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1.  Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair 
PLoS Genetics  2008;4(6):e1000110.
Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB) repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ), single-strand annealing (SSA), and homology directed repair (HDR/GC). Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI–induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy.
Author Summary
Changes to the sequence of DNA, or mutations, can disrupt cellular growth control genes, which can lead to cancer development. Such mutations likely arise from damage to DNA that is repaired in a way that fails to restore the original sequence. One type of DNA damage is a chromosomal double-strand break. We have developed assays to measure how these breaks are repaired, and also how such repair can lead to mutations. In particular, we present an assay to measure a pathway of repair that results in deletion mutations, often with evidence of short homologous sequences at the repair junctions (alt-NHEJ). We have compared the genetic requirements of this repair pathway in relation to other pathways of repair that use extensive homology. We find that factors KU and CtIP appear to affect the initial stages of repair of each of these pathways, regardless of the length of homology. However, these pathways appear to diverge at later steps, as relates to the role of the repair factors RAD52, ERCC1, and RAD51. Given that mutations observed in some cancer cells are consistent with alt-NHEJ repair, these mechanistic descriptions provide models for how such mutations could arise in cancer.
doi:10.1371/journal.pgen.1000110
PMCID: PMC2430616  PMID: 18584027
2.  The Role of ATM in the Deficiency in Nonhomologous End-Joining near Telomeres in a Human Cancer Cell Line 
PLoS Genetics  2013;9(3):e1003386.
Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.
Author Summary
The ends of chromosomes, called telomeres, prevent chromosome ends from appearing as DNA double-strand breaks (DSBs) and prevent chromosome fusion by forming a specialized nucleo-protein complex. The critical function of telomeres in end protection has a downside, in that it interferes with the repair of DSBs that occur near telomeres. DSBs are critical DNA lesions, because if they are not repaired correctly they can result in gross chromosome rearrangements (GCRs). As a result, the deficiency in DSB repair near telomeres has now been implicated in ageing, by promoting cell senescence, and cancer, by promoting telomere dysfunction due to oncogene-induced replication stress. The studies presented here demonstrate that DSBs near telomeres commonly result in GCRs in a human tumor cell line. Moreover, our results demonstrate that the mechanism of repair of telomeric DSBs is very different from the mechanism of repair of DSBs at other locations, supporting our hypothesis that the deficiency in repair of DSBs near telomeres is a result of the abnormal processing of DSBs due to the presence of telomeric proteins. Understanding the mechanism responsible for the deficiency in DSB repair near telomeres will provide important insights into critical human disease pathways.
doi:10.1371/journal.pgen.1003386
PMCID: PMC3610639  PMID: 23555296
3.  Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences 
Molecular and Cellular Biology  2004;24(21):9305-9316.
Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52−/− mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.
doi:10.1128/MCB.24.21.9305-9316.2004
PMCID: PMC522275  PMID: 15485900
4.  Histone Deacetylase Inhibitors Selectively Target Homology Dependent DNA Repair Defective Cells and Elevate Non-Homologous Endjoining Activity 
PLoS ONE  2014;9(1):e87203.
Background
We have previously used the ATAD5-luciferase high-throughput screening assay to identify genotoxic compounds with potential chemotherapeutic capabilities. The successful identification of known genotoxic agents, including the histone deacetylase inhibitor (HDACi) trichostatin A (TSA), confirmed the specificity of the screen since TSA has been widely studied for its ability to cause apoptosis in cancer cells. Because many cancers have acquired mutations in DNA damage checkpoints or repair pathways, we hypothesized that these cancers may be susceptible to treatments that target compensatory pathways. Here, we used a panel of isogenic chicken DT40 B lymphocyte mutant and human cell lines to investigate the ability of TSA to define selective pathways that promote HDACi toxicity.
Results
HDACi induced a DNA damage response and reduced viability in all repair deficient DT40 mutants although ATM-nulls were least affected. The most dramatic sensitivity was observed in mutants lacking the homology dependent repair (HDR) factor BLM or the non-homologous end-joining (NHEJ) and HDR factors, KU/RAD54, suggesting an involvement of either HDR or NHEJ in HDACi-induced cell death. To extend these findings, we measured the frequencies of HDR and NHEJ after HDACi treatment and monitored viability in human cell lines comparably deficient in HDR or NHEJ. Although no difference in HDR frequency was observed between HDACi treated and untreated cells, HDR-defective human cell lines were clearly more sensitive than wild type. Unexpectedly, cells treated with HDACis showed a significantly elevated NHEJ frequency.
Conclusions
HDACi targeting drugs induced significant increases in NHEJ activity in human cell lines but did not alter HDR frequency. Moreover, HDR is required for cellular resistance to HDACi therapy; therefore, NHEJ does not appear to be a critical axis for HDACi resistance. Rather, HDACi compounds induced DNA damage, most likely double strand breaks (DSBs), and HDR proficiency is correlated with cell survival.
doi:10.1371/journal.pone.0087203
PMCID: PMC3900704  PMID: 24466340
5.  Limiting the Persistence of a Chromosome Break Diminishes Its Mutagenic Potential 
PLoS Genetics  2009;5(10):e1000683.
To characterize the repair pathways of chromosome double-strand breaks (DSBs), one approach involves monitoring the repair of site-specific DSBs generated by rare-cutting endonucleases, such as I-SceI. Using this method, we first describe the roles of Ercc1, Msh2, Nbs1, Xrcc4, and Brca1 in a set of distinct repair events. Subsequently, we considered that the outcome of such assays could be influenced by the persistent nature of I-SceI-induced DSBs, in that end-joining (EJ) products that restore the I-SceI site are prone to repeated cutting. To address this aspect of repair, we modified I-SceI-induced DSBs by co-expressing I-SceI with a non-processive 3′ exonuclease, Trex2, which we predicted would cause partial degradation of I-SceI 3′ overhangs. We find that Trex2 expression facilitates the formation of I-SceI-resistant EJ products, which reduces the potential for repeated cutting by I-SceI and, hence, limits the persistence of I-SceI-induced DSBs. Using this approach, we find that Trex2 expression causes a significant reduction in the frequency of repair pathways that result in substantial deletion mutations: EJ between distal ends of two tandem DSBs, single-strand annealing, and alternative-NHEJ. In contrast, Trex2 expression does not inhibit homology-directed repair. These results indicate that limiting the persistence of a DSB causes a reduction in the frequency of repair pathways that lead to significant genetic loss. Furthermore, we find that individual genetic factors play distinct roles during repair of non-cohesive DSB ends that are generated via co-expression of I-SceI with Trex2.
