assay and [3H]-thymidine proliferation assay. Exposure of unstimulated mononuclear blood cells to 7 T static magnetic field alone or combined with varying gradient magnetic fields and pulsed radiofrequency fields did not induce DNA double-strand breaks, whereas irradiation with X- and γ-rays led to a dose-dependent induction of γH2AX foci. The viability assay revealed a time- and dose-dependent decrease in metabolic activity only among samples exposed to γ-radiation. Further, there was no evidence for altered proliferation response after cells were exposed to 7 T MRI or low doses of ionizing radiation (≤ 0.2 Gy). These findings confirm the acceptance of MRI as a safe non-invasive diagnostic imaging tool, but whether MRI can induce other types of DNA lesions or DNA double-strand breaks during altered conditions still needs to be investigated. PMID:26176601

  16. Sensitivity of peripheral blood lymphocytes of pilots and astronauts to gamma-radiation: induction of double-stranded DNA breaks.

    PubMed

    Vorobyova, N Yu; Osipova, A N; Pelevina, I I

    2007-10-01

    The levels of DNA breaks before and after in vitro irradiation (1 Gy) of lymphocytes from 17 donors, 41 pilots, and 8 astronauts were studied by comet assay. Seventeen donors. 41 pilots, and 8 astronauts were examined. The flights augmented individual differences in the levels of DNA breaks in blood lymphocytes and in the severity of injuries inflicted by radiation exposure to lymphocyte DNA. Dispersions in the distribution of the initial levels of DNA breaks in pilots and astronauts differed significantly from the control according to Fisher's F test. The dispersion of distribution of the levels of double-stranded DNA breaks after in vitro irradiation in the group of pilots also differed significantly from the control distribution. These results necessitate evaluation of individual sensitivity to the mission conditions during medical selection.

  17. Detection and repair of ionizing radiation-induced DNA double strand breaks: new developments in nonhomologous end joining.

    PubMed

    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. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. 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.

  19. Two-Tailed Comet Assay (2T-Comet): Simultaneous Detection of DNA Single and Double Strand Breaks.

    PubMed

    Cortés-Gutiérrez, Elva I; Fernández, José Luis; Dávila-Rodríguez, Martha I; López-Fernández, Carmen; Gosálvez, Jaime

    2017-01-01

    A modification of the original comet assay was developed for the simultaneous evaluation of DNA single strand breaks (SSBs) and double strand breaks (DSBs) in human spermatozoa. The two-dimensional perpendicular tail comet assay (2T-comet) combines non-denaturing and denaturant conditions to the same sperm nucleoid. In this case, the species-specific deproteinized sperm is first subjected to an electrophoretic field under non-denaturing conditions to mobilize isolated free discrete DNA fragments produced from DSBs; this is then followed by a second electrophoresis running perpendicular to the first one but under alkaline conditions to produce DNA denaturation, exposing SSBs on the same linear DNA chain or DNA fragments flanked by DSBs. This procedure results in a two dimensional comet tail emerging from the core where two types of original DNA affected molecule can be simultaneously discriminated. The 2T-comet is a fast, sensitive, and reliable procedure to distinguish between single and double strand DNA damage within the same cell. It is an innovative method for assessing sperm DNA integrity, which has important implications for human fertility and andrological pathology. This technique may be adapted to assess different DNA break types in other species and other cell types.

  20. Trimming of damaged 3' overhangs of DNA double-strand breaks by the Metnase and Artemis endonucleases.

    PubMed

    Mohapatra, Susovan; Yannone, Steven M; Lee, Suk-Hee; Hromas, Robert A; Akopiants, Konstantin; Menon, Vijay; Ramsden, Dale A; Povirk, Lawrence F

    2013-06-01

    Both Metnase and Artemis possess endonuclease activities that trim 3' overhangs of duplex DNA. To assess the potential of these enzymes for facilitating resolution of damaged ends during double-strand break rejoining, substrates bearing a variety of normal and structurally modified 3' overhangs were constructed, and treated either with Metnase or with Artemis plus DNA-dependent protein kinase (DNA-PK). Unlike Artemis, which trims long overhangs to 4-5 bases, cleavage by Metnase was more evenly distributed over the length of the overhang, but with significant sequence dependence. In many substrates, Metnase also induced marked cleavage in the double-stranded region within a few bases of the overhang. Like Artemis, Metnase efficiently trimmed overhangs terminated in 3'-phosphoglycolates (PGs), and in some cases the presence of 3'-PG stimulated cleavage and altered its specificity. The nonplanar base thymine glycol in a 3' overhang severely inhibited cleavage by Metnase in the vicinity of the modified base, while Artemis was less affected. Nevertheless, thymine glycol moieties could be removed by Metnase- or Artemis-mediated cleavage at sites farther from the terminus than the lesion itself. In in vitro end-joining systems based on human cell extracts, addition of Artemis, but not Metnase, effected robust trimming of an unligatable 3'-PG overhang, resulting in a dramatic stimulation of ligase IV- and XLF-dependent end joining. Thus, while both Metnase and Artemis are biochemically capable of resolving a variety of damaged DNA ends for the repair of complex double-strand breaks, Artemis appears to act more efficiently in the context of other nonhomologous end joining proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Trimming of damaged 3′ overhangs of DNA double-strand breaks by the Metnase and Artemis endonucleases

    PubMed Central

    Mohapatra, Susovan; Yannone, Steven M.; Lee, Suk-Hee; Hromas, Robert A.; Akopiants, Konstantin; Menon, Vijay; Ramsden, Dale A.; Povirk, Lawrence F.

    2013-01-01

    Both Metnase and Artemis possess endonuclease activities that trim 3′ overhangs of duplex DNA. To assess the potential of these enzymes for facilitating resolution of damaged ends during double-strand break rejoining, substrates bearing a variety of normal and structurally modified 3′ overhangs were constructed, and treated either with Metnase or with Artemis plus DNA-dependent protein kinase (DNA-PK). Unlike Artemis, which trims long overhangs to 4–5 bases, cleavage by Metnase was more evenly distributed over the length of the overhang, but with significant sequence dependence. In many substrates, Metnase also induced marked cleavage in the double-stranded region within a few bases of the overhang. Like Artemis, Metnase efficiently trimmed overhangs terminated in 3′-phosphoglycolates (PGs), and in some cases the presence of 3′-PG stimulated cleavage and altered its specificity. The nonplanar base thymine glycol in a 3′ overhang severely inhibited cleavage by Metnase in the vicinity of the modified base, while Artemis was less affected. Nevertheless, thymine glycol moieties could be removed by Metnase- or Artemis-mediated cleavage at sites farther from the terminus than the lesion itself. In in vitro end-joining systems based on human cell extracts, addition of Artemis, but not Metnase, effected robust trimming of an unligatable 3′-PG overhang, resulting in a dramatic stimulation of ligase IV- and XLF-dependent end joining. Thus, while both Metnase and Artemis are biochemically capable of resolving a variety of damaged DNA ends for the repair of complex double-strand breaks, Artemis appears to act more efficiently in the context of other nonhomologous end joining proteins. PMID:23602515

  2. Do chromatin changes around a nascent double strand DNA break spread spherically into linearly non-adjacent chromatin?

    PubMed

    Savic, Velibor

    2013-01-01

    In the last decade, a lot has been done in elucidating the sequence of events that occur at the nascent double strand DNA break. Nevertheless, the overall structure formed by the DNA damage response (DDR) factors around the break site, the repair focus, remains poorly understood. Although most of the data presented so far only address events that occur in chromatin in cis around the break, there are strong indications that in mammalian systems it may also occur in trans, analogous to the recent findings showing this if budding yeast. There have been attempts to address the issue but the final proof is still missing due to lack of a proper experimental system. If found to be true, the spatial distribution of DDR factors would have a major impact on the neighboring chromatin both in cis and in trans, significantly affecting local chromatin function; gene transcription and potentially other functions.

  3. 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.

  4. 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

  5. Gamma irradiation induces DNA double-strand breaks in fibroblasts: a model study for the development of biodosimetry

    NASA Astrophysics Data System (ADS)

    Uttayarat, P.; Tangtong, T.; Sukapirom, K.; Boonsirichai, K.

    2015-05-01

    Double-strand breaks (DSBs) of DNAs induced by ionizing radiation can pose detrimental damages on organisms which include genetic instability and cell death. It is necessary to be able to assess health risks associated with irradiation from both accidental and therapeutic exposures in a timely manner for proper medical treatments. This present study showed the first attempt to develop a biodosimetric measure in Thailand based on the quantification of phosphorylated histone H2AX (γ-H2AX) formed at DSB sites with an aim to establish a dose response curve using a two-dimensional (2D) cell culture model. Human dermal fibroblasts were grown into monolayers before irradiated by gamma rays from a Co-60 source in a custom-made lead chamber at doses 0, 0.2, 1, 2 and 4 Gy and a dose rate of 0.21 Gy/min. After 30 min post exposure, γ-H2AX proteins were immunofluorescently labelled for evaluation by confocal microscopy and flow cytometry. The accumulation of phosphorylated γ-H2AX proteins at DSBs appeared as nuclear foci with the most prominent intensity at 4 Gy. Linear regression analysis of flow cytometric data showed a linear response (R2 = 0.9862) of foci intensity in proportion to irradiation dose. In addition, the fraction of cell viability was shown to decrease at higher doses. This technique can be further developed as a quick assessment tool to identify individuals subjected to accidental radiation in parallel to other established biodosimetric measures.

  6. A multistep genomic screen identifies new genes required for repair of DNA double-strand breaks in Saccharomyces cerevisiae.

    PubMed

    McKinney, Jennifer Summers; Sethi, Sunaina; Tripp, Jennifer DeMars; Nguyen, Thuy N; Sanderson, Brian A; Westmoreland, James W; Resnick, Michael A; Lewis, L Kevin

    2013-04-15

    Efficient mechanisms for rejoining of DNA double-strand breaks (DSBs) are vital because misrepair of such lesions leads to mutation, aneuploidy and loss of cell viability. DSB repair is mediated by proteins acting in two major pathways, called homologous recombination and nonhomologous end-joining. Repair efficiency is also modulated by other processes such as sister chromatid cohesion, nucleosome remodeling and DNA damage checkpoints. The total number of genes influencing DSB repair efficiency is unknown. To identify new yeast genes affecting DSB repair, genes linked to gamma radiation resistance in previous genome-wide surveys were tested for their impact on repair of site-specific DSBs generated by in vivo expression of EcoRI endonuclease. Eight members of the RAD52 group of DNA repair genes (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, MRE11 and XRS2) and 73 additional genes were found to be required for efficient repair of EcoRI-induced DSBs in screens utilizing both MATa and MATα deletion strain libraries. Most mutants were also sensitive to the clastogenic chemicals MMS and bleomycin. Several of the non-RAD52 group genes have previously been linked to DNA repair and over half of the genes affect nuclear processes. Many proteins encoded by the protective genes have previously been shown to associate physically with each other and with known DNA repair proteins in high-throughput proteomics studies. A majority of the proteins (64%) share sequence similarity with human proteins, suggesting that they serve similar functions. We have used a genetic screening approach to detect new genes required for efficient repair of DSBs in Saccharomyces cerevisiae. The findings have spotlighted new genes that are critical for maintenance of genome integrity and are therefore of greatest concern for their potential impact when the corresponding gene orthologs and homologs are inactivated or polymorphic in human cells.

  7. Formation of carcinogenic chromosomal rearrangements in human thyroid cells after induction of double-strand DNA breaks by restriction endonucleases.

    PubMed

    Evdokimova, Viktoria; Gandhi, Manoj; Rayapureddi, Jayanagendra; Stringer, James R; Nikiforov, Yuri E

    2012-06-01

    Ionizing radiation (IR) exposure increases the risk of thyroid cancer and other cancer types. Chromosomal rearrangements, such as RET/PTC, are characteristic features of radiation-associated thyroid cancer and can be induced by radiation in vitro. IR causes double-strand breaks (DSBs), suggesting that such damage leads to RET/PTC, but the rearrangement mechanism has not been established. To study the mechanism, we explored the possibility of inducing RET/PTC by electroporation of restriction endonucleases (REs) into HTori-3 human thyroid cells. We used five REs, which induced DSB in a dose-dependent manner similar to that seen with IR. Although all but one RE caused DSB in one or more of the three genes involved in RET/PTC, rearrangement was detected only in cells electroporated with either PvuII (25 and 100  U) or StuI (100 and 250  U). The predominant rearrangement type was RET/PTC3, which is characteristic of human thyroid cancer arising early after Chernobyl-related radioactive iodine exposure. Both enzymes that produced RET/PTC had restriction sites only in one of the two fusion partner genes. Moreover, the two enzymes that produced RET/PTC had restriction sites present in clusters, which was not the case for RE that failed to induce RET/PTC. In summary, we establish a model of DSB induction by RE and report for the first time the formation of carcinogenic chromosomal rearrangements, predominantly RET/PTC3, as a result of DSB produced by RE. Our data also raise a possibility that RET/PTC rearrangement can be initiated by a complex DSB that is induced in one of the fusion partner genes.