Author Summary
A deleterious lesion in DNA is a break of both strands, or a chromosome double-strand break (DSB). DSBs can arise during normal cellular metabolism, but are also a consequence of many forms of cancer therapy. If DSBs are not repaired prior to cell division, entire segments of a chromosome can be lost. Several pathways ensure that DSBs are repaired, though some pathways are prone to causing mutations and/or chromosomal rearrangements, each of which can contribute to cancer development. In the first part of this study, we describe the roles of individual genetic factors in distinct repair pathways of DSBs generated by the I-SceI endonuclease. From these studies, we find that some factors can function in multiple repair pathways. In the second part of this study, we present a method for partially degrading the cohesive DSB overhangs that are generated by I-SceI, which we find facilitates repair products that are not prone to being re-cut by the endonuclease. As a consequence, we have limited the persistence of such breaks, which we find causes a reduction in repair pathways that lead to significant genetic loss. As well, we use this method to characterize the role of individual genetic factors during the repair of non-cohesive DSB ends.
doi:10.1371/journal.pgen.1000683
PMCID: PMC2752804  PMID: 19834534
6.  Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells 
PLoS Genetics  2010;6(2):e1000855.
The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways—the main Ku heterodimer-dependent or “classic” NHEJ (C-NHEJ) pathway and an “alternative” NHEJ (A-NHEJ) pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PKcs, XLF, and LIGIV), and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PKcs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PKcs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice.
Author Summary
Humans utilize at least two major pathways to repair DNA double-strand breaks (DSBs): homologous recombination (HR) and non-homologous end joining (NHEJ), and there are at least two genetically discrete sub-pathways of NHEJ: classical-NHEJ (C-NHEJ) and alternative-NHEJ (A-NHEJ). Since the products generated by each of these three repair (sub)pathways differ substantially from one another, it is biologically critical that certain DSBs are repaired by certain DSB repair pathways. How this pathway choice is made in human cells was unclear. In this study, knockout human cell lines that are defective in core C-NHEJ factors were generated. These cell lines are by-and-large extremely deficient in DSB repair, proving that C-NHEJ is the major DSB repair pathway in human cells. Unexpectedly, cell lines reduced for the C-NHEJ factors Ku70 or Ku86, carried out proficient DSB repair because of hyperactive A-NHEJ. In published work we have also demonstrated that Ku suppresses HR throughout the genome and at telomeres. Collectively, these data imply that Ku ensures that C-NHEJ is the major DSB repair pathway by two mechanisms: i) enabling C-NHEJ and ii) by actively suppressing HR and A-NHEJ. Thus, Ku is the critical regulator of pathway choice in human somatic cells.
doi:10.1371/journal.pgen.1000855
PMCID: PMC2829059  PMID: 20195511
7.  Saccharomyces cerevisiae DNA Ligase IV Supports Imprecise End Joining Independently of Its Catalytic Activity 
PLoS Genetics  2013;9(6):e1003599.
DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5′ resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.
Author Summary
Chromosomal rearrangements are common driver mutations in human genetic disease and cancer. The junctions observed at rearrangements typically show only a few base pairs in common between the partners, suggesting that they were formed by the end-to-end joining process, nonhomologous end joining (NHEJ). However, there is uncertainty about the mechanisms that actually create mutated junctions. DNA ligase IV catalyzes restorative double-strand break (DSB) joining in the canonical NHEJ pathway, but increasing evidence suggests that distinct NHEJ pathways that use DNA ligases I and/or III might be more important for mutations. We used yeast to study the in vivo consequence of having DNA ligase IV that was catalytically inactive but that nonetheless accumulated at DSBs normally. We detected mutated junctions in some assays that required DNA ligase IV protein but not its catalytic activity. This pattern suggests that DNA ligase I creates many mutated junctions when DNA ligase IV is present and that this can become a predominant mode of repair when DNA ligase IV activity is inefficient. Our yeast ligase IV mutations have properties similar to those observed in the human ligase IV syndrome, underscoring the relevance of these observations.
doi:10.1371/journal.pgen.1003599
PMCID: PMC3694833  PMID: 23825968
8.  ATM Release at Resected Double-Strand Breaks Provides Heterochromatin Reconstitution to Facilitate Homologous Recombination 
PLoS Genetics  2013;9(8):e1003667.
Non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two main pathways for repairing DNA double-strand breaks (DSBs). During the G2 phase of the mammalian cell cycle, both processes can operate and chromatin structure is one important factor which determines DSB repair pathway choice. ATM facilitates the repair of heterochromatic DSBs by phosphorylating and inactivating the heterochromatin building factor KAP-1, leading to local chromatin relaxation. Here, we show that ATM accumulation and activity is strongly diminished at DSBs undergoing end-resection during HR. Such DSBs remain unrepaired in cells devoid of the HR factors BRCA2, XRCC3 or RAD51. Strikingly, depletion of KAP-1 or expression of phospho-mimic KAP-1 allows repair of resected DSBs in the absence of BRCA2, XRCC3 or RAD51 by an erroneous PARP-dependent alt-NHEJ process. We suggest that DSBs in heterochromatin elicit initial local heterochromatin relaxation which is reversed during HR due to the release of ATM from resection break ends. The restored heterochromatic structure facilitates HR and prevents usage of error-prone alternative processes.
Author Summary
Double-strand breaks (DSBs) are critical DNA lesions because they can lead to cell death or, which is even more devastating, the formation of genomic rearrangements. Cells are equipped with two main pathways to repair such lesions, homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is an error-free process and completely restores the genetic information, whereas NHEJ has the potential to form genomic rearrangements. We have previously shown that the structure of the chromatin is one important factor which determines the choice between these two pathways, such that DSBs localizing to highly condensed heterochromatic regions are mainly repaired by HR and breaks in more open euchromatic DNA undergo repair by NHEJ. Here, we investigate this aspect of DSB repair pathway choice. We show that DSB end-resection, which channels DSB repair into the process of HR, counteracts the profound local relaxation which initially takes place at the break site and reconstitutes the heterochromatic structure. Cells which are genetically modified, such that they cannot reconstitute the heterochromatic structure at resected DSBs, fail to employ HR and instead repair heterochromatic DSBs by alternative NHEJ mechanisms. Thus, chromatin modifications which occur during the process of end-resection prevent error-prone repair pathways from generating genomic rearrangements.
doi:10.1371/journal.pgen.1003667
PMCID: PMC3731223  PMID: 23935532
9.  RAD50 Is Required for Efficient Initiation of Resection and Recombinational Repair at Random, γ-Induced Double-Strand Break Ends 
PLoS Genetics  2009;5(9):e1000656.
Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.
Author Summary
Double-strand breaks (DSBs) in chromosomal DNA are common sources of genomic change that may be beneficial or deleterious to an organism, from yeast to humans. While they can arise through programmed cellular events, DSBs are frequently associated with defective chromosomal replication, and they are induced by various types of DNA damaging agents such as those employed in cancer therapy, especially ionizing radiation. Elaborate systems have evolved for DSB recognition and subsequent repair, either by homologous recombination or by direct joining of ends. Although much is known about repair mechanisms associated with defined, artificially produced DSBs, there is a relative dearth of information about events surrounding random DSBs. Using a novel, yeast-based system that is applicable to other organisms, we have addressed resection at DSBs, considered a first step in repair. We provide the first direct evidence that cells possess a highly efficient system for recognition and initiation of resection at γ-radiation–induced dirty ends and that the resection is largely dependent on the Rad50/Mre11/Xrs2 complex, identified by the RAD50 gene. The system provides unique opportunities to address other components in resection and repair as well as to identify the contribution of random DSBs and resection to genome instability resulting from other DNA damaging agents.
doi:10.1371/journal.pgen.1000656
PMCID: PMC2734177  PMID: 19763170
10.  Nucleosome resection at a double-strand break during Non-Homologous Ends Joining in mammalian cells - implications from repressive chromatin organization and the role of ARTEMIS 
BMC Research Notes  2011;4:13.
Background
The S. cerevisiae mating type switch model of double-strand break (DSB) repair, utilizing the HO endonuclease, is one of the best studied systems for both Homologous Recombination Repair (HRR) and direct ends-joining repair (Non-Homologous Ends Joining - NHEJ). We have recently transposed that system to a mammalian cell culture model taking advantage of an adenovirus expressing HO and an integrated genomic target. This made it possible to compare directly the mechanism of repair between yeast and mammalian cells for the same type of induced DSB. Studies of DSB repair have emphasized commonality of features, proteins and machineries between organisms, and differences when conservation is not found. Two proteins that stand out that differ between yeast and mammalian cells are DNA-PK, a protein kinase that is activated by the presence of DSBs, and Artemis, a nuclease whose activity is modulated by DNA-PK and ATM. In this report we describe how these two proteins may be involved in a specific pattern of ends-processing at the DSB, particularly in the context of heterochromatin.
Findings
We previously published that the repair of the HO-induced DSB was generally accurate and occurred by simple rejoining of the cohesive 3'-overhangs generated by HO. During continuous passage of those cells in the absence of puromycin selection, the locus appears to have become more heterochromatic and silenced by displaying several features. 1) The site had become less accessible to cleavage by the HO endonuclease; 2) the expression of the puro mRNA, which confers resistance to puromycin, had become reduced; 3) occupancy of nucleosomes at the site (ChIP for histone H3) was increased, an indicator for more condensed chromatin. After reselection of these cells by addition of puromycin, many of these features were reversed. However, even the reselected cells were not identical in the pattern of cleavage and repair as the cells when originally created. Specifically, the pattern of repair revealed discrete deletions at the DSB that indicated unit losses of nucleosomes (or other protein complexes) before religation, represented by a ladder of PCR products reminiscent of an internucleosomal cleavage that is typically observed during apoptosis. This pattern of cleavage suggested to us that perhaps, Artemis, a protein that is believed to generate the internucleosomal fragments during apoptosis and in DSB repair, was involved in that specific pattern of ends-processing. Preliminary evidence indicates that this may be the case, since knock-down of Artemis with siRNA eliminated the laddering pattern and revealed instead an extensive exonucleolytic processing of the ends before religation.
Conclusions
e have generated a system in mammalian cells where the absence of positive selection resulted in chromatin remodeling at the target locus that recapitulates many of the features of the mating-type switching system in yeast. Specifically, just as for yeast HML and HMR, the locus had become transcriptionally repressed; accessibility to cleavage by the HO endonuclease was reduced; and processing of the ends was drastically changed. The switch was from high-fidelity religation of the cohesive ends, to a pattern of release of internucleosomal fragments, perhaps in search of micro-homology stretches for ligation. This is consistent with reports that the involvement of ATM, DNA-PK and Artemis in DSB repair is largely focused to heterochromatic regions, and not required for the majority of IR-induced DSB repair foci in euchromatin.
doi:10.1186/1756-0500-4-13
PMCID: PMC3035584  PMID: 21255428
11.  Coincident Resection at Both Ends of Random, γ–Induced Double-Strand Breaks Requires MRX (MRN), Sae2 (Ctp1), and Mre11-Nuclease 
PLoS Genetics  2013;9(3):e1003420.