  8. γH2AX as a marker of DNA double strand breaks and genomic instability in human population studies.

    PubMed

    Valdiglesias, Vanessa; Giunta, Simona; Fenech, Michael; Neri, Monica; Bonassi, Stefano

    2013-01-01

    DNA double strand breaks (DSB) are the gravest form of DNA damage in eukaryotic cells. Failure to detect DSB and activate appropriate DNA damage responses can cause genomic instability, leading to tumorigenesis and possibly accelerated aging. Phosphorylated histone H2AX (γH2AX) is used as a biomarker of cellular response to DSB and its potential for monitoring DNA damage and repair in human populations has been explored in this review. A systematic search was conducted in PubMed for articles, in English, on human studies reporting γH2AX as a biomarker of either DNA repair or DNA damage. A total of 68 publications were identified. Thirty-four studies (50.0%) evaluated the effect of medical procedures or treatments on γH2AX levels; 20 (29.4%) monitored γH2AX in specific pathological conditions with a case/control or case/case design; 5 studies (7.4%) evaluated the effect of environmental genotoxic exposures, and 9 (13.2%) were descriptive studies on cancer and aging. Peripheral blood lymphocytes (44.6%) or biopsies/tissue specimens (24.3%) were the most commonly used samples. γH2AX was scored by optical microscopy as immunostained foci (78%), or by flow cytometry (16%). Critical features affecting the reliability of the assay, including protocols heterogeneity, specimen, cell cycle, kinetics, study design, and statistical analysis, are hereby discussed. Because of its sensitivity, efficiency and mechanistic relevance, the γH2AX assay has great potential as a DNA damage biomarker; however, the technical and epidemiological heterogeneity highlighted in this review infer a necessity for experimental standardization of the assay. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Effects of chemopreventive natural products on non-homologous end-joining DNA double-strand break repair.

    PubMed

    Charles, Catherine; Nachtergael, Amandine; Ouedraogo, Moustapha; Belayew, Alexandra; Duez, Pierre

    2014-07-01

    Double-strand breaks (DSBs) may result from endogenous (e.g., reactive oxygen species, variable (diversity) joining, meiotic exchanges, collapsed replication forks, nucleases) or exogenous (e.g., ionizing radiation, chemotherapeutic agents, radiomimetic compounds) events. DSBs disrupt the integrity of DNA and failed or improper DSBs repair may lead to genomic instability and, eventually, mutations, cancer, or cell death. Non-homologous end-joining (NHEJ) is the major pathway used by higher eukaryotic cells to repair these lesions. Given the complexity of NHEJ and the number of proteins and cofactors involved, secondary metabolites from medicinal or food plants might interfere with the process, activating or inhibiting repair. Twelve natural products, arbutin, curcumin, indole-3-carbinol, and nine flavonoids (apigenin, baicalein, chalcone, epicatechin, genistein, myricetin, naringenin, quercetin, sakuranetin) were chosen for their postulated roles in cancer chemoprevention and/or treatment. The effects of these compounds on NHEJ were investigated with an in vitro protocol based on plasmid substrates. Plasmids were linearized by a restriction enzyme, generating cohesive ends, or by a combination of enzymes, generating incompatible ends; plasmids were then incubated with a nuclear extract prepared from normal human small-intestinal cells (FHS 74 Int), either treated with these natural products or untreated (controls). The NHEJ repair complex from nuclear extracts ligates linearized plasmids, resulting in plasmid oligomers that can be separated and quantified by on-chip microelectrophoresis. Some compounds (chalcone, epicatechin, myricetin, sakuranetin and arbutin) clearly activated NHEJ, whereas others (apigenin, baicalein and curcumin) significantly reduced the repair rate of both types of plasmid substrates. Although this in vitro protocol is only partly representative of the in vivo situation, the natural products appear to interfere with NHEJ repair and warrant

  10. Atrazine Triggers DNA Damage Response and Induces DNA Double-Strand Breaks in MCF-10A Cells.

    PubMed

    Huang, Peixin; Yang, John; Ning, Jie; Wang, Michael; Song, Qisheng

    2015-06-24

    Atrazine, a pre-emergent herbicide in the chloro-s-triazine family, has been widely used in crop lands and often detected in agriculture watersheds, which is considered as a potential threat to human health. Although atrazine and its metabolites showed an elevated incidence of mammary tumors in female Sprague-Dawley (SD) rats, no molecular evidence was found relevant to its carcinogenesis in humans. This study aims to determine whether atrazine could induce the expression of DNA damage response-related proteins in normal human breast epithelial cells (MCF-10A) and to examine the cytotoxicity of atrazine at a molecular level. Our results indicate that a short-term exposure of MCF-10A to an environmentally-detectable concentration of atrazine (0.1 µg/mL) significantly increased the expression of tumor necrosis factor receptor-1 (TNFR1) and phosphorylated Rad17 in the cells. Atrazine treatment increased H2AX phosphorylation (γH2AX) and the formation of γH2AX foci in the nuclei of MCF-10A cells. Atrazine also sequentially elevated DNA damage checkpoint proteins of ATM- and RAD3-related (ATR), ATRIP and phospho-Chk1, suggesting that atrazine could induce DNA double-strand breaks and trigger the DNA damage response ATR-Chk1 pathway in MCF-10A cells. Further investigations are needed to determine whether atrazine-triggered DNA double-strand breaks and DNA damage response ATR-Chk1 pathway occur in vivo.

  11. A 140-Bp-Long Palindromic Sequence Induces Double-Strand Breaks during Meiosis in the Yeast Saccharomyces Cerevisiae

    PubMed Central

    Nag, D. K.; Kurst, A.

    1997-01-01

    Palindromic sequences have the potential to form hairpin or cruciform structures, which are putative substrates for several nucleases and mismatch repair enzymes. A genetic method was developed to detect such structures in vivo in the yeast Saccharomyces cerevisiae. Using this method we previously showed that short hairpin structures are poorly repaired by the mismatch repair system in S. cerevisiae. We show here that mismatches, when present in the stem of the hairpin structure, are not processed by the repair machinery, suggesting that they are treated differently than those in the interstrand base-paired duplex DNA. A 140-bp-long palindromic sequence, on the contrary, acts as a meiotic recombination hotspot by generating a site for a double-strand break, an initiator of meiotic recombination. We suggest that long palindromic sequences undergo cruciform extrusion more readily than short ones. This cruciform structure then acts as a substrate for structure-specific nucleases resulting in the formation of a double-strand break during meiosis in yeast. In addition, we show that residual repair of the short hairpin structure occurs in an MSH2-independent pathway. PMID:9215890

  12. 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

  13. 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.

  14. Tbf1 and Vid22 promote resection and non-homologous end joining of DNA double-strand break ends.

    PubMed

    Bonetti, Diego; Anbalagan, Savani; Lucchini, Giovanna; Clerici, Michela; Longhese, Maria Pia

    2013-01-23

    The repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome stability. The Saccharomyces cerevisiae protein Tbf1, which is characterized by a Myb domain and is related to mammalian TRF1 and TRF2, has been proposed to act as a transcriptional activator. Here, we show that Tbf1 and its interacting protein Vid22 are new players in the response to DSBs. Inactivation of either TBF1 or VID22 causes hypersensitivity to DSB-inducing agents and shows strong negative interactions with mutations affecting homologous recombination. Furthermore, Tbf1 and Vid22 are recruited to an HO-induced DSB, where they promote both resection of DNA ends and repair by non-homologous end joining. Finally, inactivation of either Tbf1 or Vid22 impairs nucleosome eviction around the DSB, suggesting that these proteins promote efficient repair of the break by influencing chromatin identity in its surroundings.

  15. SWR1 and INO80 chromatin remodelers contribute to DNA double-strand break perinuclear anchorage site choice.

    PubMed

    Horigome, Chihiro; Oma, Yukako; Konishi, Tatsunori; Schmid, Roger; Marcomini, Isabella; Hauer, Michael H; Dion, Vincent; Harata, Masahiko; Gasser, Susan M

    2014-08-21

    Persistent DNA double-strand breaks (DSBs) are recruited to the nuclear periphery in budding yeast. Both the Nup84 pore subcomplex and Mps3, an inner nuclear membrane (INM) SUN domain protein, have been implicated in DSB binding. It was unclear what, if anything, distinguishes the two potential sites of repair. Here, we characterize and distinguish the two binding sites. First, DSB-pore interaction occurs independently of cell-cycle phase and requires neither the chromatin remodeler INO80 nor recombinase Rad51 activity. In contrast, Mps3 binding is S and G2 phase specific and requires both factors. SWR1-dependent incorporation of Htz1 (H2A.Z) is necessary for break relocation to either site in both G1- and S-phase cells. Importantly, functional assays indicate that mutations in the two sites have additive repair defects, arguing that the two perinuclear anchorage sites define distinct survival pathways.

  16. Measurement of intracellular DNA double-strand break induction and rejoining along the track of carbon and neon particle beams in water

    SciTech Connect

    Heilmann, J.; Taucher-Scholz, G.; Haberer, T.

    1996-02-01

    The study was aimed at the measurement of effect-depth distributions of intracellularly induced DNA damage in water as tissue equivalent after heavy ion irradiation with therapy particle beams. An assay involving embedding of Chinese hamster ovary (CHO-K1) cells in large agarose plugs and electrophoretic elution of radiation induced DNA fragments by constant field gel electrophoresis was developed. Double-strand break production was quantified by densitometric analysis of DNA-fluorescence after staining with ethidium-bromide and determination of the fraction of DNA eluted out of the agarose plugs. Intracellular double-strand break induction and the effect of a 3 h rejoining incubation were investigated following irradiation with 250 kV x-rays and 190 MeV/u carbon- and 295 MeV/u neon-ions. While the DNA damage induced by x-irradiation decreased continuously with penetration depth, a steady increase in the yield of double-strand breaks was observed for particle radiation, reaching distinct maxima at the position of the physical Bragg peaks. Beyond this, the extent of radiation damage dropped drastically. From comparison of DNA damage and calculated dose profiles, relative biological efficiencies (RBEs) for both double-strand break induction and unrejoined strand breaks after 3 h were determined. While RBE for the induction of DNA double-strand breaks decreased continuously with penetration depth, RBE maxima greater than unity were found with carbon- and neon-ions for double-strand break rejoining near the maximum range of the particles. The method presented here allows for a fast and accurate determination of depth profiles of relevant radiobiological effects for mixed particle fields in tissue equivalent. DNA DSB-induction, Strand break rejoining, CHO-K1 cells, Heavy ion therapy beams, Effect-depth distribution. 35 refs., 8 figs.

  17. Molecular Basis for DNA Double-Strand Break Annealing and Primer Extension by an NHEJ DNA Polymerase

    PubMed Central

    Brissett, Nigel C.; Martin, Maria J.; Bartlett, Edward J.; Bianchi, Julie; Blanco, Luis; Doherty, Aidan J.

    2013-01-01

    Summary Nonhomologous end-joining (NHEJ) is one of the major DNA double-strand break (DSB) repair pathways. The mechanisms by which breaks are competently brought together and extended during NHEJ is poorly understood. As polymerases extend DNA in a 5′-3′ direction by nucleotide addition to a primer, it is unclear how NHEJ polymerases fill in break termini containing 3′ overhangs that lack a primer strand. Here, we describe, at the molecular level, how prokaryotic NHEJ polymerases configure a primer-template substrate by annealing the 3′ overhanging strands from opposing breaks, forming a gapped intermediate that can be extended in trans. We identify structural elements that facilitate docking of the 3′ ends in the active sites of adjacent polymerases and reveal how the termini act as primers for extension of the annealed break, thus explaining how such DSBs are extended in trans. This study clarifies how polymerases couple break-synapsis to catalysis, providing a molecular mechanism to explain how primer extension is achieved on DNA breaks. PMID:24239356

  18. Molecular basis for DNA double-strand break annealing and primer extension by an NHEJ DNA polymerase.

    PubMed

    Brissett, Nigel C; Martin, Maria J; Bartlett, Edward J; Bianchi, Julie; Blanco, Luis; Doherty, Aidan J

    2013-11-27

    Nonhomologous end-joining (NHEJ) is one of the major DNA double-strand break (DSB) repair pathways. The mechanisms by which breaks are competently brought together and extended during NHEJ is poorly understood. As polymerases extend DNA in a 5'-3' direction by nucleotide addition to a primer, it is unclear how NHEJ polymerases fill in break termini containing 3' overhangs that lack a primer strand. Here, we describe, at the molecular level, how prokaryotic NHEJ polymerases configure a primer-template substrate by annealing the 3' overhanging strands from opposing breaks, forming a gapped intermediate that can be extended in trans. We identify structural elements that facilitate docking of the 3' ends in the active sites of adjacent polymerases and reveal how the termini act as primers for extension of the annealed break, thus explaining how such DSBs are extended in trans. This study clarifies how polymerases couple break-synapsis to catalysis, providing a molecular mechanism to explain how primer extension is achieved on DNA breaks.

  19. Large inverted repeats in the vicinity of a single double-strand break strongly affect repair in yeast diploids lacking Rad51.