Resection is an early step in homology-directed recombinational repair (HDRR) of DNA double-strand breaks (DSBs). Resection enables strand invasion as well as reannealing following DNA synthesis across a DSB to assure efficient HDRR. While resection of only one end could result in genome instability, it has not been feasible to address events at both ends of a DSB, or to distinguish 1- versus 2-end resections at random, radiation-induced “dirty” DSBs or even enzyme-induced “clean” DSBs. Previously, we quantitatively addressed resection and the role of Mre11/Rad50/Xrs2 complex (MRX) at random DSBs in circular chromosomes within budding yeast based on reduced pulsed-field gel electrophoretic mobility (“PFGE-shift”). Here, we extend PFGE analysis to a second dimension and demonstrate unique patterns associated with 0-, 1-, and 2-end resections at DSBs, providing opportunities to examine coincidence of resection. In G2-arrested WT, Δrad51 and Δrad52 cells deficient in late stages of HDRR, resection occurs at both ends of γ-DSBs. However, for radiation-induced and I-SceI-induced DSBs, 1-end resections predominate in MRX (MRN) null mutants with or without Ku70. Surprisingly, Sae2 (Ctp1/CtIP) and Mre11 nuclease-deficient mutants have similar responses, although there is less impact on repair. Thus, we provide direct molecular characterization of coincident resection at random, radiation-induced DSBs and show that rapid and coincident initiation of resection at γ-DSBs requires MRX, Sae2 protein, and Mre11 nuclease. Structural features of MRX complex are consistent with coincident resection being due to an ability to interact with both DSB ends to directly coordinate resection. Interestingly, coincident resection at clean I-SceI-induced breaks is much less dependent on Mre11 nuclease or Sae2, contrary to a strong dependence on MRX complex, suggesting different roles for these functions at “dirty” and clean DSB ends. These approaches apply to resection at other DSBs. Given evolutionary conservation, the observations are relevant to DNA repair in human cells.
Author Summary
DNA double-strand breaks (DSBs) can cause genome instability and cancer. While repair can occur through recombination, coincident events at both ends—while assumed—have not been directly addressable at unique or random damage-induced DSBs. Here, we describe pulse-field gel electrophoresis approaches that for the first time distinguish resection at 0, 1, or both ends of DSBs. Resection, an early step in DSB end-processing, is efficiently initiated at both ends of random, radiation-induced DSBs in wild-type budding yeast and in cells deficient in late steps of recombinational repair. However, 0- and 1-end resections predominate in MRX-null, Sae2, and Mre11 nuclease mutants, suggesting new roles for the cancer-related proteins (Ctp1 and MRN in humans) in repair, namely, efficient and coincident resection at both ends of a DSB. We suggest that the structural features of the MRX complex are consistent with coincident resection being due to an ability to interact with both DSB ends to directly coordinate resection. Interestingly, we provide direct evidence that coincident resection at a clean I-SceI-induced break is much less dependent on the Mre11 nuclease or Sae2, contrary to the strong dependence on the MRX complex. These results suggest a differential role for these functions at “dirty” and clean DSB ends.
doi:10.1371/journal.pgen.1003420
PMCID: PMC3610664  PMID: 23555316
12.  Nampt is involved in DNA double-strand break repair 
Chinese Journal of Cancer  2012;31(8):392-398.
DNA double-strand break (DSB) is the most severe form of DNA damage, which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer. Nicotinamide phosphoribosyltransferase (Nampt), which is involved in nicotinamide adenine dinucleotide metabolism, is overexpressed in a variety of tumors. In this report, we found that Nampt physically associated with CtIP and DNA-PKcs/Ku80, which are key factors in HR and NHEJ, respectively. Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair. Furthermore, the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining, indicating a delay in the onset of cellular senescence in normal human fibroblasts. Taken together, our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair, contributing to the acceleration of cellular senescence.
doi:10.5732/cjc.012.10089
PMCID: PMC3777511  PMID: 22704488
Nampt; DNA-PKcs/Ku80; CtIP; DNA repair; cellular senescence
13.  The Forkhead Box M1 protein regulates BRIP1 expression and DNA damage repair in epirubicin treatment 
Oncogene  2012;32(39):10.1038/onc.2012.491.
FOXM1 is implicated in genotoxic drug resistance but its role and mechanism of action remain unclear. Here, we establish that γH2AX foci, indicative of DNA double strand breaks, accumulate in a time-dependent manner in the drug sensitive MCF-7 cells but not in the resistant counterparts in response to epirubicin. We find that FOXM1 expression is associated with epirubicin sensitivity and double strand break (DSB) repair. Ectopic expression of FOXM1 can increase cell viability and abrogate DSBs sustained by MCF-7 cells following epirubicin, owing to an enhancement in repair efficiency. Conversely, alkaline comet and γH2AX foci formation assays show that Foxm1-null cells are hypersensitive to DNA damage, epirubicin and γ-irradiation. Furthermore, we find that FOXM1 is required for DNA repair by homologous recombination (HR) but not non-homologous end joining (NHEJ), using HeLa cell lines habouring an integrated direct repeat green fluorescent protein (DR-GFP) reporter for DSB repair. We also identify BRIP1 as a direct transcription target of FOXM1 by promoter analysis and chromatin-immunoprecipitation assay. In agreement, depletion of FOXM1 expression by siRNA down-regulates BRIP1 expression at the protein and mRNA levels in MCF-7 and the epirubicin resistant MCF-7 EpiR cells. Remarkably, the requirement for FOXM1 for DSB repair can be circumvented by reintroduction of BRIP1, suggesting that BRIP1 is an important target of FOXM1 in DSB repair. Indeed, like FOXM1, BRIP1 is needed for HR. These data suggest that FOXM1 regulates BRIP1 expression to modulate epirubicin-induced DNA damage repair and drug resistance.
doi:10.1038/onc.2012.491
PMCID: PMC3874579  PMID: 23108394
FOXM1; BRIP1; DNA damage; epirubicin; resistance; breast cancer
14.  Release of Ku and MRN from DNA Ends by Mre11 Nuclease Activity and Ctp1 Is Required for Homologous Recombination Repair of Double-Strand Breaks 
PLoS Genetics  2011;7(9):e1002271.