    PubMed

    Downing, Brandon; Morgan, Rachel; VanHulle, Kelly; Deem, Angela; Malkova, Anna

    2008-10-14

    DNA double-strand breaks (DSBs) are critical lesions that can lead to cell death or chromosomal rearrangements. Rad51 is necessary for most mitotic and meiotic DSB repair events, although a number of RAD51-independent pathways exist. Previously, we described DSB repair in rad51Delta yeast diploids that was stimulated by a DNA region termed "facilitator of break-induced replication" (FBI) located approximately 30kb from the site of an HO-induced DSB. Here, we demonstrate that FBI is a large inverted DNA repeat that channels the repair of DSBs into the single-strand annealing-gross chromosomal rearrangements (SSA-GCR) pathway. Further, analysis of DSB repair in rad54Delta cells allowed us to propose that the SSA-GCR repair pathway is suppressed in the presence of Rad51p. Therefore, an additional role of Rad51 might be to protect eukaryotic genomes from instabilities by preventing chromosomal rearrangements.

  20. The ancestral role of ATP hydrolysis in type II topoisomerases: prevention of DNA double-strand breaks

    PubMed Central

    Bates, Andrew D.; Berger, James M.; Maxwell, Anthony

    2011-01-01

    Type II DNA topoisomerases (topos) catalyse changes in DNA topology by passing one double-stranded DNA segment through another. This reaction is essential to processes such as replication and transcription, but carries with it the inherent danger of permanent double-strand break (DSB) formation. All type II topos hydrolyse ATP during their reactions; however, only DNA gyrase is able to harness the free energy of hydrolysis to drive DNA supercoiling, an energetically unfavourable process. A long-standing puzzle has been to understand why the majority of type II enzymes consume ATP to support reactions that do not require a net energy input. While certain type II topos are known to ‘simplify’ distributions of DNA topoisomers below thermodynamic equilibrium levels, the energy required for this process is very low, suggesting that this behaviour is not the principal reason for ATP hydrolysis. Instead, we propose that the energy of ATP hydrolysis is needed to control the separation of protein–protein interfaces and prevent the accidental formation of potentially mutagenic or cytotoxic DSBs. This interpretation has parallels with the actions of a variety of molecular machines that catalyse the conformational rearrangement of biological macromolecules. PMID:21525132

  1. Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells.

    PubMed

    Lo, Anthony W I; Sprung, Carl N; Fouladi, Bijan; Pedram, Mehrdad; Sabatier, Laure; Ricoul, Michelle; Reynolds, Gloria E; Murnane, John P

    2002-07-01

    Telomeres are essential for protecting the ends of chromosomes and preventing chromosome fusion. Telomere loss has been proposed to play an important role in the chromosomal rearrangements associated with tumorigenesis. To determine the relationship between telomere loss and chromosome instability in mammalian cells, we investigated the events resulting from the introduction of a double-strand break near a telomere with I-SceI endonuclease in mouse embryonic stem cells. The inactivation of a selectable marker gene adjacent to a telomere as a result of the I-SceI-induced double-strand break involved either the addition of a telomere at the site of the break or the formation of inverted repeats and large tandem duplications on the end of the chromosome. Nucleotide sequence analysis demonstrated large deletions and little or no complementarity at the recombination sites involved in the formation of the inverted repeats. The formation of inverted repeats was followed by a period of chromosome instability, characterized by amplification of the subtelomeric region, translocation of chromosomal fragments onto the end of the chromosome, and the formation of dicentric chromosomes. Despite this heterogeneity, the rearranged chromosomes eventually acquired telomeres and were stable in most of the cells in the population at the time of analysis. Our observations are consistent with a model in which broken chromosomes that do not regain a telomere undergo sister chromatid fusion involving nonhomologous end joining. Sister chromatid fusion is followed by chromosome instability resulting from breakage-fusion-bridge cycles involving the sister chromatids and rearrangements with other chromosomes. This process results in highly rearranged chromosomes that eventually become stable through the addition of a telomere onto the broken end. We have observed similar events after spontaneous telomere loss in a human tumor cell line, suggesting that chromosome instability resulting from

  2. DNA double-strand breaks as potential indicators for the biological effects of ionising radiation exposure from cardiac CT and conventional coronary angiography: a randomised, controlled study.

    PubMed

    Geisel, Dominik; Zimmermann, Elke; Rief, Matthias; Greupner, Johannes; Laule, Michael; Knebel, Fabian; Hamm, Bernd; Dewey, Marc

    2012-08-01

    To prospectively compare induced DNA double-strand breaks by cardiac computed tomography (CT) and conventional coronary angiography (CCA). 56 patients with suspected coronary artery disease were randomised to undergo either CCA or cardiac CT. DNA double-strand breaks were assessed in fluorescence microscopy of blood lymphocytes as indicators of the biological effects of radiation exposure. Radiation doses were estimated using dose-length product (DLP) and dose-area product (DAP) with conversion factors for CT and CCA, respectively. On average there were 0.12 ± 0.06 induced double-strand breaks per lymphocyte for CT and 0.29 ± 0.18 for diagnostic CCA (P < 0.001). This relative biological effect of ionising radiation from CCA was 1.9 times higher (P < 0.001) than the effective dose estimated by conversion factors would have suggested. The correlation between the biological effects and the estimated radiation doses was excellent for CT (r = 0.951, P < 0.001) and moderate to good for CCA (r = 0.862, P < 0.001). One day after radiation, a complete repair of double-strand breaks to background levels was found in both groups. Conversion factors may underestimate the relative biological effects of ionising radiation from CCA. DNA double-strand break assessment may provide a strategy for individualised assessments of radiation. • Radiation dose causes concern for both conventional coronary angiography and cardiac CT. • Estimations of the biological effects of ionising radiation may become feasible. • Fewer DNA double-strand breaks are induced by cardiac CT than CCA. • Conversion factors may underestimate the relative effects of ionising radiation from CCA.

  3. [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).

  4. Yields of single-strand breaks in double-stranded calf thymus DNA irradiated in aqueous solution in the presence of oxygen and scavengers

    SciTech Connect

    Udovicic, Lj.; Mark, F.; Bothe, E.

    1994-11-01

    Yields of radiation-induced single-strand breaks in double-stranded calf thymus DNA have been measured as a function of OH scavenger concentration in N{sub 2}O/O{sub 2}-saturated aqueous solution. The experimental data are well represented by a theoretical model based on non-homogeneous reaction kinetics, without the need to adjust any parameter. The good agreement between experimental and theoretical data is taken as evidence that, in the presence of oxygen, the main effect of added scavengers with respect to the formation of single-strand breaks in double-stranded DNA is OH radical scavenging. 30 refs., 3 figs., 1 tab.

  5. The Deubiquitylating Enzyme USP4 Cooperates with CtIP in DNA Double-Strand Break End Resection.

    PubMed

    Liu, Hailong; Zhang, Haoxing; Wang, Xiaohui; Tian, Qingsong; Hu, Zhaohua; Peng, Changmin; Jiang, Pei; Wang, TingTing; Guo, Wei; Chen, Yali; Li, Xinzhi; Zhang, Pumin; Pei, Huadong

    2015-10-06

    DNA end resection is a highly regulated and critical step in DNA double-stranded break (DSB) repair. In higher eukaryotes, DSB resection is initiated by the collaborative action of CtIP and the MRE11-RAD50-NBS1 (MRN) complex. Here, we find that the deubiquitylating enzyme USP4 directly participates in DSB resection and homologous recombination (HR). USP4 confers resistance to DNA damage-inducing agents. Mechanistically, USP4 interacts with CtIP and MRN via a specific, conserved region and the catalytic domain of USP4, respectively, and regulates CtIP recruitment to sites of DNA damage. We also find that USP4 autodeubiquitylation is essential for its HR functions. Collectively, our findings identify USP4 as a key regulator of DNA DSB end resection.

  6. 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

  7. 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

  8. An inhibitor of nonhomologous end-joining abrogates double-strand break repair and impedes cancer progression.

    PubMed

    Srivastava, Mrinal; Nambiar, Mridula; Sharma, Sheetal; Karki, Subhas S; Goldsmith, G; Hegde, Mahesh; Kumar, Sujeet; Pandey, Monica; Singh, Ram K; Ray, Pritha; Natarajan, Renuka; Kelkar, Madhura; De, Abhijit; Choudhary, Bibha; Raghavan, Sathees C

    2012-12-21

    DNA Ligase IV is responsible for sealing of double-strand breaks (DSBs) during nonhomologous end-joining (NHEJ). Inhibiting Ligase IV could result in amassing of DSBs, thereby serving as a strategy toward treatment of cancer. Here, we identify a molecule, SCR7 that inhibits joining of DSBs in cell-free repair system. SCR7 blocks Ligase IV-mediated joining by interfering with its DNA binding but not that of T4 DNA Ligase or Ligase I. SCR7 inhibits NHEJ in a Ligase IV-dependent manner within cells, and activates the intrinsic apoptotic pathway. More importantly, SCR7 impedes tumor progression in mouse models and when coadministered with DSB-inducing therapeutic modalities enhances their sensitivity significantly. This inhibitor to target NHEJ offers a strategy toward the treatment of cancer and improvement of existing regimens. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. An in vitro DNA double-strand break repair assay based on end-joining of defined duplex oligonucleotides.

    PubMed

    Datta, Kamal; Purkayastha, Shubhadeep; Neumann, Ronald D; Winters, Thomas A

    2012-01-01

    DNA double-strand breaks (DSBs) are caused by endogenous cellular processes such as oxidative metabolism, or by exogenous events like exposure to ionizing radiation or other genotoxic agents. Repair of these DSBs is essential for the maintenance of cellular genomic integrity. In human cells, and cells of other higher eukaryotes, DSBs are primarily repaired by the nonhomologous end-joining (NHEJ) DSB repair pathway. Most in vitro assays that have been designed to measure NHEJ activity employ linear plasmid DNA as end-joining substrates, and such assays have made significant contributions to our understanding of the biochemical mechanisms of NHEJ. Here we describe an in vitro end-joining assay employing linear oligonucleotides that has distinct advantages over plasmid-based assays for the study of structure-function relationships between the proteins of the NHEJ pathway and synthetic DNA end-joining substrates possessing predetermined DSB configurations and chemistries.

  10. Analysis of double-strand break repair by nonhomologous DNA end joining in cell-free extracts from mammalian cells.

    PubMed

    Pfeiffer, Petra; Odersky, Andrea; Goedecke, Wolfgang; Kuhfittig-Kulle, Steffi

    2014-01-01

    Double-strand breaks (DSB) in genomic DNA are induced by ionizing radiation or radiomimetic drugs but also occur spontaneously during the cell cycle at quite significant frequencies. In vertebrate cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of DSB repair which is able to rejoin two broken DNA termini directly end-to-end irrespective of sequence and structure. Genetic studies in various radiosensitive and DSB repair-deficient cell lines yielded insight into the factors involved in NHEJ. Studies in cell-free systems derived from Xenopus eggs and mammalian cells allowed the dissection of the underlying mechanisms. In the present chapter, we describe a protocol for the preparation of whole cell extracts from mammalian cells and a plasmid-based in vitro assay which permits the easy analysis of the efficiency and fidelity of DSB repair via NHEJ in different cell types.

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

    PubMed

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

    2012-01-01

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

  12. Long noncoding RNA LINP1 regulates repair of DNA double-strand breaks in triple-negative breast cancer.

    PubMed

    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

    2016-06-01

    Long noncoding RNAs (lncRNAs) play critical roles during tumorigenesis by functioning as scaffolds that regulate protein-protein, protein-DNA or protein-RNA interactions. Using a clinically guided genetic screening approach, we identified lncRNA in nonhomologous end joining (NHEJ) pathway 1 (LINP1), which is overexpressed in human triple-negative breast cancer. We found that LINP1 enhances repair of DNA double-strand breaks by serving as a scaffold linking Ku80 and DNA-PKcs, thereby coordinating the NHEJ pathway. Importantly, blocking LINP1, which is regulated by p53 and epidermal growth factor receptor (EGFR) signaling, increases the sensitivity of the tumor-cell response to radiotherapy in breast cancer.

  13. Application of laser-accelerated protons to the demonstration of DNA double-strand breaks in human cancer cells

    SciTech Connect

    Yogo, A.; Nishikino, M.; Mori, M.; Ogura, K.; Sagisaka, A.; Orimo, S.; Nishiuchi, M.; Pirozhkov, A. S.; Ikegami, M.; Tampo, M.; Sakaki, H.; Suzuki, M.; Daito, I.; Kiriyama, H.; Okada, H.; Kanazawa, S.; Kondo, S.; Shimomura, T.; Nakai, Y.; Bolton, P. R.

    2009-05-04

    We report the demonstrated irradiation effect of laser-accelerated protons on human cancer cells. In vitro (living) A549 cells are irradiated with quasimonoenergetic proton bunches of 0.8-2.4 MeV with a single bunch duration of 15 ns. Irradiation with the proton dose of 20 Gy results in a distinct formation of {gamma}-H2AX foci as an indicator of DNA double-strand breaks generated in the cancer cells. This is a pioneering result that points to future investigations of the radiobiological effects of laser-driven ion beams. Unique high-current and short-bunch features make laser-driven proton bunches an excitation source for time-resolved determination of radical yields.