The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5′-to-3′ resection of DNA ends, generating 3′ single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair.
Author Summary
A double-strand break (DSB) is a devastating form of DNA damage. Fortunately, cells are equipped with two DSB repair pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ). The Mre11-Rad50-Nbs1 (MRN) protein complex recognizes DSBs and initiates HR repair. The Mre11 subunit harbors a nuclease domain that is essential for repair in fission yeast and mammals, although the function is unknown. Here we show that Mre11 nuclease activity is required to release the Ku complex from DNA ends in fission yeast. While the initiation of repair, i.e. the generation of single-stranded DNA (ssDNA) overhangs in Mre11-nuclease dead mutants, is unaffected, we find that an essential downstream step involving the localization of Replication Protein A (RPA) to ssDNA is substantially decreased due to the inability to release Ku and MRN from the DNA end. In contrast, a DNA processing factor called Ctp1, which binds to Nbs1, is essential for the initiation of repair as well as the release of Ku and MRN from DNA ends. Importantly, we find that efficient localization of RPA, which is essential for efficient DSB repair by HR, requires the release of Ku and MRN from the DNA by the combined action of Mre11 nuclease and Ctp1.
doi:10.1371/journal.pgen.1002271
PMCID: PMC3169521  PMID: 21931565
15.  Involvement of Nucleotide Excision and Mismatch Repair Mechanisms in Double Strand Break Repair 
Current Genomics  2009;10(4):250-258.
Living organisms are constantly threatened by environmental DNA-damaging agents, including UV and ionizing radiation (IR). Repair of various forms of DNA damage caused by IR is normally thought to follow lesion-specific repair pathways with distinct enzymatic machinery. DNA double strand break is one of the most serious kinds of damage induced by IR, which is repaired through double strand break (DSB) repair mechanisms, including homologous recombination (HR) and non-homologous end joining (NHEJ). However, recent studies have presented increasing evidence that various DNA repair pathways are not separated, but well interlinked. It has been suggested that non-DSB repair mechanisms, such as Nucleotide Excision Repair (NER), Mismatch Repair (MMR) and cell cycle regulation, are highly involved in DSB repairs. These findings revealed previously unrecognized roles of various non-DSB repair genes and indicated that a successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems. One of our recent studies found that suppressed expression of non-DSB repair genes, such as XPA, RPA and MLH1, influenced the yield of IR induced micronuclei formation and/or chromosome aberrations, suggesting that these genes are highly involved in DSB repair and DSB-related cell cycle arrest, which reveals new roles for these gene products in the DNA repair network. In this review, we summarize current progress on the function of non-DSB repair-related proteins, especially those that participate in NER and MMR pathways, and their influence on DSB repair. In addition, we present our developing view that the DSB repair mechanisms are more complex and are regulated by not only the well known HR/NHEJ pathways, but also a systematically coordinated cellular network.
doi:10.2174/138920209788488544
PMCID: PMC2709936  PMID: 19949546
Ionizing radiation (IR); DNA damage; DSB repair; NER; MMR and cell cycle.
16.  The interaction between CtIP and BRCA1 is not essential for resection-mediated DNA repair or tumor suppression 
The Journal of Cell Biology  2013;201(5):693-707.
In mammalian cells, the phospho-dependent interaction between BRCA1 and CtIP is not required for homology-directed DNA repair or tumor suppression.
The CtIP protein facilitates homology-directed repair (HDR) of double-strand DNA breaks (DSBs) by initiating DNA resection, a process in which DSB ends are converted into 3′-ssDNA overhangs. The BRCA1 tumor suppressor, which interacts with CtIP in a phospho-dependent manner, has also been implicated in DSB repair through the HDR pathway. It was recently reported that the BRCA1–CtIP interaction is essential for HDR in chicken DT40 cells. To examine the role of this interaction in mammalian cells, we generated cells and mice that express Ctip polypeptides (Ctip-S326A) that fail to bind BRCA1. Surprisingly, isogenic lines of Ctip-S326A mutant and wild-type cells displayed comparable levels of HDR function and chromosomal stability. Although Ctip-S326A mutant cells were modestly sensitive to topoisomerase inhibitors, mice expressing Ctip-S326A polypeptides developed normally and did not exhibit a predisposition to cancer. Thus, in mammals, the phospho-dependent BRCA1–CtIP interaction is not essential for HDR-mediated DSB repair or for tumor suppression.
doi:10.1083/jcb.201302145
PMCID: PMC3664708  PMID: 23712259
17.  Alternative Pathways for the Repair of RAG-Induced DNA Breaks 
Molecular and Cellular Biology  2006;26(1):131-139.
RAG1 and RAG2 cleave DNA to generate blunt signal ends and hairpin coding ends at antigen receptor loci in lymphoid cells. During V(D)J recombination, repair of these RAG-generated double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway contributes substantially to the antigen receptor diversity necessary for immune system function, although recent evidence also supports the ability of RAG-generated breaks to undergo homology-directed repair (HDR). We have determined that RAG-generated chromosomal breaks can be repaired by pathways other than NHEJ in mouse embryonic stem (ES) cells, although repair by these pathways occurs at a significantly lower frequency than NHEJ. HDR frequency was estimated to be ≥40-fold lower than NHEJ frequency for both coding end and signal end reporters. Repair by single-strand annealing was estimated to occur at a comparable or lower frequency than HDR. As expected, V(D)J recombination was substantially impaired in cells deficient for the NHEJ components Ku70, XRCC4, and DNA-PKcs. Concomitant with decreased NHEJ, RAG-induced HDR was increased in each of the mutants, including cells lacking DNA-PKcs, which has been implicated in hairpin opening. HDR was increased to the largest extent in Ku70−/− cells, implicating the Ku70/80 DNA end-binding protein in regulating pathway choice. Thus, RAG-generated DSBs are typically repaired by the NHEJ pathway in ES cells, but in the absence of NHEJ components, a substantial fraction of breaks can be efficiently channeled into alternative pathways in these cells.
doi:10.1128/MCB.26.1.131-139.2006
PMCID: PMC1317616  PMID: 16354685
18.  MicroRNAs down-regulate homologous recombination in the G1 phase of cycling cells to maintain genomic stability 
eLife  2014;3:e02445.