  14. 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.

  15. 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.

  16. DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain.

    PubMed

    Unal, Elçin; Arbel-Eden, Ayelet; Sattler, Ulrike; Shroff, Robert; Lichten, Michael; Haber, James E; Koshland, Douglas

    2004-12-22

    The postreplicative repair of double-strand breaks (DSBs) is thought to require sister chromatid cohesion, provided by the cohesin complex along the chromosome arms. A further specialized role for cohesin in DSB repair is suggested by its de novo recruitment to regions of DNA damage in mammals. Here, we show in budding yeast that a single DSB induces the formation of a approximately 100 kb cohesin domain around the lesion. Our analyses suggest that the primary DNA damage checkpoint kinases Mec1p and Tel1p phosphorylate histone H2AX to generate a large domain, which is permissive for cohesin binding. Cohesin binding to the phospho-H2AX domain is enabled by Mre11p, a component of a critical repair complex, and Scc2p, a component of the cohesin loading machinery that is necessary for sister chromatid cohesion. We also provide evidence that the DSB-induced cohesin domain functions in postreplicative repair.

  17. Vilya, a component of the recombination nodule, is required for meiotic double-strand break formation in Drosophila.

    PubMed

    Lake, Cathleen M; Nielsen, Rachel J; Guo, Fengli; Unruh, Jay R; Slaughter, Brian D; Hawley, R Scott

    2015-10-09

    Meiotic recombination begins with the induction of programmed double-strand breaks (DSBs). In most organisms only a fraction of DSBs become crossovers. Here we report a novel meiotic gene, vilya, which encodes a protein with homology to Zip3-like proteins shown to determine DSB fate in other organisms. Vilya is required for meiotic DSB formation, perhaps as a consequence of its interaction with the DSB accessory protein Mei-P22, and localizes to those DSB sites that will mature into crossovers. In early pachytene Vilya localizes along the central region of the synaptonemal complex and to discrete foci. The accumulation of Vilya at foci is dependent on DSB formation. Immuno-electron microscopy demonstrates that Vilya is a component of recombination nodules, which mark the sites of crossover formation. Thus Vilya links the mechanism of DSB formation to either the selection of those DSBs that will become crossovers or to the actual process of crossing over.

  18. 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

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

    PubMed Central

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

    2015-01-01

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

  20. The TopoVIB-Like protein family is required for meiotic DNA double-strand break formation.

    PubMed

    Robert, T; Nore, A; Brun, C; Maffre, C; Crimi, B; Bourbon, H-M; de Massy, B

    2016-02-26

    Meiotic recombination is induced by the formation of DNA double-strand breaks (DSBs) catalyzed by SPO11, the ortholog of subunit A of TopoVI DNA topoisomerase (TopoVIA). TopoVI activity requires the interaction between A and B subunits. We identified a conserved family of plant and animal proteins [the TOPOVIB-Like (TOPOVIBL) family] that share strong structural similarity to the TopoVIB subunit of TopoVI DNA topoisomerase. We further characterize the meiotic recombination proteins Rec102 (Saccharomyces cerevisiae), Rec6 (Schizosaccharomyces pombe), and MEI-P22 (Drosophila melanogaster) as homologs to the transducer domain of TopoVIB. We demonstrate that the mouse TOPOVIBL protein interacts and forms a complex with SPO11 and is required for meiotic DSB formation. We conclude that meiotic DSBs are catalyzed by a complex involving SPO11 and TOPOVIBL.

  1. Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination.

    PubMed

    Panizza, Silvia; Mendoza, Marco A; Berlinger, Marc; Huang, Lingzhi; Nicolas, Alain; Shirahige, Katsuhiko; Klein, Franz

    2011-08-05

    Meiotic recombination between homologous chromosomes initiates via programmed DNA double-strand breaks (DSBs), generated by complexes comprising Spo11 transesterase plus accessory proteins. DSBs arise concomitantly with the development of axial chromosome structures, where the coalescence of axis sites produces linear arrays of chromatin loops. Recombining DNA sequences map to loops, but are ultimately tethered to the underlying axis. How and when such tethering occurs is currently unclear. Using ChIPchip in yeast, we show that Spo11-accessory proteins Rec114, Mer2, and Mei4 stably interact with chromosome axis sequences, upon phosphorylation of Mer2 by S phase Cdk. This axis tethering requires meiotic axis components (Red1/Hop1) and is modulated in a domain-specific fashion by cohesin. Loss of Rec114, Mer2, and Mei4 binding correlates with loss of DSBs. Our results strongly suggest that hotspot sequences become tethered to axis sites by the DSB machinery prior to DSB formation.

  2. The requirement of Artemis in double-strand break repair depends on the type of DNA damage.

    PubMed

    Kurosawa, Aya; Koyama, Hideki; Takayama, Shinichi; Miki, Kensuke; Ayusawa, Dai; Fujii, Michihiko; Iiizumi, Susumu; Adachi, Noritaka

    2008-01-01

    Artemis is a recently identified factor involved in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Here, we performed targeted disruption of the Artemis gene (ARTEMIS) in the human pre-B cell line Nalm-6. Unexpectedly, we found that cells lacking Artemis exhibit increased sensitivity to low doses, but not high doses, of ionizing radiation. We also show that ARTEMIS-deficient cells are hypersensitive to the topoisomerase II inhibitor etoposide, but to a much lesser extent than cells lacking DNA ligase IV, a critical component of NHEJ. Unlike DNA ligase IV-deficient cells, ARTEMIS-deficient cells were not hypersensitive to ICRF-193, a topoisomerase II inhibitor that does not stabilize topoisomerase II-DNA cleavable complexes. Collectively, our results suggest that Artemis only partially participates in the NHEJ pathway to repair DSBs in human somatic cells.

  3. Evidence that single-stranded DNA breaks are a normal feature of koala sperm chromatin, while double-stranded DNA breaks are indicative of DNA damage.

    PubMed

    Zee, Yeng Peng; López-Fernández, Carmen; Arroyo, F; Johnston, Stephen D; Holt, William V; Gosalvez, Jaime

    2009-08-01

    In this study, we have used single and double comet assays to differentiate between single- and double-stranded DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa. We have also investigated the likelihood that single-stranded DNA breakage is part of the natural spermiogenic process in koalas, where its function would be the generation of structural bends in the DNA molecule so that appropriate packaging and compaction can occur. Koala spermatozoa were examined using the sperm chromatin dispersion test (SCDt) and comet assays to investigate non-orthodox double-stranded DNA. Comet assays were conducted under 1) neutral conditions; and 2) neutral followed by alkaline conditions (double comet assay); the latter technique enabled simultaneous visualisation of both single-stranded and double-stranded DNA breaks. Following the SCDt, there was a continuum of nuclear morphotypes, ranging from no apparent DNA fragmentation to those with highly dispersed and degraded chromatin. Dispersion morphotypes were mirrored by a similar diversity of comet morphologies that could be further differentiated using the double comet assay. The majority of koala spermatozoa had nuclei with DNA abasic-like residues that produced single-tailed comets following the double comet assay. The ubiquity of these residues suggests that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with 'true' DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA with a diffuse single tail to nuclei that exhibited both single- and double-stranded breaks with two comet tails.

  4. 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.

  5. Atrazine Triggers DNA Damage Response and Induces DNA Double-Strand Breaks in MCF-10A Cells

    PubMed Central

    Huang, Peixin; Yang, John; Ning, Jie; Wang, Michael; Song, Qisheng

    2015-01-01

    Atrazine, a pre-emergent herbicide in the chloro-s-triazine family, has been widely used in crop lands and often detected in agriculture watersheds, which is considered as a potential threat to human health. Although atrazine and its metabolites showed an elevated incidence of mammary tumors in female Sprague–Dawley (SD) rats, no molecular evidence was found relevant to its carcinogenesis in humans. This study aims to determine whether atrazine could induce the expression of DNA damage response-related proteins in normal human breast epithelial cells (MCF-10A) and to examine the cytotoxicity of atrazine at a molecular level. Our results indicate that a short-term exposure of MCF-10A to an environmentally-detectable concentration of atrazine (0.1 µg/mL) significantly increased the expression of tumor necrosis factor receptor-1 (TNFR1) and phosphorylated Rad17 in the cells. Atrazine treatment increased H2AX phosphorylation (γH2AX) and the formation of γH2AX foci in the nuclei of MCF-10A cells. Atrazine also sequentially elevated DNA damage checkpoint proteins of ATM- and RAD3-related (ATR), ATRIP and phospho-Chk1, suggesting that atrazine could induce DNA double-strand breaks and trigger the DNA damage response ATR-Chk1 pathway in MCF-10A cells. Further investigations are needed to determine whether atrazine-triggered DNA double-strand breaks and DNA damage response ATR-Chk1 pathway occur in vivo. PMID:26114388

  6. 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. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks

    PubMed Central

    Sommers, Joshua A.; Brosh, Robert M.; Sharma, Sudha

    2013-01-01

    Genomic instability is a known precursor to cancer and aging. The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in maintaining genome stability in all living organisms. Human RecQ homologs include RECQ1, BLM, WRN, RECQ4, and RECQ5β, three of which have been linked to diseases with elevated risk of cancer and growth defects (Bloom Syndrome and Rothmund-Thomson Syndrome) or premature aging (Werner Syndrome). RECQ1, the first RecQ helicase discovered and the most abundant in human cells, is the least well understood of the five human RecQ homologs. We have previously described that knockout of RECQ1 in mice or knockdown of its expression in human cells results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased load of DNA damage and heightened sensitivity to ionizing radiation. We have now obtained evidence implicating RECQ1 in the nonhomologous end-joining pathway of DNA double-strand break repair. We show that RECQ1 interacts directly with the Ku70/80 subunit of the DNA-PK complex, and depletion of RECQ1 results in reduced end-joining in cell free extracts. In vitro, RECQ1 binds and unwinds the Ku70/80-bound partial duplex DNA substrate efficiently. Linear DNA is co-bound by RECQ1 and Ku70/80, and DNA binding by Ku70/80 is modulated by RECQ1. Collectively, these results provide the first evidence for an interaction of RECQ1 with Ku70/80 and a role of the human RecQ helicase in double-strand break repair through nonhomologous end-joining. PMID:23650516

  8. 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.

  9. 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

  10. Endonuclease G plays a role in immunoglobulin class switch DNA recombination by introducing double-strand breaks in switch regions.

    PubMed

    Zan, Hong; Zhang, Jinsong; Al-Qahtani, Ahmed; Pone, Egest J; White, Clayton A; Lee, Derrik; Yel, Leman; Mai, Thach; Casali, Paolo

    2011-01-01

    Immunoglobulin (Ig) class switch DNA recombination (CSR) is the crucial mechanism diversifying the biological effector functions of antibodies. Generation of double-strand DNA breaks (DSBs), particularly staggered DSBs, in switch (S) regions of the upstream and downstream CH genes involved in the specific recombination process is an absolute requirement for CSR. Staggered DSBs would be generated through deamination of dCs on opposite DNA strands by activation-induced cytidine deaminase (AID), subsequent dU deglycosylation by uracil DNA glycosylase (Ung) and abasic site nicking by apurinic/apyrimidic endonuclease. However, consistent with the findings that significant amounts of DSBs can be detected in the IgH locus in the absence of AID or Ung, we have shown in human and mouse B cells that AID generates staggered DSBs not only by cleaving intact double-strand DNA, but also by processing blunt DSB ends generated in an AID-independent fashion. How these AID-independent DSBs are generated is still unclear. It is possible that S region DNA may undergo AID-independent cleavage by structure-specific nucleases, such as endonuclease G (EndoG). EndoG is an abundant nuclease in eukaryotic cells. It cleaves single and double-strand DNA, primarily at dG/dC residues, the preferential sites of DSBs in S region DNA. We show here that EndoG can localize to the nucleus of B cells undergoing CSR and binds to S region DNA, as shown by specific chromatin immunoprecipitation assays. Using knockout EndoG(-/-) mice and EndoG(-/-) B cells, we found that EndoG deficiency resulted in a two-fold reduction in CSR in vivo and in vitro, as demonstrated by reduced cell surface IgG1, IgG2a, IgG3 and IgA, reduced secreted IgG1, reduced circle Iγ1-Cμ, Iγ3-Cμ, Iɛ-Cμ, Iα-Cμ transcripts, post-recombination Iμ-Cγ1, Iμ-Cγ3, Iμ-Cɛ and Iμ-Cα transcripts. In addition to reduced CSR, EndoG(-/-) mice showed a significantly altered spectrum of mutations in IgH J(H)-iEμ DNA. Impaired CSR in

  11. Endonuclease G plays a role in immunoglobulin class switch DNA recombination by introducing double-strand breaks in switch regions

    PubMed Central

    Zan, Hong; Zhang, Jinsong; Al-Qahtani, Ahmed; Pone, Egest J.; White, Clayton A.; Lee, Derrik; Yel, Leman; Mai, Thach; Casali, Paolo