Homologous recombination (HR)-mediated repair of DNA double-strand break (DSB)s is restricted to the post-replicative phases of the cell cycle. Initiation of HR in the G1 phase blocks non-homologous end joining (NHEJ) impairing DSB repair. Completion of HR in G1 cells can lead to the loss-of-heterozygosity (LOH), which is potentially carcinogenic. We conducted a gain-of-function screen to identify miRNAs that regulate HR-mediated DSB repair, and of these miRNAs, miR-1255b, miR-148b*, and miR-193b* specifically suppress the HR-pathway in the G1 phase. These miRNAs target the transcripts of HR factors, BRCA1, BRCA2, and RAD51, and inhibiting miR-1255b, miR-148b*, and miR-193b* increases expression of BRCA1/BRCA2/RAD51 specifically in the G1-phase leading to impaired DSB repair. Depletion of CtIP, a BRCA1-associated DNA end resection protein, rescues this phenotype. Furthermore, deletion of miR-1255b, miR-148b*, and miR-193b* in independent cohorts of ovarian tumors correlates with significant increase in LOH events/chromosomal aberrations and BRCA1 expression.
DOI: http://dx.doi.org/10.7554/eLife.02445.001
eLife digest
The DNA in a cell is damaged thousands of times every day. One of the most serious types of damage involves something breaking both of the strands in the double helix. Such a double-strand break can delete genes or even kill the cell. In fact, conventional cancer therapy kills cancer cells by causing irreparable double-strand breaks. Conversely, a normal cell that is constantly exposed to DNA damaging agents can become a tumor if double-strand breaks are incorrectly repaired. An efficient and accurate double-strand break repair system needs to be in place to prevent this transformation. Therefore, an in-depth understanding of double-strand break repair and the factors involved are important for both gaining insight into the cause of cancer and to improve cancer therapy.
Cells have evolved several different ways to detect and repair double-strand breaks. A method called homologous recombination, for example, uses an undamaged DNA molecule as a template that can be copied to make new DNA. Since it needs a readily available DNA template, this method only works in phases of the cell growth cycle where there are many copies of DNA—that is, in the post-DNA replication phases. In particular, homologous recombination does not work during the pre-replication, G1 phase. If homologous recombination is attempted during G1, it will block the other methods employed by cells to repair broken strands of DNA.
An important challenge is to understand how homologous recombination is restricted to particular parts of the cell cycle. Although certain proteins associated with the early stages of double-strand repair are thought to determine the type of DNA repair that occurs, the details of this process are not fully understood.
One group of molecules that are thought to be involved are microRNAs, which normally limit the number of proteins produced from certain genes. However, since a single microRNA molecule can be associated with several proteins, and since a single protein can be associated with several microRNA molecules, it has proved difficult to establish the exact effects of a specific microRNA molecule.
Choi et al. now show that seven microRNA molecules can control homologous recombination, and three microRNAs in particular restrict homologous recombination during the G1 phase of the cell cycle. If these microRNAs are inhibited during the G1 phase, which allows homologous recombination to start, and counter-intuitively more double-stranded breaks are seen. However, if a gene involved in starting homologous repair–called CtIP—is silenced while the microRNAs are inhibited, then the DNA breaks are repaired. Exactly, how the microRNA molecules produce different effects during different phases of the cell cycle will be need to be investigated by future studies.
DOI: http://dx.doi.org/10.7554/eLife.02445.002
doi:10.7554/eLife.02445
PMCID: PMC4031983  PMID: 24843000
DNA repair; BRCA1; cell cycle; human; mouse
19.  Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate 
Mutagenesis  2011;26(6):795-803.
Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments.
doi:10.1093/mutage/ger049
PMCID: PMC3198890  PMID: 21811007
20.  The UIM-containing protein RAP80 interacts with BRCA1 and functions in DNA damage repair response 
Cancer research  2007;67(14):6647-6656.
In this study, we examine the potential role of receptor-associated protein 80 (RAP80), a nuclear protein containing two ubiquitin-interacting motifs (UIMs), in DNA damage response and double-strand break (DSB) repair. We demonstrate that following ionizing radiation (IR) and treatment with DNA-damaging agents RAP80 translocates to discrete nuclear foci that co-localize with those of γ-H2AX. The UIMs and the region between aa 204–304 are critical for the re-localization of RAP80 to IR-induced foci (IRIF). These observations suggest that RAP80 becomes part of a DNA-repair complex at the sites of IRIF. We also demonstrate that RAP80 forms a complex with the tumor repressor BRCA1 and that this interaction is mediated through the BRCT repeats of BRCA1. The UIMs are not required for the interaction of RAP80 with BRCA1. Knockdown of RAP80 in HEK293 cells significantly reduced DSB-induced homology-directed recombination (HDR). Moreover, inhibition RAP80 expression by siRNA increased radiosensitivity, whereas increased radioresistance was observed in human breast cancer MCF-7 cells over-expression of RAP80. Taken together, our data suggest that RAP80 plays an important role in DNA damage response signaling and HDR-mediated DSB repair. We further demonstrate that RAP80 can function as a substrate of the ataxia-telangiectasia mutated (ATM) protein kinase in vitro which phosphorylates RAP80 at Ser205 and Ser402. We show that this phosphorylation is not required for the migration of RAP80 to IRIF.
doi:10.1158/0008-5472.CAN-07-0924
PMCID: PMC2391092  PMID: 17621610
RAP80; UIMC1; ubiquitin-interacting motif; DNA damage; ATM; BRCA1; BRCT
21.  TDP2–Dependent Non-Homologous End-Joining Protects against Topoisomerase II–Induced DNA Breaks and Genome Instability in Cells and In Vivo 
PLoS Genetics  2013;9(3):e1003226.