    2012-01-01

    Immunoglobulin (Ig) class switch DNA recombination (CSR) is the crucial mechanism diversifying the biological effector functions of antibodies. Generation of double-strand DNA breaks (DSBs), particularly staggered DSBs, in switch (S) regions of the upstream and downstream CH genes involved in the specific recombination process is an absolute requirement for CSR. Staggered DSBs would be generated through deamination of dCs on opposite DNA strands by activation-induced cytidine deaminase (AID), subsequent dU deglycosylation by uracil DNA glycosylase (Ung) and abasic site nicking by apurinic/apyrimidic endonuclease. However, consistent with the findings that significant amounts of DSBs can be detected in the IgH locus in the absence of AID or Ung, we have shown in human and mouse B cells that AID generates staggered DSBs not only by cleaving intact double-strand DNA, but also by processing blunt DSB ends generated in an AID-independent fashion. How these AID-independent DSBs are generated is still unclear. It is possible that S region DNA may undergo AID-independent cleavage by structure-specific nucleases, such as endonuclease G (EndoG). EndoG is an abundant nuclease in eukaryotic cells. It cleaves single- and double-strand DNA, primarily at dG/dC residues, the preferential sites of DSBs in S region DNA. We show here that EndoG can localize to the nucleus of B cells undergoing CSR and binds to S region DNA, as shown by specific chromatin immunoprecipitation assays. Using knockout EndoG−/− mice and EndoG−/− B cells, we found that EndoG deficiency resulted in defective CSR in vivo and in vitro, as demonstrated by reduced cell surface IgG1, IgG2a, IgG3 and IgA, reduced secreted IgG1, reduced circle Iγ1-Cμ, Iγ3-Cμ, Iε-Cμ, Iα-Cμ transcripts, post-recombination Iμ-Cγ1, Iμ-Cγ3, Iμ-Cε and Iμ-Cα transcripts. In addition to reduced CSR, EndoG−/− mice showed a significantly altered spectrum of mutations in IgH JH-iEμ DNA. Impaired CSR in Endo

  12. Promyelocytic leukemia protein interacts with werner syndrome helicase and regulates double-strand break repair in γ-irradiation-induced DNA damage responses.

    PubMed

    Liu, Jilai; Song, Yi; Qian, Junjie; Liu, Bin; Dong, Yan; Tian, Baolei; Sun, Zhixian

    2011-05-01

    We show here that γ-irradiation leads to the translocation of endogenous Werner syndrome helicase (WRN) from nucleoli to nucleoplasmic DNA double strand breaks (DSBs), and WRN plays a role in damage repair. The relocation of WRN after irradiation was perturbed by promyelocytic leukemia protein (PML) knockdown and enhanced by PML IV overexpression. PML IV physically interacted with WRN after irradiation. Amino acids (a.a.) 394 to 433 of PML were necessary for this interaction and the nucleoplasmic translocation of WRN and were involved in DSB repair and cellular sensitivity to γ-irradiation. Taken together, our results provide molecular support for a model in which PML IV physically interacts with and regulates the translocation of WRN for DNA damage repair through its 394-433 a.a. domain.

  13. Telomere attrition induces a DNA double-strand break damage signal that reactivates p53 transcription in HTLV-I leukemic cells.

    PubMed

    Datta, A; Nicot, C

    2008-02-14

    Persistent inhibition of telomerase induces a severe telomere shortening in human T-cell leukemia virus type-1-infected cells which signals a DNA double-strand break damage response, formation of telomere dysfunction-induced foci and activates the ATM pathway. In turn, activation of ATM and its downstream effectors led to an increased phosphorylation and acetylation on specific residues of p53 known to be involved in transcriptional activation. Disruption of Mdm2-p53 complexes coupled with increased proteasomal degradation of MDMX further enhanced reactivation of p53 transcription, ultimately leading to senescence of tumor cells. Induction of senescence in these T-cells was associated with an increased expression of p21, p16 and activation of GSK3beta. Our results support the cancer-aging model and demonstrate that the halt of aging in cancer cells can be reversed through reactivation of p53.

  14. 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.

  15. Genetic recombination induced by DNA double-strand break in bacteriophage T4: nature of the left/right bias.

    PubMed

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

    2008-06-01

    The experimental system combining double-strand breaks (DSBs), produced site-specifically by SegC endonuclease, with the famous advantages of the bacteriophage T4 rII mutant recombination analysis was used here to elucidate the origin of the recombination bias on two sides of the DSB, especially pronounced in gene 39 (topoisomerase II) and gene 59 (41-helicase loader) mutants. Three sources were found to contribute to the bias: (1) the SegC endonuclease may remain bound to the end of the broken DNA and thus protect it from exonuclease degradation; (2) in heteroduplex heterozygotes (HHs), arising as the recombinant products in the left-hand crosses, the transcribed strands are of rII mutant phenotype, so they, in contrast to the right-hand HHs, do not produce plaques on the lawn of the lambda-lysogenic host; and (3) the intrinsic polarity of T4 chromosome, reflected in transcription, may be a cause for discrimination of promoter-proximal and promoter-distal DNA sequences. It is shown that the apparent recombination bias does not imply one-sidedness of the DSB repair but just reflects a different depth of the end processing. It is inferred that the cause, underlying the "intrinsic" bias, might be interference between strand exchange and transcription. Topoisomerase and helicase functions are necessary to turn the process in favor of strand exchange. The idea is substantiated that the double-stranded to single-stranded DNA transition edge (not ss-DNA tip) serves as an actual recombinogenic element.

  16. Dual Roles for DNA Polymerase Theta in Alternative End-Joining Repair of Double-Strand Breaks in Drosophila

    PubMed Central

    McVey, Mitch

    2010-01-01

    DNA double-strand breaks are repaired by multiple mechanisms that are roughly grouped into the categories of homology-directed repair and non-homologous end joining. End-joining repair can be further classified as either classical non-homologous end joining, which requires DNA ligase 4, or “alternative” end joining, which does not. Alternative end joining has been associated with genomic deletions and translocations, but its molecular mechanism(s) are largely uncharacterized. Here, we report that Drosophila melanogaster DNA polymerase theta (pol theta), encoded by the mus308 gene and previously implicated in DNA interstrand crosslink repair, plays a crucial role in DNA ligase 4-independent alternative end joining. In the absence of pol theta, end joining is impaired and residual repair often creates large deletions flanking the break site. Analysis of break repair junctions from flies with mus308 separation-of-function alleles suggests that pol theta promotes the use of long microhomologies during alternative end joining and increases the likelihood of complex insertion events. Our results establish pol theta as a key protein in alternative end joining in Drosophila and suggest a potential mechanistic link between alternative end joining and interstrand crosslink repair. PMID:20617203

  17. Bridging of double-stranded breaks by the nonhomologous end-joining ligation complex is modulated by DNA end chemistry.

    PubMed

    Reid, Dylan A; Conlin, Michael P; Yin, Yandong; Chang, Howard H; Watanabe, Go; Lieber, Michael R; Ramsden, Dale A; Rothenberg, Eli

    2017-02-28

    The nonhomologous end-joining (NHEJ) pathway is the primary repair pathway for DNA double strand breaks (DSBs) in humans. Repair is mediated by a core complex of NHEJ factors that includes a ligase (DNA Ligase IV; L4) that relies on juxtaposition of 3΄ hydroxyl and 5΄ phosphate termini of the strand breaks for catalysis. However, chromosome breaks arising from biological sources often have different end chemistries, and how these different end chemistries impact the way in which the core complex directs the necessary transitions from end pairing to ligation is not known. Here, using single-molecule FRET (smFRET), we show that prior to ligation, differences in end chemistry strongly modulate the bridging of broken ends by the NHEJ core complex. In particular, the 5΄ phosphate group is a recognition element for L4 and is critical for the ability of NHEJ factors to promote stable pairing of ends. Moreover, other chemical incompatibilities, including products of aborted ligation, are sufficient to disrupt end pairing. Based on these observations, we propose a mechanism for iterative repair of DSBs by NHEJ. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Single- and double-strand DNA breaks in rat brain cells after acute exposure to radiofrequency electromagnetic radiation.

    PubMed

    Lai, H; Singh, N P

    1996-04-01

    We investigated the effects of acute (2-h) exposure to pulsed (2-micros pulse width, 500 pulses s(-1)) and continuous wave 2450-MHz radiofrequency electromagnetic radiation on DNA strand breaks in brain cells of rat. The spatial averaged power density of the radiation was 2mW/cm2, which produced a whole-body average-specific absorption rate of 1.2W/kg. Single- and double-strand DNA breaks in individual brain cells were measured at 4h post-exposure using a microgel electrophoresis assay. An increase in both types of DNA strand breaks was observed after exposure to either the pulsed or continuous-wave radiation, No significant difference was observed between the effects of the two forms of radiation. We speculate that these effects could result from a direct effect of radiofrequency electromagnetic energy on DNA molecules and/or impairment of DNA-damage repair mechanisms in brain cells. Our data further support the results of earlier in vitro and in vivo studies showing effects of radiofrequency electromagnetic radiation on DNA.

  19. The ratio of single- to double-strand DNA breaks and their absolute values determine cell death pathway

    PubMed Central

    Tounekti, O; Kenani, A; Foray, N; Orlowski, S; Mir, L M

    2001-01-01

    Bleomycin is a cytotoxic antibiotic that generates DNA double-strand breaks (DSB) and DNA single-strand breaks (SSB). It is possible to introduce known quantities of bleomycin molecules into cells. Low amounts kill the cells by a slow process termed mitotic cell death, while high amounts produce a fast process that has been termed pseudoapoptosis. We previously showed that these types of cell death are a direct consequence of the DSB generated by bleomycin. Here, we use deglyco-bleomycin, a bleomycin derivative lacking the carbohydrate moiety. Although this molecule performs the same nucleophilic attacks on DNA as bleomycin, we show that deglyco-bleomycin is at least 100 times less toxic to Chinese hamster fibroblasts than bleomycin. In fact, deglyco-bleomycin treatment results in apoptosis induction. In contrast, however, deglyco-bleomycin was found to generate almost exclusively SSB. Our results suggest that more than 150 000 SSB per cell are required to trigger apoptosis in Chinese hamster fibroblasts and that SSB are 300 times less toxic than DSB. Taken together with previous studies on bleomycin, our data demonstrates that cells can die by apoptosis, mitotic cell death, or pseudoapoptosis, depending on the number of DNA breaks and on the ratio of SSB to DSB. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11336481

  20. Factors That Affect the Location and Frequency of Meiosis-Induced Double-Strand Breaks in Saccharomyces Cerevisiae

    PubMed Central

    Wu, T. C.; Lichten, M.

    1995-01-01

    Double-strand DNA breaks (DSBs) initiate meiotic recombination in Saccharomyces cerevisiae. DSBs occur at sites that are hypersensitive in nuclease digests of chromatin, suggesting a role for chromatin structure in determining DSB location. We show here that the frequency of DSBs at a site is not determined simply by DNA sequence or by features of chromatin structure. An arg4-containing plasmid was inserted at several different locations in the yeast genome. Meiosis-induced DSBs occurred at similar sites in pBR322-derived portions of the construct at all insert loci, and the frequency of these breaks varied in a manner that mirrored the frequency of meiotic recombination in the arg4 portion of the insert. However, DSBs did not occur in the insert-borne arg4 gene at a site that is frequently broken at the normal ARG4 locus, even though the insert-borne arg4 gene and the normal ARG4 locus displayed similar DNase I hypersensitivity patterns. Deletions that removed active DSB sites from an insert at HIS4 restored breaks to the insert-borne arg4 gene and to a DSB site in flanking chromosomal sequences. We conclude that the frequency of DSB at a site can be affected by sequences several thousands nucleotides away and suggest that this is because of competition between DSB sites for locally limited factors. PMID:7635308

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

    PubMed Central

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

    2009-01-01

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

  2. Poor recognition of O6-isopropyl dG by MGMT triggers double strand break-mediated cell death and micronucleus induction in FANC-deficient cells

    PubMed Central

    Hashimoto, Kiyohiro; Sharma, Vyom; Sasanuma, Hiroyuki; Tian, Xu; Takata, Minoru; Takeda, Shunichi; Swenberg, James A.; Nakamura, Jun

    2016-01-01

    Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that dysfunction of the FANC pathway resulted in higher sensitivity to IPMS compared to EMS or MMS. O6-alkyl dG is primarily repaired by methyl guanine methyltransferase (MGMT), while isopropyl dG is less likely to be a substrate for MGMT. Comparison of the cytotoxic potential of IPMS and its isomer n-propyl methanesulfonate (nPMS) revealed that the isopropyl moiety avoids recognition by MGMT and leads to higher cytotoxicity. Next, the micronucleus (MN) assay showed that FANC deficiency increases the sensitivity of DT40 cells to MN induction by IPMS. Pretreatment with O6-benzyl guanine (OBG), an inhibitor of MGMT, increased the MN frequency in DT40 cells treated with nPMS, but not IPMS. Lastly, IPMS induced more double strand breaks in FANC-deficient cells compared to wild-type cells in a time-dependent manner. All together, these results suggest that IPMS-derived O6-isopropyl dG escapes recognition by MGMT, and the unrepaired DNA damage leads to double strand breaks, resulting in MN induction. FANC, therefore, plays a pivotal role in preventing MN induction and cell death caused by IPMS. PMID:27486975

  3. Rad50 Is Not Essential for the Mre11-Dependent Repair of DNA Double-Strand Breaks in Halobacterium sp. Strain NRC-1▿

    PubMed Central

    Kish, A.; DiRuggiero, J.