Anticancer topoisomerase “poisons” exploit the break-and-rejoining mechanism of topoisomerase II (TOP2) to generate TOP2-linked DNA double-strand breaks (DSBs). This characteristic underlies the clinical efficacy of TOP2 poisons, but is also implicated in chromosomal translocations and genome instability associated with secondary, treatment-related, haematological malignancy. Despite this relevance for cancer therapy, the mechanistic aspects governing repair of TOP2-induced DSBs and the physiological consequences that absent or aberrant repair can have are still poorly understood. To address these deficits, we employed cells and mice lacking tyrosyl DNA phosphodiesterase 2 (TDP2), an enzyme that hydrolyses 5′-phosphotyrosyl bonds at TOP2-associated DSBs, and studied their response to TOP2 poisons. Our results demonstrate that TDP2 functions in non-homologous end-joining (NHEJ) and liberates DSB termini that are competent for ligation. Moreover, we show that the absence of TDP2 in cells impairs not only the capacity to repair TOP2-induced DSBs but also the accuracy of the process, thus compromising genome integrity. Most importantly, we find this TDP2-dependent NHEJ mechanism to be physiologically relevant, as Tdp2-deleted mice are sensitive to TOP2-induced damage, displaying marked lymphoid toxicity, severe intestinal damage, and increased genome instability in the bone marrow. Collectively, our data reveal TDP2-mediated error-free NHEJ as an efficient and accurate mechanism to repair TOP2-induced DSBs. Given the widespread use of TOP2 poisons in cancer chemotherapy, this raises the possibility of TDP2 being an important etiological factor in the response of tumours to this type of agent and in the development of treatment-related malignancy.
Author Summary
DNA double-strand breaks (DSBs) are dangerous because they can lead to cellular death and tissue degeneration if not repaired, or to genome rearrangements, which are a common hallmark of cancer, if repaired incorrectly. Although required for all chromosomal transitions in cells, transient DNA cleavage by topoisomerase II (TOP2) is a potential endogenous source of DSBs, which are characteristic in that TOP2 remains covalently bound to the DNA termini. In addition, numerous chemotherapeutic regimes rely on compounds that “poison” TOP2 activity, stimulating the formation of DSBs that target tumour cells. However, these compounds also affect healthy tissue and confer undesirable side effects, including the stimulation of genome rearrangements that can trigger secondary malignancies (mainly acute leukemia). Identifying the factors that participate in the repair of TOP2-induced DSBs and fully understanding their mechanism of action are therefore important for the design of chemotherapeutic regimes that are more effective and safer. Here we demonstrate that TDP2, a recently identified protein that can liberate DSB termini from blocked TOP2, functions as part of established cellular DSB repair processes and is required to safeguard genome integrity upon treatment with TOP2 poisons, both in cells and in mice. These results can therefore have important implications in cancer treatment.
doi:10.1371/journal.pgen.1003226
PMCID: PMC3592926  PMID: 23505375
22.  Alkylation Base Damage Is Converted into Repairable Double-Strand Breaks and Complex Intermediates in G2 Cells Lacking AP Endonuclease 
PLoS Genetics  2011;7(4):e1002059.
DNA double-strand breaks (DSBs) are potent sources of genome instability. While there is considerable genetic and molecular information about the disposition of direct DSBs and breaks that arise during replication, relatively little is known about DSBs derived during processing of single-strand lesions, especially for the case of single-strand breaks (SSBs) with 3′-blocked termini generated in vivo. Using our recently developed assay for detecting end-processing at random DSBs in budding yeast, we show that single-strand lesions produced by the alkylating agent methyl methanesulfonate (MMS) can generate DSBs in G2-arrested cells, i.e., S-phase independent. These derived DSBs were observed in apn1/2 endonuclease mutants and resulted from aborted base excision repair leading to 3′ blocked single-strand breaks following the creation of abasic (AP) sites. DSB formation was reduced by additional mutations that affect processing of AP sites including ntg1, ntg2, and, unexpectedly, ogg1, or by a lack of AP sites due to deletion of the MAG1 glycosylase gene. Similar to direct DSBs, the derived DSBs were subject to MRX (Mre11, Rad50, Xrs2)-determined resection and relied upon the recombinational repair genes RAD51, RAD52, as well as on the MCD1 cohesin gene, for repair. In addition, we identified a novel DNA intermediate, detected as slow-moving chromosomal DNA (SMD) in pulsed field electrophoresis gels shortly after MMS exposure in apn1/2 cells. The SMD requires nicked AP sites, but is independent of resection/recombination processes, suggesting that it is a novel structure generated during processing of 3′-blocked SSBs. Collectively, this study provides new insights into the potential consequences of alkylation base damage in vivo, including creation of novel structures as well as generation and repair of DSBs in nonreplicating cells.