    2008-01-01

    The genome of the halophilic archaeon Halobacterium sp. strain NRC-1 encodes homologs of the eukaryotic Mre11 and Rad50 proteins, which are involved in the recognition and end processing of DNA double-strand breaks in the homologous recombination repair pathway. We have analyzed the phenotype of Halobacterium deletion mutants lacking mre11 and/or rad50 after exposure to UV-C radiation, an alkylating agent (N-methyl-N′-nitro-N-nitrosoguanidine), and γ radiation, none of which resulted in a decrease in survival of the mutant strains compared to that of the background strain. However, a decreased rate of repair of DNA double-strand breaks in strains lacking the mre11 gene was observed using pulsed-field gel electrophoresis. These observations led to the hypothesis that Mre11 is essential for the repair of DNA double-strand breaks in Halobacterium, whereas Rad50 is dispensable. This is the first identification of a Rad50-independent function for the Mre11 protein, and it represents a shift in the Archaea away from the eukaryotic model of homologous recombination repair of DNA double-strand breaks. PMID:18502851

  4. Rad50 is not essential for the Mre11-dependent repair of DNA double-strand breaks in Halobacterium sp. strain NRC-1.

    PubMed

    Kish, A; DiRuggiero, J

    2008-08-01

    The genome of the halophilic archaeon Halobacterium sp. strain NRC-1 encodes homologs of the eukaryotic Mre11 and Rad50 proteins, which are involved in the recognition and end processing of DNA double-strand breaks in the homologous recombination repair pathway. We have analyzed the phenotype of Halobacterium deletion mutants lacking mre11 and/or rad50 after exposure to UV-C radiation, an alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), and gamma radiation, none of which resulted in a decrease in survival of the mutant strains compared to that of the background strain. However, a decreased rate of repair of DNA double-strand breaks in strains lacking the mre11 gene was observed using pulsed-field gel electrophoresis. These observations led to the hypothesis that Mre11 is essential for the repair of DNA double-strand breaks in Halobacterium, whereas Rad50 is dispensable. This is the first identification of a Rad50-independent function for the Mre11 protein, and it represents a shift in the Archaea away from the eukaryotic model of homologous recombination repair of DNA double-strand breaks.

  5. 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.

  6. The Caenorhabditis elegans Homolog of Gen1/Yen1 Resolvases Links DNA Damage Signaling to DNA Double-Strand Break Repair

    PubMed Central

    Bailly, Aymeric P.; Alpi, Arno; Lilley, David M. J.; Ahmed, Shawn; Gartner, Anton

    2010-01-01

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53–mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair. PMID:20661466

  7. 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.

  8. 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.

  9. 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

  10. Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining.

    PubMed

    Jessulat, Matthew; Malty, Ramy H; Nguyen-Tran, Diem-Hang; Deineko, Viktor; Aoki, Hiroyuki; Vlasblom, James; Omidi, Katayoun; Jin, Ke; Minic, Zoran; Hooshyar, Mohsen; Burnside, Daniel; Samanfar, Bahram; Phanse, Sadhna; Freywald, Tanya; Prasad, Bhanu; Zhang, Zhaolei; Vizeacoumar, Franco; Krogan, Nevan J; Freywald, Andrew; Golshani, Ashkan; Babu, Mohan

    2015-07-01

    The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

    PubMed Central

    Kostyrko, Kaja; Mermod, Nicolas

    2016-01-01

    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. PMID:26657630

  12. Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

    PubMed Central

    Jessulat, Matthew; Malty, Ramy H.; Nguyen-Tran, Diem-Hang; Deineko, Viktor; Aoki, Hiroyuki; Vlasblom, James; Omidi, Katayoun; Jin, Ke; Minic, Zoran; Hooshyar, Mohsen; Burnside, Daniel; Samanfar, Bahram; Phanse, Sadhna; Freywald, Tanya; Prasad, Bhanu; Zhang, Zhaolei; Vizeacoumar, Franco; Krogan, Nevan J.; Freywald, Andrew

    2015-01-01

    The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals. PMID:25963654

  13. RSC facilitates Rad59-dependent homologous recombination between sister chromatids by promoting cohesin loading at DNA double-strand breaks.

    PubMed

    Oum, Ji-Hyun; Seong, Changhyun; Kwon, Youngho; Ji, Jae-Hoon; Sid, Amy; Ramakrishnan, Sreejith; Ira, Grzegorz; Malkova, Anna; Sung, Patrick; Lee, Sang Eun; Shim, Eun Yong

    2011-10-01

    Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.

  14. 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.

  15. Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks

    PubMed Central

    van Attikum, Haico; Fritsch, Olivier; Gasser, Susan M

    2007-01-01

    INO80 and SWR1 are two closely related ATP-dependent chromatin remodeling complexes that share several subunits. Ino80 was reported to be recruited to the HO endonuclease-induced double-strand break (DSB) at the budding yeast mating-type locus, MAT. We find Swr1 similarly recruited in a manner dependent on the phosphorylation of H2A (γH2AX). This is not unique to cleavage at MAT; both Swr1 and Ino80 bind near an induced DSB on chromosome XV. Whereas Swr1 incorporates the histone variant H2A.Z into chromatin at promoters, H2A.Z levels do not increase at DSBs. Instead, H2A.Z, γH2AX and core histones are coordinately removed near the break in an INO80-dependent, but SWR1-independent, manner. Mutations in INO80-specific subunits Arp8 or Nhp10 impair the binding of Mre11 nuclease, yKu80 and ATR-related Mec1 kinase at the DSB, resulting in defective end-processing and checkpoint activation. In contrast, Mre11 binding, end-resection and checkpoint activation were normal in the swr1 strain, but yKu80 loading and error-free end-joining were impaired. Thus, these two related chromatin remodelers have distinct roles in DSB repair and checkpoint activation. PMID:17762868

  16. The cytotoxicity of (–)-lomaiviticin A arises from induction of double-strand breaks in DNA

    PubMed Central

    Colis, Laureen C.; Woo, Christina M.; Hegan, Denise C.; Li, Zhenwu; Glazer, Peter M.; Herzon, Seth B.

    2014-01-01

    The metabolite (–)-lomaiviticin A, which contains two diazotetrahydrobenzo[b]fluorene (diazofluorene) functional groups, inhibits the growth of cultured human cancer cells at nanomolar–picomolar concentrations; however, the mechanism responsible for the potent cytotoxicity of this natural product is not known. Here we report that (–)-lomaiviticin A nicks and cleaves plasmid DNA by an ROS- and iron-independent pathway and that the potent cytotoxicity of (–)-lomaiviticin A arises from induction of DNA double-strand breaks (dsbs). In a plasmid cleavage assay, the ratio of single-strand breaks (ssbs) to dsbs is 5.3±0.6:1. Labeling studies suggest this cleavage occurs via a radical pathway. The structurally related isolates (–)-lomaiviticin C and (–)-kinamycin C, which contain one diazofluorene, are demonstrated to be much less effective DNA cleavage agents, thereby providing an explanation for the enhanced cytotoxicity of (–)-lomaiviticin A compared to other members of this family. PMID:24848236

  17. 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

  18. 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

  19. Multiple heterologies increase mitotic double-strand break-induced allelic gene conversion tract lengths in yeast.

    PubMed Central

    Nickoloff, J A; Sweetser, D B; Clikeman, J A; Khalsa, G J; Wheeler, S L

    1999-01-01

    Spontaneous and double-strand break (DSB)-induced allelic recombination in yeast was investigated in crosses between ura3 heteroalleles inactivated by an HO site and a +1 frameshift mutation, with flanking markers defining a 3.4-kbp interval. In some crosses, nine additional phenotypically silent RFLP mutations were present at approximately 100-bp intervals. Increasing heterology from 0.2 to 1% in this interval reduced spontaneous, but not DSB-induced, recombination. For DSB-induced events, 75% were continuous tract gene conversions without a crossover in this interval; discontinuous tracts and conversions associated with a crossover each comprised approximately 7% of events, and 10% also converted markers in unbroken alleles. Loss of heterozygosity was seen for all markers centromere distal to the HO site in 50% of products; such loss could reflect gene conversion, break-induced replication, chromosome loss, or G2 crossovers. Using telomere-marked strains we determined that nearly all allelic DSB repair occurs by gene conversion. We further show that most allelic conversion results from mismatch repair of heteroduplex DNA. Interestingly, markers shared between the sparsely and densely marked interval converted at higher rates in the densely marked interval. Thus, the extra markers increased gene conversion tract lengths, which may reflect mismatch repair-induced recombination, or a shift from restoration- to conversion-type repair. PMID:10511547

  20. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.

    PubMed

    Zhu, Zhu; Chung, Woo-Hyun; Shim, Eun Yong; Lee, Sang Eun; Ira, Grzegorz

    2008-09-19

    Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA-damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remain unknown. We monitored 5'-strand resection at inducible DSB ends in yeast and identified proteins required for two stages of resection: initiation and long-range 5'-strand resection. We show that the Mre11-Rad50-Xrs2 complex (MRX) initiates 5' degradation, whereas Sgs1 and Dna2 degrade 5' strands exposing long 3' strands. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single-strand annealing between distant repeats while the remaining long-range resection activity depends on the exonuclease Exo1. In exo1Deltasgs1Delta double mutants, the MRX complex together with Sae2 nuclease generate, in a stepwise manner, only few hundred nucleotides of ssDNA at the break, resulting in inefficient gene conversion and G2/M damage checkpoint arrest. These results provide important insights into the early steps of DSB repair in eukaryotes.

  1. Molecular Analysis of Base Damage Clustering Associated with a Site-Specific Radiation-Induced DNA Double-Strand Break

    PubMed Central

    Datta, Kamal; Jaruga, Pawel; Dizdaroglu, Miral; Neumann, Ronald D.; Winters, Thomas A.

    2010-01-01

    Base damage flanking a radiation-induced DNA double-strand break (DSB) may contribute to DSB complexity and affect break repair. However, to date, an isolated radiation-induced DSB has not been assessed for such structures at the molecular level. In this study, an authentic site-specific radiation-induced DSB was produced in plasmid DNA by triplex forming oligonucleotide-targeted 125I decay. A restriction fragment terminated by the DSB was isolated and probed for base damage with the E. coli DNA repair enzymes, endonuclease III and formamidopyrimidine-DNA glycosylase. Our results demonstrate base damage clustering within 8 bases of the 125I-targeted base in the DNA duplex. An increased yield of base damage (purine>pyrimidine) was observed for DSBs formed by irradiation in the absence of DMSO. An internal control fragment 1354 bp upstream from the targeted base was insensitive to enzymatic probing, indicating the damage detected proximal to the DSB was produced by the 125I decay that formed the DSB. Gas chromatography-mass spectrometry identified three types of damaged bases in the ~32 bp region proximal to the DSB. These base lesions were 8-hydroxyguanine, 8-hydroxyadenine, and 5-hydroxycytosine. Finally, evidence is presented for base damage >24 bp upstream from the 125I-decay site that may form via a charge migration mechanism. PMID:17067210

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

    PubMed Central

    Davis, Luther; Maizels, Nancy

    2014-01-01

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

  3. 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.

  4. TU-CD-303-02: Beyond Radiation Induced Double Strand Breaks - a New Horizon for Radiation Therapy Research

    SciTech Connect

    Chang, S.

    2015-06-15

    Recent advances in cancer research have shed new light on the complex processes of how therapeutic radiation initiates changes at cellular, tissue, and system levels that may lead to clinical effects. These new advances may transform the way we use radiation to combat certain types of cancers. For the past two decades many technological advancements in radiation therapy have been largely based on the hypothesis that direct radiation-induced DNA double strand breaks cause cell death and thus tumor control and normal tissue damage. However, new insights have elucidated that in addition to causing cellular DNA damage, localized therapeutic radiation also initiates cascades of complex downstream biological responses in tissue that extend far beyond where therapeutic radiation dose is directly deposited. For instance, studies show that irradiated dying tumor cells release tumor antigens that can lead the immune system to a systemic anti-cancer attack throughout the body of cancer patient; targeted irradiation to solid tumor also increases the migration of tumor cells already in bloodstream, the seeds of potential metastasis. Some of the new insights may explain the long ago discovered but still unexplained non-localized radiation effects (bystander effect and abscopal effect) and the efficacy of spatially fractionated radiation therapy (microbeam radiation therapy and GRID therapy) where many “hot” and “cold” spots are intentionally created throughout the treatment volume. Better understanding of the mechanisms behind the non-localized radiation effects creates tremendous opportunities to develop new and integrated cancer treatment strategies that are based on radiotherapy, immunology, and chemotherapy. However, in the multidisciplinary effort to advance new radiobiology, there are also tremendous challenges including a lack of multidisciplinary researchers and imaging technologies for the microscopic radiation-induced responses. A better grasp of the essence of

  5. Production of single- and double-strand breaks in plasmid DNA by ozone

    SciTech Connect

    Hamelin, C.

    1985-02-01

    Agarose gel electrophoresis and electron microscopy were used to determine the type of lesions produced in DNA by ozone. This strong oxidizing agent was found to relax, linearize, then degrade native plasmid (pAT153) DNA molecules in solution. Ozone, like ionizing radiation, thus produced DNA breakage. To ascertain this point, wild-type and radiosensitive strains of Escherichia coli were transfected with control or ozonated plasmid DNA, and the host cells were selected for antibiotic resistance. A significant reduction in the transforming ability of pAT153 was observed following ozonation. Mutants deficient in the repair of DNA single-strand breaks yielded less ampicillin- or tetracycline-resistant clones than repair-proficient strains. In E. coli, the same gene products are probably involved in the repair of both radiation- and ozone-induced DNA breaks.

  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. Reduced contribution of thermally-labile sugar lesions to DNA double-strand break formation after exposure to neutrons.

    PubMed

    Singh, Satyendra K; Wu, Wenqi; Stuschke, Martin; Bockisch, Andreas; Iliakis, George

    2012-12-01

    In cells exposed to ionizing radiation, double-strand breaks (DSBs) form within clustered damage sites from lesions disrupting the DNA sugar-phosphate backbone. It is commonly assumed that DSBs form promptly and are immediately detected and processed by the cellular DNA damage response apparatus. However, DSBs also form by delayed chemical conversion of thermally-labile sugar lesions (TLSL) to breaks. We recently reported that conversion of thermally-labile sugar lesions to breaks occurs in cells maintained at physiological temperatures. Here, we investigate the influence of radiation quality on the formation of thermally-labile sugar lesions dependent DSBs. We show that, although the yields of total DSBs are very similar after exposure to neutrons and X rays, the yields of thermally-labile sugar lesions dependent DSBs from neutrons are decreased in comparison to that from X rays. Thus, the yields of prompt DSBs for neutrons are greater than for X rays. Notably, after neutron irradiation the decreased yield of thermally-labile sugar lesion dependent DSBs is strongly cell line dependent, likely reflecting subtle differences in DNA organization. We propose that the higher ionization density of neutrons generates with higher probability prompt DSBs within ionization clusters and renders the ensuing chemical evolution of thermally-labile sugar lesions inconsequential to DNA integrity. Modification of thermally-labile sugar lesion evolution may define novel radiation protection strategies aiming at decreasing DSB formation by chemically preserving thermally-labile sugar lesions until other DSB contributing lesions within the clustered damage site are removed by non-DSB repair pathways.

  9. Loss of p15/Ink4b accompanies tumorigenesis triggered by complex DNA double-strand breaks

    PubMed Central

    Camacho, Cristel V.; Mukherjee, Bipasha; McEllin, Brian; Ding, Liang-Hao; Hu, Burong; Habib, Amyn A.; Xie, Xian-Jin; Nirodi, Chaitanya S.; Saha, Debabrata; Story, Michael D.; Balajee, Adayabalam S.; Bachoo, Robert M.; Boothman, David A.; Burma, Sandeep

    2010-01-01

    DNA double-strand breaks (DSBs) are the most deleterious lesion inflicted by ionizing radiation. Although DSBs are potentially carcinogenic, it is not clear whether complex DSBs that are refractory to repair are more potently tumorigenic compared with simple breaks that can be rapidly repaired, correctly or incorrectly, by mammalian cells. We previously demonstrated that complex DSBs induced by high-linear energy transfer (LET) Fe ions are repaired slowly and incompletely, whereas those induced by low-LET gamma rays are repaired efficiently by mammalian cells. To determine whether Fe-induced DSBs are more potently tumorigenic than gamma ray-induced breaks, we irradiated ‘sensitized’ murine astrocytes that were deficient in Ink4a and Arf tumor suppressors and injected the surviving cells subcutaneously into nude mice. Using this model system, we find that Fe ions are potently tumorigenic, generating tumors with significantly higher frequency and shorter latency compared with tumors generated by gamma rays. Tumor formation by Fe-irradiated cells is accompanied by rampant genomic instability and multiple genomic changes, the most interesting of which is loss of the p15/Ink4b tumor suppressor due to deletion of a chromosomal region harboring the CDKN2A and CDKN2B loci. The additional loss of p15/Ink4b in tumors derived from cells that are already deficient in p16/Ink4a bolsters the hypothesis that p15 plays an important role in tumor suppression, especially in the absence of p16. Indeed, we find that reexpression of p15 in tumor-derived cells significantly attenuates the tumorigenic potential of these cells, indicating that p15 loss may be a critical event in tumorigenesis triggered by complex DSBs. PMID:20663777

  10. Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break.

    PubMed

    Brown, M Scott; Grubb, Jennifer; Zhang, Annie; Rust, Michael J; Bishop, Douglas K

    2015-12-01

    The Eukaryotic RecA-like proteins Rad51 and Dmc1 cooperate during meiosis to promote recombination between homologous chromosomes by repairing programmed DNA double strand breaks (DSBs). Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci. Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm. Paired co-foci remain prevalent when DSBs are dramatically reduced or when strand exchange or synapsis is blocked. Super-resolution dSTORM microscopy reveals that individual foci observed by conventional light microscopy are often composed of two or more substructures. The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt) Rad51 filaments and also by one or more short Dmc1 filaments.

  11. A single subexcitation-energy electron can induce a double-strand break in DNA modified by platinum chemotherapeutic drugs.

    PubMed

    Rezaee, Mohammad; Alizadeh, Elahe; Cloutier, Pierre; Hunting, Darel J; Sanche, Léon

    2014-06-01

    The sensitization of malignant cells to ionizing radiation is the clinical rationale for the use of platinum-drug-based concurrent chemoradiotherapy (CCRT) for cancer treatment; however, the specific mechanisms of radiosensitization and their respective contributions still remain unknown. Biological mechanisms such as inhibition of DNA repair may contribute to the efficacy of CCRT; nevertheless, there is a dearth of information on the possible contribution of nanoscopic mechanisms to the generation of lethal DNA lesions, such as double-strand breaks (DSB). The present study demonstrates that the abundant near zero-eV (0.5 eV) electrons, created by ionizing radiation during radiotherapy, induce DSB in supercoiled plasmid DNA modified by platinum-containing anticancer drugs (Pt drugs), but not in unmodified DNA. They do so more efficiently than other types of radiation, including soft X-rays and 10 eV electrons. The formation of DSB by 0.5 eV electrons is found to be a single-hit process. These findings reveal insights into the radiosensitization mechanism of Pt drugs that can have implications for the development of optimal clinical protocols for platinum-based CCRT and the deployment of in situ sources of subexcitation-energy electrons (e.g., Auger electron-emitting radionuclides) to efficiently enhance DSB formation in DNA modified by Pt drugs in malignant cells.

  12. 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.

  13. MEI4 – a central player in the regulation of meiotic DNA double-strand break formation in the mouse.

    PubMed

    Kumar, Rajeev; Ghyselinck, Norbert; Ishiguro, Kei-ichiro; Watanabe, Yoshinori; Kouznetsova, Anna; Höög, Christer; Strong, Edward; Schimenti, John; Daniel, Katrin; Toth, Attila; de Massy, Bernard

    2015-05-01

    The formation of programmed DNA double-strand breaks (DSBs) at the beginning of meiotic prophase marks the initiation of meiotic recombination. Meiotic DSB formation is catalyzed by SPO11 and their repair takes place on meiotic chromosome axes. The evolutionarily conserved MEI4 protein is required for meiotic DSB formation and is localized on chromosome axes. Here, we show that HORMAD1, one of the meiotic chromosome axis components, is required for MEI4 localization. Importantly, the quantitative correlation between the level of axis-associated MEI4 and DSB formation suggests that axis-associated MEI4 could be a limiting factor for DSB formation. We also show that MEI1, REC8 and RAD21L are important for proper MEI4 localization. These findings on MEI4 dynamics during meiotic prophase suggest that the association of MEI4 to chromosome axes is required for DSB formation, and that the loss of this association upon DSB repair could contribute to turning off meiotic DSB formation.

  14. 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.

  15. 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.

  16. 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.

  17. DNA double-strand breaks, but not crossovers, are required for the reorganization of meiotic nuclei in Tetrahymena.

    PubMed

    Mochizuki, Kazufumi; Novatchkova, Maria; Loidl, Josef

    2008-07-01

    During meiosis, the micronuclei of the ciliated protist Tetrahymena thermophila elongate dramatically. Within these elongated nuclei, chromosomes are arranged in a bouquet-like fashion and homologous pairing and recombination takes place. We studied meiotic chromosome behavior in Tetrahymena in the absence of two genes, SPO11 and a homolog of HOP2 (HOP2A), which have conserved roles in the formation of meiotic DNA double-strand breaks (DSBs) and their repair, respectively. Single-knockout mutants for each gene display only a moderate reduction in chromosome pairing, but show a complete failure to form chiasmata and exhibit chromosome missegregation. The lack of SPO11 prevents the elongation of meiotic nuclei, but it is restored by the artificial induction of DSBs. In the hop2ADelta mutant, the transient appearance of gamma-H2A.X and Rad51p signals indicates the formation and efficient repair of DSBs; but this repair does not occur by interhomolog crossing over. In the absence of HOP2A, the nuclei are elongated, meaning that DSBs but not their conversion to crossovers are required for the development of this meiosis-specific morphology. In addition, by in silico homology searches, we compiled a list of likely Tetrahymena meiotic proteins as the basis for further studies of the unusual synaptonemal complex-less meiosis in this phylogenetically remote model organism.

  18. Tel1 and Rif2 Regulate MRX Functions in End-Tethering and Repair of DNA Double-Strand Breaks

    PubMed Central

    Wang, Weibin; Niu, Hengyao; Clerici, Michela; Sung, Patrick; Longhese, Maria Pia

    2016-01-01

    The cellular response to DNA double-strand breaks (DSBs) is initiated by the MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast; Mre11-Rad50-Nbs1 in mammals), which recruits the checkpoint kinase Tel1/ATM to DSBs. In Saccharomyces cerevisiae, the role of Tel1 at DSBs remains enigmatic, as tel1Δ cells do not show obvious hypersensitivity to DSB-inducing agents. By performing a synthetic phenotype screen, we isolated a rad50-V1269M allele that sensitizes tel1Δ cells to genotoxic agents. The MRV1269MX complex associates poorly to DNA ends, and its retention at DSBs is further reduced by the lack of Tel1. As a consequence, tel1Δ rad50-V1269M cells are severely defective both in keeping the DSB ends tethered to each other and in repairing a DSB by either homologous recombination (HR) or nonhomologous end joining (NHEJ). These data indicate that Tel1 promotes MRX retention to DSBs and this function is important to allow proper MRX-DNA binding that is needed for end-tethering and DSB repair. The role of Tel1 in promoting MRX accumulation to DSBs is counteracted by Rif2, which is recruited to DSBs. We also found that Rif2 enhances ATP hydrolysis by MRX and attenuates MRX function in end-tethering, suggesting that Rif2 can regulate MRX activity at DSBs by modulating ATP-dependent conformational changes of Rad50. PMID:26901759

  19. Tel1 and Rif2 Regulate MRX Functions in End-Tethering and Repair of DNA Double-Strand Breaks.

    PubMed

    Cassani, Corinne; Gobbini, Elisa; Wang, Weibin; Niu, Hengyao; Clerici, Michela; Sung, Patrick; Longhese, Maria Pia

    2016-02-01

    The cellular response to DNA double-strand breaks (DSBs) is initiated by the MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast; Mre11-Rad50-Nbs1 in mammals), which recruits the checkpoint kinase Tel1/ATM to DSBs. In Saccharomyces cerevisiae, the role of Tel1 at DSBs remains enigmatic, as tel1Δ cells do not show obvious hypersensitivity to DSB-inducing agents. By performing a synthetic phenotype screen, we isolated a rad50-V1269M allele that sensitizes tel1Δ cells to genotoxic agents. The MRV1269MX complex associates poorly to DNA ends, and its retention at DSBs is further reduced by the lack of Tel1. As a consequence, tel1Δ rad50-V1269M cells are severely defective both in keeping the DSB ends tethered to each other and in repairing a DSB by either homologous recombination (HR) or nonhomologous end joining (NHEJ). These data indicate that Tel1 promotes MRX retention to DSBs and this function is important to allow proper MRX-DNA binding that is needed for end-tethering and DSB repair. The role of Tel1 in promoting MRX accumulation to DSBs is counteracted by Rif2, which is recruited to DSBs. We also found that Rif2 enhances ATP hydrolysis by MRX and attenuates MRX function in end-tethering, suggesting that Rif2 can regulate MRX activity at DSBs by modulating ATP-dependent conformational changes of Rad50.