Author Summary
DNA double-strand breaks (DSBs) are an important source of genome instability that can lead to severe biological consequences including tumorigenesis and cell death. Although much is known about DSBs induced directly by ionizing radiation and radiomimetic cancer drugs, there is a relative dearth of information about the formation of derived DSBs that arise from processing of single-strand lesions. Since as many as 10,000–200,000 single-strand lesions have been estimated to occur each day in mammalian cells, conversion of even a small percentage of such lesions to DSBs could dramatically affect genome stability. Here we addressed the mechanism of formation and repair of derived DSBs in vivo during the processing of DNA methylation damage in yeast that are defective in base excision repair (BER) due to a lack of AP endonucleases. Armed with a technique developed in our lab that detects resection at DSBs, a first step in DSB repair, we demonstrated formation of DSBs in G2 cells and the role of recombinational repair in subsequent chromosome restitution. Furthermore, we have identified a novel repair intermediate that can be generated if abasic sites are nicked by AP lyases, providing additional insights into the processing of 3′-blocked groups at single-strand breaks.
doi:10.1371/journal.pgen.1002059
PMCID: PMC3084215  PMID: 21552545
23.  53BP1 regulates DSB repair using Rif1 to control 5’ end resection 
Science (New York, N.Y.)  2013;339(6120):700-704.
The choice between double-strand break (DSB) repair by either homology-directed repair (HDR) or non-homologous end-joining (NHEJ) is tightly regulated. Defects in this regulation can induce genome instability and cancer. 53BP1 is critical for the control of DSB repair, promoting NHEJ and inhibiting the 5’ end resection needed for HDR. Using dysfunctional telomeres and genome-wide DSBs, we identify Rif1 as the main factor used by 53BP1 to impair 5’ end resection. Rif1 inhibits resection involving CtIP, BLM, and Exo1, limits accumulation of BRCA1/BARD1 complexes at sites of DNA damage, and defines one of the mechanisms by which 53BP1 causes chromosomal abnormalities in Brca1-deficient cells. These data establish Rif1 as an important contributor to the control of DSB repair by 53BP1.
doi:10.1126/science.1231573
PMCID: PMC3664841  PMID: 23306437
24.  Translesion DNA synthesis-assisted non-homologous end-joining of complex double-strand breaks prevents loss of DNA sequences in mammalian cells 
Nucleic Acids Research  2009;37(20):6737-6745.
Double strand breaks (DSB) are severe DNA lesions, and if not properly repaired, may lead to cell death or cancer. While there is considerable data on the repair of simple DSB (sDSB) by non-homologous end-joining (NHEJ), little is known about the repair of complex DSBs (cDSB), namely breaks with a nearby modification, which precludes ligation without prior processing. To study the mechanism of cDSB repair we developed a plasmid-based shuttle assay for the repair of a defined site-specific cDSB in cultured mammalian cells. Using this assay we found that repair efficiency and accuracy of a cDSB with an abasic site in a 5′ overhang was reduced compared with a sDSB. Translesion DNA synthesis (TLS) across the abasic site located at the break prevented loss of DNA sequences, but was highly mutagenic also at the template base next to the abasic site. Similar to sDSB repair, cDSB repair was totally dependent on XrccIV, and altered in the absence of Ku80. In contrast, Artemis appears to be specifically involved in cDSB repair. These results may indicate that mammalian cells have a damage control strategy, whereby severe deletions are prevented at the expense of the less deleterious point mutations during NHEJ.
doi:10.1093/nar/gkp703
PMCID: PMC2777433  PMID: 19762482
25.  COM-1 Promotes Homologous Recombination during Caenorhabditis elegans Meiosis by Antagonizing Ku-Mediated Non-Homologous End Joining 
PLoS Genetics  2013;9(2):e1003276.
Successful completion of meiosis requires the induction and faithful repair of DNA double-strand breaks (DSBs). DSBs can be repaired via homologous recombination (HR) or non-homologous end joining (NHEJ), yet only repair via HR can generate the interhomolog crossovers (COs) needed for meiotic chromosome segregation. Here we identify COM-1, the homolog of CtIP/Sae2/Ctp1, as a crucial regulator of DSB repair pathway choice during Caenorhabditis elegans gametogenesis. COM-1–deficient germ cells repair meiotic DSBs via the error-prone pathway NHEJ, resulting in a lack of COs, extensive chromosomal aggregation, and near-complete embryonic lethality. In contrast to its yeast counterparts, COM-1 is not required for Spo11 removal and initiation of meiotic DSB repair, but instead promotes meiotic recombination by counteracting the NHEJ complex Ku. In fact, animals defective for both COM-1 and Ku are viable and proficient in CO formation. Further genetic dissection revealed that COM-1 acts parallel to the nuclease EXO-1 to promote interhomolog HR at early pachytene stage of meiotic prophase and thereby safeguards timely CO formation. Both of these nucleases, however, are dispensable for RAD-51 recruitment at late pachytene stage, when homolog-independent repair pathways predominate, suggesting further redundancy and/or temporal regulation of DNA end resection during meiotic prophase. Collectively, our results uncover the potentially lethal properties of NHEJ during meiosis and identify a critical role for COM-1 in NHEJ inhibition and CO assurance in germ cells.
Author Summary
Sexually reproducing animals create germ cells via meiosis, a cell division program that requires the induction and faithful repair of DNA double-strand breaks (DSBs). Meiotic DSBs are typically repaired via homologous recombination (HR), an error-free repair pathway that generates transient links between homologous chromosomes, named crossovers (COs), which are needed for proper chromosome segregation. To date, it is unclear how germ cells channel these programmed DSBs into HR and not into error-prone DSB repair pathways such as non-homologous end joining (NHEJ). We used the genetically tractable animal model Caenorhabditis elegans to study the mechanisms underlying the strong HR bias in germ cells. Here, we identify COM-1, the worm homolog of CtIP, as a crucial regulator of meiotic DSB repair pathway choice: COM-1 effectively blocks the action of the NHEJ complex Ku, thereby assuring correct repair via HR. In addition, we show that unscheduled NHEJ activity during meiosis leads to a lack of COs, extensive chromosomal aggregation, and near-complete embryonic lethality. Further genetic dissection also revealed a redundant and stage-specific role for COM-1 in meiotic HR. Our work thus establishes COM-1/CtIP as a caretaker of germline genome stability and unveils meiotic NHEJ as a potent source of chromosomal aberrations in newborns.
doi:10.1371/journal.pgen.1003276
PMCID: PMC3567172  PMID: 23408909

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