  20. Age-dependent change of HMGB1 and DNA double-strand break accumulation in mouse brain.

    PubMed

    Enokido, Yasushi; Yoshitake, Ayaka; Ito, Hikaru; Okazawa, Hitoshi

    2008-11-07

    HMGB1 is an evolutionarily conserved non-histone chromatin-associated protein with key roles in maintenance of nuclear homeostasis; however, the function of HMGB1 in the brain remains largely unknown. Recently, we found that the reduction of nuclear HMGB1 protein level in the nucleus associates with DNA double-strand break (DDSB)-mediated neuronal damage in Huntington's disease [M.L. Qi, K. Tagawa, Y. Enokido, N. Yoshimura, Y. Wada, K. Watase, S. Ishiura, I. Kanazawa, J. Botas, M. Saitoe, E.E. Wanker, H. Okazawa, Proteome analysis of soluble nuclear proteins reveals that HMGB1/2 suppress genotoxic stress in polyglutamine diseases, Nat. Cell Biol. 9 (2007) 402-414]. In this study, we analyze the region- and cell type-specific changes of HMGB1 and DDSB accumulation during the aging of mouse brain. HMGB1 is localized in the nuclei of neurons and astrocytes, and the protein level changes in various brain regions age-dependently. HMGB1 reduces in neurons, whereas it increases in astrocytes during aging. In contrast, DDSB remarkably accumulates in neurons, but it does not change significantly in astrocytes during aging. These results indicate that HMGB1 expression during aging is differentially regulated between neurons and astrocytes, and suggest that the reduction of nuclear HMGB1 might be causative for DDSB in neurons of the aged brain.

  1. Epigenetic Modifications and Accumulation of DNA Double-Strand Breaks in Oral Lichen Planus Lesions Presenting Poor Response to Therapy

    PubMed Central

    Dillenburg, Caroline S.; Martins, Marco A.T.; Almeida, Luciana O.; Meurer, Luise; Squarize, Cristiane H.; Martins, Manoela D.; Castilho, Rogerio M.

    2015-01-01

    Abstract Epigenetics refers to changes in cell characteristics that occur independently of modifications to the deoxyribonucleic acid (DNA) sequence. Alterations mediated by epigenetic mechanisms are important factors in cancer progression. Although an exciting prospect, the identification of early epigenetic markers associated with clinical outcome in premalignant and malignant disorders remains elusive. We examined alterations in chromatin acetylation in oral lichen planus (OLP) with distinct clinical behavior and compared the alterations to the levels of DNA double-strand breaks (DSBs). We analyzed 42 OLP patients, who had different responses to therapy, for acetyl-histone H3 at lys9 (H3K9ac), which is associated with enhanced transcription and nuclear decondensation, and the presence of DSBs, as determined by accumulation of phosphorylated γH2AX foci. Patients with high levels of H3K9ac acetylation failed to respond to therapy or experienced disease recurrence shortly after therapy. Similar to H3K9ac, patients who responded poorly to therapy had increased accumulation of DNA DSB, indicating genomic instability. These findings suggest that histone modifications occur in OLP, and H3K9ac and γH2AX histones may serve as epigenetic markers for OLP recurrence. PMID:26222871

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

    PubMed Central

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

    2016-01-01

    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. PMID:26609070

  3. Impaired resection of meiotic double-strand breaks channels repair to nonhomologous end joining in Caenorhabditis elegans.

    PubMed

    Yin, Yizhi; Smolikove, Sarit

    2013-07-01

    Repair of double-strand DNA breaks (DSBs) by the homologous recombination (HR) pathway results in crossovers (COs) required for a successful first meiotic division. Mre11 is one member of the MRX/N (Mre11, Rad50, and Xrs2/Nbs1) complex required for meiotic DSB formation and for resection in Saccharomyces cerevisiae. In Caenorhabditis elegans, evidence for the MRX/N role in DSB resection is limited. We report the first separation-of-function allele, mre-11(iow1) in C. elegans, which is specifically defective in meiotic DSB resection but not in formation. The mre-11(iow1) mutants displayed chromosomal fragmentation and aggregation in late prophase I. Recombination intermediates and crossover formation was greatly reduced in mre-11(iow1) mutants. Irradiation-induced DSBs during meiosis failed to be repaired from early to middle prophase I in mre-11(iow1) mutants. In the absence of a functional HR, our data suggest that some DSBs in mre-11(iow1) mutants are repaired by the nonhomologous end joining (NHEJ) pathway, as removing NHEJ partially suppressed the meiotic defects shown by mre-11(iow1). In the absence of NHEJ and a functional MRX/N, meiotic DSBs are channeled to EXO-1-dependent HR repair. Overall, our analysis supports a role for MRE-11 in the resection of DSBs in middle meiotic prophase I and in blocking NHEJ.

  4. Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining.

    PubMed

    Lu, Guangqing; Duan, Jinzhi; Shu, Sheng; Wang, Xuxiang; Gao, Linlin; Guo, Jing; Zhang, Yu

    2016-02-02

    In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated. Surprisingly, we found that both Lig1- and Lig3-containing complexes could efficiently catalyze A-EJ for class switching recombination (CSR) in the IgH locus and chromosomal deletions between DSBs generated by CRISPR/Cas9 in cis-chromosomes. However, only deletion of nuclear Lig3, but not Lig1, could significantly reduce the interchromosomal translocations in Lig4(-/-) cells, suggesting the unique role of Lig3 in catalyzing chromosome translocation. Additional sequence analysis of chromosome translocation junction microhomology revealed the specificity of different ligase-containing complexes. The data suggested the existence of multiple DNA ligase-containing complexes in A-EJ.

  5. Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair.

    PubMed

    Reid, Dylan A; Keegan, Sarah; Leo-Macias, Alejandra; Watanabe, Go; Strande, Natasha T; Chang, Howard H; Oksuz, Betul Akgol; Fenyo, David; Lieber, Michael R; Ramsden, Dale A; Rothenberg, Eli

    2015-05-19

    Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.

  6. Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

    PubMed Central

    Reid, Dylan A.; Keegan, Sarah; Leo-Macias, Alejandra; Watanabe, Go; Strande, Natasha T.; Chang, Howard H.; Oksuz, Betul Akgol; Fenyo, David; Lieber, Michael R.; Ramsden, Dale A.; Rothenberg, Eli

    2015-01-01

    Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation. PMID:25941401

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

    PubMed Central

    Westmoreland, James W.; Resnick, Michael A.

    2016-01-01

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

  8. Repair rates of DNA double-strand breaks under different doses of proton and γ-ray irradiation

    NASA Astrophysics Data System (ADS)

    Wu, Jingwen; Fu, Qibin; Quan, Yi; Wang, Weikang; Mei, Tao; Li, Jia; Yang, Gen; Ren, Xiaotang; Xue, Jianming; Wang, Yugang

    2012-04-01

    It is known that DNA double-strand breaks (DSBs), which can be induced by a variety of treatments including ionizing radiation (IR), can cause most deleterious consequences among all kinds of DNA lesions. However, it is still under debate about whether DSBs repair is equally efficient after low and high-LET radiation, especially the basic biological responses after exposure to high-LET particles. In present study, synchronous fibroblast normal Human lung fibroblast (NHLF) cells were irradiated with graded doses of proton and γ-ray. Then γ-H2AX foci assay was used to monitor DSBs induction and repair at 0.5, 1, 2, 4, and 18 h post irradiation. The results showed that the γ-ray irradiation could produce more γ-H2AX foci than proton irradiation at the same dose. However, compared to low LET radiation with γ-ray, the results also showed a much slower DSBs repair rate after high LET radiation with protons, suggesting that the cellular ability to eliminate DSBs after low and high-LET ionizing radiation is quite different.

  9. Vilya, a component of the recombination nodule, is required for meiotic double-strand break formation in Drosophila

    PubMed Central

    Lake, Cathleen M; Nielsen, Rachel J; Guo, Fengli; Unruh, Jay R; Slaughter, Brian D; Hawley, R Scott

    2015-01-01

    Meiotic recombination begins with the induction of programmed double-strand breaks (DSBs). In most organisms only a fraction of DSBs become crossovers. Here we report a novel meiotic gene, vilya, which encodes a protein with homology to Zip3-like proteins shown to determine DSB fate in other organisms. Vilya is required for meiotic DSB formation, perhaps as a consequence of its interaction with the DSB accessory protein Mei-P22, and localizes to those DSB sites that will mature into crossovers. In early pachytene Vilya localizes along the central region of the synaptonemal complex and to discrete foci. The accumulation of Vilya at foci is dependent on DSB formation. Immuno-electron microscopy demonstrates that Vilya is a component of recombination nodules, which mark the sites of crossover formation. Thus Vilya links the mechanism of DSB formation to either the selection of those DSBs that will become crossovers or to the actual process of crossing over. DOI: http://dx.doi.org/10.7554/eLife.08287.001 PMID:26452093

  10. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks.

    PubMed

    Dantuma, Nico P; Pfeiffer, Annika

    2016-01-01

    Ubiquitin and the ubiquitin-like modifier SUMO are intimately connected with the cellular response to various types of DNA damage. A striking feature is the local accumulation of these proteinaceous post-translational modifications in the direct vicinity to DNA double-strand breaks, which plays a critical role in the formation of ionizing radiation-induced foci. The functional significance of these modifications is the coordinated recruitment and removal of proteins involved in DNA damage signaling and repair in a timely manner. The central orchestrators of these processes are the ubiquitin and SUMO ligases that are responsible for accurately tagging a broad array of chromatin and chromatin-associated proteins thereby changing their behavior or destination. Despite many differences in the mode of action of these enzymes, they share some striking features that are of direct relevance for their function in the DNA damage response. In this review, we outline the molecular mechanisms that are responsible for the recruitment of ubiquitin and SUMO ligases and discuss the importance of chromatin proximity in this process.

  11. Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli

    PubMed Central

    SOLANKY, DIPESH; HAYDEL, SHELLEY E.

    2012-01-01

    This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which primarily associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions consisting of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to the CB clay resulted in significantly longer comet lengths, compared to water and kanamycin exposures, suggesting that the induction of DNA DSBs contributes to the killing activity of this antibacterial clay mineral mixture. The comet assay protocol described herein provides a general technique for evaluating soluble antimicrobial-derived DNA damage and for comparing DNA fragmentation between experimental and control assays. PMID:22940101

  12. Non-redundant Functions of ATM and DNA-PKcs in Response to DNA Double-Strand Breaks.

    PubMed

    Caron, Pierre; Choudjaye, Jonathan; Clouaire, Thomas; Bugler, Béatrix; Daburon, Virginie; Aguirrebengoa, Marion; Mangeat, Thomas; Iacovoni, Jason S; Álvarez-Quilón, Alejandro; Cortés-Ledesma, Felipe; Legube, Gaëlle

    2015-11-24

    DNA double-strand breaks (DSBs) elicit the so-called DNA damage response (DDR), largely relying on ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PKcs), two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI) combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within "repair foci." Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival.

  13. Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells

    SciTech Connect

    Bauerschmidt, Christina; Helleday, Thomas

    2011-02-01

    Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.

  14. Single- and Double-Strand Breaks of Dry DNA Exposed to Protons at Bragg-Peak Energies.

    PubMed

    Souici, Mounir; Khalil, Talat T; Muller, Dominique; Raffy, Quentin; Barillon, Rémi; Belafrites, Abdelfettah; Champion, Christophe; Fromm, Michel

    2017-01-26

    Ultrathin layers (<20 nm) of pBR322 plasmid DNA were deposited onto 2.5 μm thick polyester films and exposed to proton Bragg-peak energies (90-3000 keV) at various fluences. A quantitative analysis of radio-induced DNA damage is reported here in terms of single- and double-strand breaks (SSB and DSB, respectively). The corresponding yields as well as G-values and the cross sections exhibit fairly good agreement with the rare available data, stemming from close experimental conditions, namely, based on α particle irradiation. SSB/DSB rates appear to be linear when plotted against linear energy transfer (LET) in the whole energy range studied. All the data present a maximum in the 150-200 keV energy range; as for LET, it peaks at 90 keV. We also show that fragmentation starts to be significant for proton fluences greater than 1 × 10(11) cm(-2) at the Bragg-peak energies. Finally, we determine the average proton track radial extension, rmax, corresponding to an occupation probability of 100% DSB in the Bragg-peak region. The rmax values determined are in excellent agreement with the radial extensions of proton tracks determined by simulation approaches in water. When plotted as a function of LET, both SSB and DSB cross sections bend back at high LETs.

  15. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks

    PubMed Central

    Dantuma, Nico P.; Pfeiffer, Annika

    2016-01-01

    Ubiquitin and the ubiquitin-like modifier SUMO are intimately connected with the cellular response to various types of DNA damage. A striking feature is the local accumulation of these proteinaceous post-translational modifications in the direct vicinity to DNA double-strand breaks, which plays a critical role in the formation of ionizing radiation-induced foci. The functional significance of these modifications is the coordinated recruitment and removal of proteins involved in DNA damage signaling and repair in a timely manner. The central orchestrators of these processes are the ubiquitin and SUMO ligases that are responsible for accurately tagging a broad array of chromatin and chromatin-associated proteins thereby changing thei